CN102648977A

CN102648977A - Application of follistatin-related protein 1 in adjusting Na, K-ATPase activity

Info

Publication number: CN102648977A
Application number: CN2011100469896A
Authority: CN
Inventors: 张旭; 鲍岚; 李开诚; 张方雄; 陆莹瑾
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 2011-02-25
Filing date: 2011-02-25
Publication date: 2012-08-29
Anticipated expiration: 2031-02-25
Also published as: CN102648977B

Abstract

The invention relates to an application of follistatin-related protein 1 (FSTL1) in adjusting Na, K-ATPase activity, in particular to an application of FSTL1 in improving the Na, K-ATPase activity so as to treat, release or improve diseases or symptoms related to the activity, or an application of FSTL1 inhibitor in reducing the Na, K-ATPase activity so as to treat, release or improve diseases or symptoms related to the activity. The invention further provides a drug for adjusting Na, K-ATPase activity and a novel screening method of the drug. The polypeptide, nucleotide, composition and method have a great application prospect in treating, releasing or improving diseases or symptoms related to the Na, K-ATPase activity.

Description

The purposes of Progynon inhibin appearance albumen 1 in regulating the na-k-atp enzyme activity

Technical field

The invention belongs to biological technical field, more specifically, the present invention relates to Progynon inhibin appearance albumen 1 (being FSTL1) thus improving the active purposes that is used for treatment, alleviates or improve relevant disease or symptom (especially pain) of na-k-atp enzyme.

Background technology

Present discovers: na-k-atp enzyme has important effect in the homergy of human body, be distributed widely on the cell membrane of various histoorgans, like kidney, heart, lung and nervous system etc.The activity of sodiumpotassium pump (being na-k-atp enzyme) and some disease or symptom are closely related, and its active raising or reduction produce significant effects to these diseases or symptom.

For example; Following disease or symptom are relevant with the hyperactivity of sodiumpotassium pump; Maybe can be eased through the activity that suppresses sodiumpotassium pump or treat: research shows that sodiumpotassium pump possibly have certain effect in the pathogenesis of renal hypertension; The activity of sodiumpotassium pump increases the promotion ion transport, thereby encourages hypertension (Wang etc., CurrOpin Nephrol Hypertens.2009Sep; 18 (5): 412-20; Ferrari etc., Cell Mol Biol (Noisy-le-grand) .2006Dec 30; 52 (8): 15-8); Bibliographical information α subunit expression of sodiumpotassium pump in the mankind's MC obviously raise (Mathieu etc., J Cell Mol Med.2009Feb 20. [Epubahead of print]) is arranged; The growth and the growth of tumor of the sensory neuron projection of cultivating are also relevant with the hyperactivity of sodiumpotassium pump with generation; Inhibitor such as digoxin that the bibliographical information sodiumpotassium pump is arranged; The growth of the sensory neuron projection that can obviously suppress to cultivate and suppress growth of tumor and generation (Penniyainen etc., NeurosciBehav Physiol.2009Mar; 39 (3): 301-4.; Mijatovic etc., Expert Opin Ther Targets.2008Nov; 12 (11): 1403-17.).The treatment target spot of bibliographical information sodiumpotassium pump α 1 for treatment glioma and nonsmall-cell lung cancer arranged; Sodiumpotassium pump α 1 inhibitor be hopeful to be used to treatment (like a kind of new derivatives 2 of cardiac glycoside " oxovoruscharin (UNBS1450)) treatment glioblastoma multiforme and nonsmall-cell lung cancer (KissR etc. .Neoplasia.2008Mar; 10 (3): 198-206; Kiss R etc., J Pathol.2007Jun; 212 (2): 170-9.).

Following disease or symptom are active on the low side relevant with sodiumpotassium pump; Maybe can be eased through the activity that improves sodiumpotassium pump or treat: at familial hemiplegia type migraine disease philtrum; Having found has more deletion mutation on the sodiumpotassium pump, confirmed that these sudden changes and clinical symptoms have confidential relation; Also have some patients epilepsy symptom (Aperia, J Intern Med.2007 Jan can occur; 261 (1): 44-52.); The activity of in the animal model of diabetes, finding sodiumpotassium pump all has reduction with expressing; Wherein the C polypeptide in the erythrocyte also has tangible minimizing; External source is replenished the C polypeptide can obviously improve the activity of sodiumpotassium pump and symptom (Vague etc., the ExpDiabesity Res.2004Jan-Mar of diabetes-alleviating; 5 (1): 37-50.); Sodiumpotassium pump in some serious symptom patients with depression on the erythrocyte is active obviously to be reduced, and gives lithium salts treatment back and takes a turn for the better; The activity of the inhibition sodiumpotassium pump of moderate also can cause manic generation (Lichtstein and Rosen, Neurochem Res.2001 Sep; 26 (8-9): 971-8.); The digitalism of seeing clinically be exactly since excessively suppress the activity of sodiumpotassium pump cause cardiac muscle be overexcited due to (Vivo etc., Am JMed Sci.2008Nov; 336 (5): 423-8.); Former studies confirms that also the activity that improves the alveolar sodiumpotassium pump can obviously improve injury of lung, the symptom of pulmonary edema (Factor etc., J Clin Invest.1998 Oct 1; 102 (7): 1421-30.; Lecuona etc., J Bioenerg Biomembr.2007Dec; 39 (5-6): 391-5.; Sznajder etc., J Appl Physiol.2002 Nov; 93 (5): 1860-6).

(Dorsal Root Ganglia, small neuron DRG) are responsible for transmitting the thermo aesthesia and the pain sensation to spinal cord dorsal root ganglion.When outer circumference end received physics or chemical irritation, the centripetal fiber end of these small neurons discharged excitatory transmitter glutamic acid and neuropeptide in synaptic vesicle or the big densified core bubble (LDCV) at cornu dorsale medullae spinalis slightly through the exocytosis form.Think that at present the transmission of this irritability information can be comprised neuropeptide material opioid peptide, neurotransmitter GABA (GBGA), glycine (Glycine) and 5-hydroxy tryptamine adjustings such as (Serotonin) by the inhibition factor of the relay cell of cornu dorsale medullae spinalis and downlink release.This inhibition is regulated for normal somesthesia and pain sensation modulation all has the significance on the function.

Had been found that receptor and the passage of some selectivity high expresseds at dorsal root ganglion small neuron surface of cell membrane, this has deepened the understanding of people to the sensory information pass through mechanism.These receptors and passage comprise the P2X of ATP gate ₃Receptor, transient receptor potential channel V1 (TRPV1) and A1 (TRPA1), valtage-gated Na _v1.8 the relevant g protein coupled receptor of passage with Mas.These membrane surface molecules are relevant with the facilitation process that the sensitization and the nocuity of elementary sensation transmission, sensory neuron react.

Although at present this area had been found that more above-mentioned in sensory information transmits acting molecule; Yet also press for research and in the sensory information transmittance process, also have those molecules in action; With the understanding that promotes sensory information is transmitted, the new analgesia strategy analgesic new with exploitation proposed.

Summary of the invention

Thereby main purpose of the present invention is to provide Progynon inhibin appearance albumen 1 improving the active purposes that is used for treating, alleviates or improve in relevant disease or the symptom (especially improve somesthesia, particularly pain is felt or thermo aesthesia) of na-k-atp enzyme.

Another object of the present invention is to provide a kind of bioactive fragment of Progynon inhibin appearance albumen 1, described active fragment has the somesthetic effect of good inhibition.

In first aspect of the present invention, the purposes of Progynon inhibin appearance albumen 1 in the preparation compositions is provided, said compositions is used for treating, alleviating or improvement and na-k-atp enzyme active relevant disease or symptom through improving the na-k-atp enzyme activity.

In an embodiment of the invention, said disease or symptom are selected from: pain, familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung or pulmonary edema.

In yet another embodiment of the present invention, said compositions is used for treatment, alleviates or improve pain.In a preference, said pain is body source property pain, preferred neuropathic pain or nociceptive pain.

In yet another embodiment of the present invention, said neurogenic is selected from bitterly: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.In another preference, said neuropathic pain is the pain that causes of damage or pathological change by maincenter or peripheral nervous system that is selected from down group: lumbago and backache, neuralgia, fibromyalgia, neuralgia that diabetes are relevant, neuralgia that multiple sclerosis is relevant, PHN and with the related neurogenic neuralgia of HIV.

In yet another embodiment of the present invention, said nociceptive pain is selected from: body nociceptive pain or internal organs nociceptive pain.In a preference, said nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.

In yet another embodiment of the present invention, said Progynon inhibin appearance albumen 1 is:

(a) protein of aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:2;

(b) the proteinic conservative variant protein matter or its active fragment that are limited in (a); Or

(c) replacement, disappearance or the interpolation of aminoacid sequence in (a) through 1-20 (preferred 1-10, more preferably 1-5) amino acid residue formed, and it is active by (a) deutero-protein to have a na-k-atp enzyme of raising.

In yet another embodiment of the present invention, said (b) is selected from:

(1) has the polypeptide of 156-173 aminoacid sequence among the SEQ ID NO:1;

(2) has 158-175 amino acids polypeptide of sequence among the SEQ ID NO:2;

(3) by the formed polypeptide of sequential amino acid deletion Progynon inhibin domain (being the FS domain) of SEQ ID NO:1 or SEQ ID NO:2;

(4) by the formed polypeptide in sequential amino acid deletion Von Willebrand type C-structure territory (being the VWC domain) of SEQ ID NO:1 or SEQ ID NO:2;

(5) by the formed polypeptide of sequential amino acid deletion EF hand-type II domain of SEQ ID NO:1 or SEQ ID NO:2; Or

(6) by the formed polypeptide in sequential amino acid deletion EF hand-type syndeton territory of SEQ ID NO:1 or SEQ ID NO:2.

In second aspect of the present invention, a kind of separated polypeptide is provided, said polypeptide is selected from:

(1) have the polypeptide of 156-173 amino acids sequence NGDSHLDSSEFLKFVEQN among the SEQ ID NO:1, condition is that this polypeptide is not the protein shown in the SEQ ID NO:1;

(2) have the polypeptide of 158-175 amino acids sequence of N GDSRLDSSEFLKFVEQN among the SEQ ID NO:2, condition is that said polypeptide is not the protein shown in the SEQ ID NO:2;

In the third aspect of the invention, a kind of isolating polynucleotide are provided, the protein shown in aforesaid polypeptide of said polynucleotide encoding the present invention or SEQ ID NO:1 or the SEQ ID NO:2.

In fourth aspect of the present invention, a kind of carrier is provided, said carrier contains the aforesaid polynucleotide of the present invention.

In aspect the of the present invention the 5th, a kind of genetically engineered host cell is provided, said host cell contains the foregoing carrier of the present invention; Or be integrated with the foregoing polynucleotide of the present invention in its genome.

In aspect the of the present invention the 6th, the proteinic method for preparing of foregoing polypeptide of the present invention or SEQ ID NO:1 or SEQ ID NO:2 is provided, this method comprises:

(a) be fit to cultivate the foregoing host cell of the present invention under the condition of expressing;

(b) from culture, isolate the protein of foregoing polypeptide of the present invention or SEQID and NO:1 or SEQID and NO:2.

In aspect the of the present invention the 7th; Provide a kind of and treated, alleviate or improvement and the active relevant disease of na-k-atp enzyme or the pharmaceutical composition of symptom through improving the na-k-atp enzyme activity, it contains: (a) protein of the polypeptide of the present invention of safe and effective amount, SEQ ID NO:1 or SEQ ID NO:2; And (b) pharmaceutically acceptable carrier.

In an embodiment of the invention, pharmaceutical composition of the present invention is used to treat, alleviate or improves disease or the symptom that is selected from down group: familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung, pulmonary edema or pain.In a preference, described pain is body source property pain, preferred nociceptive pain or neuropathic pain.

In yet another embodiment of the present invention, described neurogenic is selected from bitterly: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.In another embodiment of the present invention, said neuropathic pain is the pain that causes of damage or pathological change by maincenter or peripheral nervous system that is selected from down group: lumbago and backache, neuralgia, fibromyalgia, neuralgia that diabetes are relevant, neuralgia that multiple sclerosis is relevant, PHN and with the related neurogenic neuralgia of HIV.

In yet another embodiment of the present invention, said nociceptive pain is selected from: body nociceptor property pain and internal organs nociceptor property pain.In a preference of the present invention, said nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.

In eight aspect of the present invention, provide the protein shown in protein shown in polypeptide of the present invention, the SEQ ID NO:1 and/or the SEQ ID NO:2 to be used for purposes through the pharmaceutical composition that improves the na-k-atp enzyme activity is treated, alleviated or improvement is relevant with the na-k-atp enzyme activity disease or symptom in preparation.

In an embodiment of the invention, said pharmaceutical composition is used to treat, alleviate or improves disease or the symptom that is selected from down group: familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung, pulmonary edema or pain.In a preference, described pain is body source property pain, preferred nociceptive pain or neuropathic pain.

In yet another embodiment of the present invention, described neurogenic is selected from bitterly: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.In another embodiment of the present invention, said neuropathic pain is the pain that causes of damage or pathological change by maincenter or peripheral nervous system that is selected from down group: lumbago and backache, neuralgia, fibromyalgia, neuralgia that diabetes are relevant, neuralgia that multiple sclerosis is relevant, PHN and with the related neuropathic pain of HIV.

In aspect the of the present invention the 9th, provide polypeptide of the present invention to be used for treating the purposes of the pharmaceutical composition of rheumatoid arthritis and/or ischemic heart desease in preparation.

In aspect the of the present invention the tenth, the method that provides a kind of screening can pass through to improve na-k-atp enzyme active treatment, alleviation or improve the potential material of disease relevant or symptom with this activity, said method comprises:

(1) candidate substances is contacted with the system of expressing Progynon inhibin appearance albumen 1;

(2) detect the influence of candidate substances to Progynon inhibin appearance albumen 1;

If said candidate substances can improve expression, activity and/or the secretion of Progynon inhibin appearance albumen 1, show that then this candidate substances is to can be used for treating, alleviate or improve the potential material of disease relevant with this activity or symptom through improving the na-k-atp enzyme activity.

In a preference, said disease or symptom are selected from: familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung, pulmonary edema or pain.In another preference, described pain is the neurogenic pain, preferred dorsal root ganglion neuropathic pain or trigeminal ganglion neuropathic pain.

In an embodiment of the invention, step (1) comprising: in testing group, candidate substances is joined in the system of expressing Progynon inhibin appearance albumen 1; And/or step (2) comprising: the expression of Progynon inhibin appearance albumen 1, activity and/or secretion in the system of detection testing group; And with matched group relatively, wherein said matched group is the system of not adding the expression Progynon inhibin appearance albumen 1 of said candidate substances; (preferably be significantly higher than, more than 20%, preferable is high more than 50% as high if the expression of Progynon inhibin appearance albumen 1 in the testing group, activity and/or secretion are higher than on statistics; Better is high more than 80%) matched group, just show that this candidate substances is to can be used for treating, alleviate or improve the potential material of disease relevant with this activity or symptom through improving the na-k-atp enzyme activity.

In another preference, described system is selected from: cell system (like the small neuron cell) (or cell culture objects system), subcellular fraction system, solution system, organizational framework, organ systems or animal system.

In another preference, said method also comprises: the potential material to obtaining carries out further cell experiment and/or animal experiment, from candidate substances, further to select and to define the material of usefulness.

In the tenth one side of the present invention, a kind of method of screening treatment or lenitive potential material is provided, described method comprises:

(1) candidate substances is contacted with the system of na-k-atp enzyme;

(2) detect the influence of candidate substances to na-k-atp enzyme;

If said candidate substances can improve the activity of na-k-atp enzyme, show that then this candidate substances is to can be used for treatment or lenitive potential material.

In aspect the of the present invention the 12, the inhibitor that Progynon inhibin appearance albumen 1 is provided preparation through reduce the na-k-atp enzyme activity and treat, alleviate or the compositions of disease that improvement is relevant with the na-k-atp enzyme activity or symptom in purposes.

In an embodiment of the invention, said disease or symptom are selected from: renal hypertension, tumor (preferred melanoma, glioblastoma multiforme or nonsmall-cell lung cancer).

In yet another embodiment of the present invention, said inhibitor is selected from: the antibody of anti-Progynon inhibin appearance albumen 1 or its active fragment; Antisense oligonucleotide or siRNA to Progynon inhibin appearance albumen 1 coded sequence; Or derive from the peptide section of na-k-atp enzyme.

In yet another embodiment of the present invention, said inhibitor is selected from:

(i) has the polypeptide of the aminoacid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or the SEQ ID NO:8;

Conservative variation polypeptide or its active fragment of the polypeptide that is (ii) limited in (i); Or

(iii) (i) or (ii) in the arbitrarily combination of polypeptide or active fragment.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1 .FSTL1 is high expressed in the DRG small neuron

Figure 1A. through the express spectra of candidate gene in the adult rat tissue and the ratio of dorsal root ganglion signal intensity and other tissue signal intensity of gene chip detection.Among the figure, the log2 proportion of ratio shows with different tones among gene signal and the DRG in various tissues.Avil represents advillin; Calca represents CGRP α; Htr3a represents 5-hydroxytryptamine receptor 3A (5-hydroxytryptamine receptor 3A); The Plekha4 representative contains the member 4 (pleckstrin homologydomain containing, family A member 4) of the A of family of platelet homology domain; Mpz represents myelin protein 0 (myelin proteinzero); P2rx3 represents the P2X3 receptor; Prph1 represents peripheral protein 1 (peripherin 1); Nef3 represents neurofilament 3 (neurofilament 3); The valtage-gated type VII α of Scn7a represent sodium ion channel (sodiumchannel, voltage-gated type VII, α); The positive contrast of dorsal root ganglion GAPDH signal intensity.

Figure 1B .RNA blotting detects the expression of Fstl1mRNA in rat grows up nervous system, contrasts as applied sample amount with ribosome 18s RNA;

Fig. 1 C. immunoblotting detects FSTL1 albumen, and (～37kD) the expression in rat grows up nervous system contrasts as applied sample amount with actin;

Fig. 1 D. SABC and immunoblotting show the expression of rat FSTL1 in period of embryo, birth dorsal root ganglion in back and adult period.The embryo that is expressed in of FSTL1 began in 15 days, reached top level in back 28 days in birth.Contrast as applied sample amount with actin;

The two mark methods of Fig. 1 E. immunofluorescence detect in dorsal root ganglion and cornu dorsale medullae spinalis, FSTL1 respectively with the common location situation of CGRP or IB4, FSTL1 is present in the CGRP positive or the male neuron of IB4.Wherein, green expression FSTL1 expresses; Red expression CGRP or IB4 express;

Fig. 1 F. immunofluorescence detects FSTL1 and Na _v1.8 being expressed in, the expression of channel protein, FSTL1 contain Na _v1.8 in the DRG neuron of channel protein.Wherein last figure medium green color table reaches FSTL1 expresses, red expression Na among the middle figure _v1.8 channel protein is expressed, figure below is the stack (merge) of last figure and middle figure.

The distribution situation of Fig. 1 G.FSTL1 in different DRG neuron types;

Fig. 1 H. Dorsal root ganglion neuron sum situation, and the male neuron situation of Dorsal root ganglion FSTL1.

The two mark methods of Fig. 1 I. immunofluorescence detect FSTL1 and P expression of Substance and locate situation altogether.Green expression FSTL1 expresses, red expression P material expression, and the arrow indication is the vesicle that FSTL1 expresses;

The two mark methods of Fig. 1 J. immunofluorescence detect FSTL1 respectively with TRPV1 or P2X ₃Common location situation.Wherein, red expression FSTL1 expresses; Green expression TRPV1 or P2X ₃Express;

Fig. 1 K.RNA blotting detects the expression of Fstl1 mRNA in each tissue of rat, contrasts as applied sample amount with ribosome 18s RNA.

Fig. 2. prove that FSTL1 is by the excretory experimental result of little clear vesicle

The two mark methods of Fig. 2 A. immunofluorescence detect the vesicle distribution (shown in the arrow) of FSTL1 in the peptidergic neuron of rat DRG and the positive small neuron of IB4; FSTL1 is not in containing the big densified core bubble (LDCV) of CGRP; (synaptoporin is not Synpr) in the male vesicle at synaptoporin yet for major part.Wherein, green expression FSTL1 expresses, and red expression CGRP, IB4 or Synpr express;

Fig. 2 B. immuno-gold labeling detects the location situation of FSTL1 in the DRG small neuron cell space, and FSTL1 is arranged near the little clear vesicle (arrow) the golgi's network outside;

Fig. 2 C. is with the different vesicle crowds of method isolated from rat cornu dorsale medullae spinalis of SDGC separation subcellular components, and immunoblotting detects the expression of FSTL1, Synpr and CGRP among the isolated vesicle crowd.FSTL1 mainly is present among the vesicle crowd of containing Synpr, but is not present among the vesicle crowd who contains CGRP;

The situation that contains the FSTL1 vesicle in the centripetal fiber tip of the centripetal fiber of Fig. 2 D. immuno-gold labeling detection Dorsal root and cornu dorsale medullae spinalis I, II layer.FSTL1 is arranged in the little clear vesicle (shown in the arrow) of centripetal fiber tip of centripetal fiber and cornu dorsale medullae spinalis I, the II layer of Dorsal root, and the vesicle that contains FSTL1 shown in the arrow is positioned near the presynaptic membrane.Triangle sign indication postsynaptic compact zone;

The sketch map of the back ligation of Fig. 2 E. mice.Back of the ligation mice forward transported with the blocking-up vesicle in 24 hours, can cause centripetal fiber gathering near DRG one end FSTL1;

Fig. 2 F. immunoblot experiment detects the situation of the rat drg neuron secretion FSTL1 that under non-stimulated condition, cultivates.Total expression situation with FSTL1 in the cell pyrolysis liquid contrasts as applied sample amount, and wherein bar diagram is the quantitative analysis to each swimming lane FSTL1 secretion situation.Can see that the FSTL1 secretory volume increases in time in the culture fluid of rat drg neuron.Compare with matched group, ^*P＜0.05, ^*P＜0.01;

Fig. 2 G. immunoblot experiment detects at high potassium to stimulate, and (55mM KCl is 1h) with the situation that has or not the rat drg neuron secretion FSTL1 that cultivates under the extracellular Ca2 condition.Total expression situation with FSTL1 in the cell pyrolysis liquid contrasts as applied sample amount, and wherein bar diagram is the quantitative analysis to each swimming lane FSTL1 secretion situation.Do not compare with increasing potassium stimulated control group, ^*P＜0.05 (high potassium adds calcium stimulates (being post 2) to compare with the matched group of not increasing potassium but adding calcium stimulation (being post 1)), ^#P＜0.05 (high potassium adds calcium to stimulate (being post 2) and increase the matched group that potassium do not add calcium stimulation (being post 4) and compare);

Fig. 2 H. immunoblotting detects at high potassium stimulates (55mM KCl, 15min) the secretion situation of rat spinal cord brain sheet FSTL1 under the condition.High potassium stimulates significantly increases the secretory volume of FSTL1 in the spinal cord thin slice, contrasts as applied sample amount with the expression of actin in the cell pyrolysis liquid.In identical sample, show that through immunoblotting and HPLC detection method CGRP and glutamic acid (Glu) level raise.Do not compare with increasing potassium stimulated control group, ^*P＜0.05;

Fig. 2 I. immunoblotting detects the secretion situation that stimulates and have or not FSTL1 in the synaptic glomerulus of rat spinal cord dorsal horn under the extracellular Ca2 condition at high potassium.High potassium stimulates significantly increases the secretory volume of FSTL1 in the synaptic glomerulus of cornu dorsale medullae spinalis; Expression with actin in the cell pyrolysis liquid contrasts as applied sample amount; Wherein bar diagram is the quantitative analysis to each swimming lane FSTL1 secretion situation, also representes that detecting FSTL1 through immunoblotting is divided into the CGRP existence and secretes simultaneously.Do not compare with increasing potassium stimulated control group, ^*P＜0.05, ^*P＜0.01 (the high potassium stimulating group (being post 3 and post 4) that adds calcium);

Fig. 2 J. immunoblot experiment detect KCl, capsaicin (Capsaicin, Cap) and/or capsaizepine (CPZ) exist down, with the excretory FSTL1 level of neuronal cell of young rats DRG cultivation.Total expression situation with FSTL1 in the cell pyrolysis liquid contrasts as applied sample amount, and wherein bar diagram is the quantitative analysis to each swimming lane secretion situation.Do not compare with adding the stimulated control group, ^*P＜0.01 (only add KCl or Cap stimulate and do not add any stimulated control group and compare), ^##P＜0.01 (only add Cap stimulate with add Cap with CPZ stimulation compare).

The inhibitory action that Fig. 3 .FSTL1 transmits for sensation

The expression plasmid of Fig. 3 A.FSTL1 and mutant M1 thereof (EF hand-type domain disappearance) and M2 (the 165th becomes A by E) makes up sketch map, and the C end of FSTL1 and mutant thereof and Myc and His label merge;

Fig. 3 B. carries out full cell record on adult rat spinal cord thin slice, write down 31 spinal cord II layer neurons at embedding system voltage-70mV (V _H) under sEPSC.In 16 neurons, reorganization FSTL1 has reduced the frequency and the amplitude of spontaneous EPSC (sEPSC) simultaneously.The anti-total length FSTL1 antibody of rabbit (1: 1000) can strengthen power frequency and the amplitude of sEPSC, and preimmune serum (1: 1000) is with comparing.The data that perfusion FSTL1 collects in the 3min are adopted in all quantitative analyses.Compare with matched group that does not add FSTL1 or preimmune serum group, ^*P＜0.05, ^*P＜0.01;

Fig. 3 is (intensity: 210-650 μ A) stimulate Dorsal root to induce the single synapse eEPSC of glutamic acid ability of C fiber, average amplitude 166 ± 23pA (37-459pA C.0.2Hz; V _H=-70mV).In the spinal cord II layer neuron that 24 quilts write down, single synapse that the C fiber excites is brought out sexual excitation property postsynaptic currents (eEPSC) by FSTL1 (60nM, n=14; 300nM n=5) suppresses, but is not suppressed by M1 (n=4) and M2 (n=5).After the peak current level that is standardized as contrast eEPSCs, the damping time constant of repressed eEPSCs does not have significant change.FSTL1 antibody (1: 1000) also strengthens the eEPSC (n=5) of C fiber.Compare with matched group that does not add FSTL1 or preimmune serum group, ^*P＜0.05, ^*P＜0.01;

Fig. 3 D.FSTL1 (60nM) only reduces the frequency of smallness EPSC (mEPSC).Compare with the matched group that does not add FSTL1, ^*P＜0.01;

Fig. 3 E. immunoblotting detects FSTL1, M1 and M2 Recombinant Protein Expression situation;

The people of Fig. 3 F. purification recombinates the Progynon inhibin to not influence of the sEPSC that writes down on the adult rat spinal cord thin slice.The experimental group that has added FSTL1 is compared with the matched group that does not add FSTL1, ^*P＜0.01; Compare with the group that adds FSTL1, ^#P＜0.05.

Fig. 4. conditionality knocks out the Fstl1 gene in the DRG neuron

The structure sketch map of Fig. 4 A.Fstl1 conditional gene knockout mice.With two loxP sequences (grey color triangle, Fstl1 ^F) the exon 2 deletion of the FSTL1 signal peptide of will encoding.Neo in the intron zone is also deleted by loxP.PCR detects and shows the heterozygote (Fstl1 that contains single copy Fstl1 ^F/+) comprise 399bp and two bands of 505bp; Homozygote (Fstl1 ^F/F) a 505bp band only arranged; And wild type (Fstl1 ^{+ /+}) band of 399bp arranged.Through hybridizing with the SNS-Cre mice, neo and exon 2 are removed;

Fig. 4 B. immunoblotting detects Fstl1 ^{+ /+}, Fstl1 ^+/-, Fstl1 ^-/-The expression of FSTL1 among the mice DRG.Show that FSTL1 is at Fstl1 ^-/-Basically do not exist among the DRG;

Fig. 4 C. is at Fstl1 ^-/-Among the DRG of mice, it is about 10% that the male neuron of FSTL1 immunoreation is reduced to, and the expression of some main molecules does not change.

The disappearance of Fig. 5 .FSTL1 induces somesthesia ultra quick

The neuronic sEPSC of the full cell record spinal cord of Fig. 5 A. II layer shows to be increased in Fstl1 by the inductive sEPSC frequency of 15mM KCl (1min) ^-/-In the mice than at Fstl1 ^{+ /+}More remarkable in the mice.Stimulate as contrast not add KCl, ^*P＜0.01 (Fstl1 ^-/-Mice);

Fig. 5 B. is at the wide power neuron reaction (WDR) of body record mouse spinal cord dorsal horn, Fstl1 ^-/-The WDR neuron of the cornu dorsale medullae spinalis of mice (n=6) is more responsive (with Fstl1 for thermostimulation (left side) and mechanical stimulus (right side) ^{+ /+}Mice is compared, ^*P＜0.05).Give (i.t.) FSTL1 in the sheath and can reverse Fstl1 ^-/-This enhanced WDR neuron reaction of mice (n=3, ^*P＜0.05);

Fig. 5 C.Fstl1 ^{+ /+}, Fstl1 ^F/F, Fstl1 ^-/-The contract comparison on sufficient threshold value (right side) of time (left side) and mechanical stimulus is delayed in contract foot reaction of the thermal stimulus of mice.Fstl1 ^-/-For heat (left side) and the more responsive (n=12 of mechanical stimulus (right side); With Fstl1 ^{+ /+}Mice and Fstl1 ^F/FMice is compared, ^*P＜0.01);

Fig. 5 D.Fstl1 ^{+ /+}, Fstl1 ^F/F, Fstl1 ^-/-Mice falls the comparative result (n=6) of the time of delaying in quickening rotation test;

Fig. 5 E.Fstl1 ^{+ /+}, Fstl1 ^F/F, Fstl1 ^-/-Mice hind paw injection of formalin induces mice in the inflammatory pain model to lick the comparison of foot time.Fstl1 ^-/-Mice shows more tangible pain reaction, and (n=6 is with Fstl1 ^{+ /+}Mice and Fstl1 ^F/FMice is compared, ^*P＜0.05);

Fig. 5 F.Fstl1 ^-/-To the sufficient influence of delaying the time of contracting of thermostimulation, matched group is injected carrier (0.01M PBS) behind mice intrathecal injection (i.t.) the variable concentrations FSTL1.FSTL1 reduces Fstl1 ^-/-Mice is to the reaction of thermostimulation.Fstl1 with the injection carrier ^-/-Mice is compared, ^*P＜0.05, ^*P＜0.01;

Antibody of the anti-FSTL1 full-length proteins of Fig. 5 G. adult rat intrathecal injection (1: 100,20 μ l) or preimmune serum are observed rat the foot that contracts of thermostimulation are delayed change of time.Give behind the antibody rat to thermostimulation more responsive (n=6 compares with the mice of serum before the injecting immune, ^*P＜0.05, ^*P＜0.01).

The decline that Fig. 6 .FSTL1 expresses has contribution to neuropathic pain

Fig. 6 A. in situ hybridization shows Fstl1 mRNA level, and sciatic nerve cuts off the small neuron number that contains Fstl1 mRNA after 14 days among the rat waist 4-5 sections DRG significantly to be reduced, and the control sides of not cutting off with rat is contrast, ^*P＜0.01;

Fig. 6 B. immunoblotting shows that rat sciatic nerve cuts off the variation of back Dorsal root ganglion different time FSTL1.Contrast as applied sample amount with GAPDH;

The immune labeled sciatic nerve that is presented at of Fig. 6 C. cuts off the positive DRG quantity minimizing of FSTL1 after 14 days.The two mark methods of immunofluorescence show, FSTL1 disappearance in the DRG neuron [with transcriptional factors 3 (ATF3) labelling] of damage.Sciatic nerve cut off after 14 days, and FSTL1 significantly descends in the centripetal fiber in waist 5 sections (indicating with the triangle arrow);

Fig. 6 D. is cut off in the neurogenic pain model cause by sciatic nerve branch, and the number that cuts off FSTL1 positive neuron among 14 days low back 5DRG significantly descends.The control sides of not cutting off with rat is contrast, ^*P＜0.01;

Fig. 6 E. cuts off in the neurogenic pain model cause in sciatic nerve branch, and intrathecal injection FSTL1 albumen is for the contract influence of sufficient threshold value of rat mechanical stimulus;

Fig. 7 .FSTL1 albumen combines with α 1 sodium-potassium ATP enzyme (NKA) subunit

Fig. 7 A. uses the FSTL 1-myc of reorganization to hatch the rat Dorsal root ganglion neuron of cultivation, has added the cross-linking agent (BS of impermeable membrane simultaneously ³), with the myc antibody test to FSTL1 at nearly 150kD place a visible band, test repetition 3 times;

The same sample of handling of Fig. 7 B. and Fig. 7 A detects with the monoclonal antibody of α 1NKA subunit, and visible part α 1NKA molecular weight subunit is increased to 150kD from 110kD, and α 3NKA subunit is not seen this phenomenon, experiment repetition 3 times;

Fig. 7 C. immunohistochemical staining shows that α 1NKA subunit mainly is positioned with FSTL1 common location is arranged in the neuronic minicell of Dorsal root ganglion;

The experiment of the co-immunoprecipitation of Fig. 7 D. rat Dorsal root ganglion shows, can detect α 1NKA subunit in the protein that gets off with the co-precipitation of FSTL1 antibody mediated immunity, tests repetition 3 times;

The α 1NKA subunit of Fig. 7 E. corotation zone flag label in the COS7 cell and FSTL1-myc (can detect FSTL1-myc (experiment repetition 3 times) in the protein under the co-precipitation of flag antibody mediated immunity);

Fig. 7 F. Scatchard analysis shows ¹²⁵I-FSTL1 dose dependent ground combines the COS7 cell (Kd=43nM) of coexpression α 1NKA subunit and β 1 subunit;

Fig. 7 G. is in the COS7 cell of expressing FSTL1-myc and α 1NKA subunit, and co-immunoprecipitation is tested to be presented in the protein that the co-precipitation of flag antibody mediated immunity gets off and had FSTL1-myc, at coexpression FSTL1-myc and α 1NKA subunit ^E314G-flag or α 1NKA subunit ^T889N-flag or α 1NKA subunit ^{E314G, T889N}In the cell of-flag, the FSTL1-myc that exists in the protein that the co-precipitation of flag antibody mediated immunity is got off significantly descends, and FSTL1-myc does not combine with α 3NKA subunit, but with α 3NKA subunit ^{G304E, N879T}-flag combines, experiment repetition 3 times.

Fig. 8 .FSTL1 activates α 1NKA subunit

Fig. 8 A. is corotation α 1NKA-flag and β 1 subunit of being with the myc label in the COS7 cell, can detect the activity that FSTL1 dose dependent ground raises NKA, and corotation α then can not during 3NKA-flag.In the Dorsal root ganglion neuron of cultivating, also can detect FSTL1 and be the activity that dose dependent ground raises NKA;

Fig. 8 B.FSTL1 is at corotation α 1NKA subunit ^E314G-flag or α 1NKA subunit ^T889N-flag or α 1NKA subunit ^{E314G, T889N}Can not increase the activity of NKA in the COS7 cell of-flag and β 1 subunit, FSTL1 ^E165ACan not improve the activity of NKA in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit.Express alpha 1NKA subunit is compared with the COS7 cell of β 1 subunit together, ^*P＜0.05, ^*P＜0.01;

The full cell record of Fig. 8 C.COS7 is observed FSTL1 (30nM) cell (n=15) of corotation α 1NKA-flag and β 1-myc subunit is significantly reduced cell membrane potential, to the not influence of cell (n=8) transmembrane potential of cells transfected (n=12) or corotation α 3NKA-flag and β 1-myc subunit.Compare with cells transfected not, ^*P＜0.01;

Fig. 8 D. in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit, preincubate M3M4 (EC ₅₀=3.6 μ M) or M7M8 (EC ₅₀=2.9 μ M) the peptide section can make ¹²⁵The combination rate of I-FSTL1 significantly descends, and M1M2 peptide section can not reduce;

Fig. 8 E. is in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit, and preincubate M3M4 or M7M8 peptide section can weaken the NKA increased activity effect that FSTL1 causes.Compare with the contrast that does not add FSTL1, ^*P＜0.01;

The adult rat Dorsal root ganglion small neuron of the full cell record acute isolation of Fig. 8 F.; FSTL1 (30nM) reduces the frequency of action potential; And induce film hyperpolarization (write down 16 cells have 7 effect is arranged); This influence was reversed when specific inhibitor ouabain (the OB 100 μ M) time that gives NKA simultaneously, and the effect of the film hyperpolarization that FSTL1 causes can be overturn by ouabain, also can be by the 2nd born of the same parents' outer shroud M3M4 peptide section institute antagonism of preincubate α 1NKA.

The expression of Fig. 8 G. α 1NKA in cornu dorsale medullae spinalis, SABC show that α 1NKA and FSTL1 coexist as in the primary afferent termination;

The full cell record of Fig. 8 H. spinal cord brain sheet II layer neuron shows that FSTL1 (60nM) reduces the frequency of sEPSC, and this influence was reversed when specific inhibitor ouabain (the OB 100 μ M) time that gives NKA simultaneously.Compare with the contrast that does not add FSTL1, ^*P＜0.01 (n=5);

The full cell record of Fig. 8 I. spinal cord brain sheet II layer neuron shows M3M4, and (20 μ M, 3min) the peptide section is hatched the frequency that increases sEPSC.Compare with the contrast that does not add the peptide section, ^*P＜0.05 (n=6);

The full cell record of Fig. 8 J. spinal cord brain sheet II layer neuron shows M3M4, and (20 μ M, 3min) the peptide section is hatched the eEPSC amplitude that the stimulation of C fiber causes that increases.Compare with the contrast that does not add the peptide section, ^*P＜0.05 (n=5).

Proteic each functional area of Fig. 9 .FSTL1 is for the influence of intracellular Ca2+ and α 1NKA enzymatic activity

Obtain removing the recombiant protein that FS, VWC, EF hand-type domain and the 165th amino acids are mutated into the eukaryotic expression of alanine respectively; On the Primary Sensory Neuron of acute isolation, detect protein to capsaicin (Capsaicin with the calcium method for imaging; Cap) influence that calcium raises in the DRG neuronal cell that causes; Total length FSTL1 significantly reduces the intracellular Ca2+ rising that Cap causes; Disappearance FS and VWC domain are to the not influence of effect of FSTL1, but the function of disappearance EF hand-type domain and the 165th the equal appreciable impact FSTL1 of sudden change.On the COS7 cell of stable transfection α 1NKA and β 1NKA subunit, detect α 1NKA enzymatic activity; The FSTL1 total length can significantly activate the activity of α 1NKA enzyme, and the synthetic polypeptide (SP76) of 156-173 amino acids sequence also can significantly activate the activity of α 1NKA enzyme in the EF1 hand-type domain.

The specific embodiment

The inventor is through extensive and deep research; (Follistatin-Like 1 to have proved Progynon inhibin appearance albumen 1 first; FSTL1) or its bioactive fragment can improve the activity of na-k-atp enzyme, thereby can be used for treating, alleviating or improve disease relevant or symptom with this activity.For example, at the sensory information transmittance process, downward modulation FSTL1 makes the active decline of na-k-atp enzyme can cause sensitivity to pain to increase, and this regulating action is that a kind of brand-new sensory information transmits regulatory mechanism.Therefore, FSTL1 itself or adjustment can na-k-atp enzyme is active to be used to suppress mammalian body sensation (as reducing the mammiferous pain sensation) through improving on it.

As used herein, " isolating " is meant that material separates (if crude, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " with active relevant disease or the symptom of na-k-atp enzyme " is meant through regulating (comprise and raise or reduce) na-k-atp enzyme activity can obtain disease or the symptom treating, alleviate or improve.With na-k-atp enzyme active reduce or cross low relevant disease or symptom include but not limited to: familial hemiplegia type migraine (Familial hemiplegic migraine), diabetes (diabetes), depression (depression), digitalism (digoxin toxicity), injury of lung (lung injury), pulmonary edema (lung edema) or mammalian body sensation (obvious excessively like the pain sensation or thermo aesthesia), these diseases or symptom can obtain medical treatment, alleviate or improve through improving na-k-atp enzyme active (in the present invention can through for example increasing FSTL1 quantity or improving the realization of FSTL1 activity).Improve or too high relevant disease or symptom include but not limited to na-k-atp enzyme is active: renal hypertension, tumor (preferred melanoma, glioblastoma multiforme and nonsmall-cell lung cancer), these diseases or symptom can through reduce na-k-atp enzyme active (in the present invention can through for example reduce FSTL1 quantity, reduce FSTL1 active, suppress FSTL activate na-k-atp enzyme or block FSTL combine to realize with na-k-atp enzyme) obtain medical treatment, alleviate or improve.

As used herein, described " somesthesia " is meant the sensory reaction of mammal for external stimulus (like mechanical damage) or intrinsic stimuli generation, and it is transmitted by nervous system (comprising dorsal root ganglion) usually.Describedly feel to include, but is not limited to: pain is felt, thermo aesthesia.Described pain comprises neuropathic pain, like dorsal root ganglion neuropathic pain or trigeminal ganglion neuropathic pain.Described thermo aesthesia is meant body is touched the sensory reaction behind high temperature or the low temperature.

With regard to pain, it can be divided into body source property (somatogenic) pain and psychogenic (psychogenic) pain.Body source property pain can be divided nociceptive pain (nociceptive pain) and neuropathic pain (Neuropathic Pai) again.Neuropathic pain is because the damage or the pathological change of maincenter or peripheral nervous system cause; Include but not limited to the relevant neuralgia of lumbago and backache (lower back pain), neuralgia (neuralgia), fibromyalgia (Fibromyalgia), diabetes (diabetic neuropathic pain, DNP), neuralgia (pain associated with multiple sclerosis), PHN (PHN) and HIV related (neurogenic) neuralgia (HIVNP) etc. that multiple sclerosis is relevant.Nociceptor property pain is because the intravital nociceptor of people receives machinery, heat, chemical stimulation or damage and causes; Body nociceptor property pain and internal organs nociceptor property pain be can be divided into, cancer pain (cancer-relatedpain), arthritis pain (arthritic pain), postoperative pain (post-operative pain) and HIV related (nociceptor property) neuralgia (HIVNP) etc. included but not limited to.Wherein, neuralgia (neuralgia) includes but not limited to trigeminal neuralgia (Trigeminal neuralgia), sciatica (sciatic neuralgia) etc.

As used herein, have comprised " containing ", " having " or " comprising " " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute "; " mainly by ... constitute ", " basically by ... constitute " belong to the subordinate concept of " containing ", " having " or " comprising " with " by ... formation ".

FSTL1 albumen or its bioactive fragment

In the process of seeking specific expressed new functional molecular on the DRG neuron, the inventor finds FSTL1 selectivity high expressed on the DRG small neuron.

FSTL1 is a kind of glycoprotein, is found to be a kind of transforming growth factor (TGF) β 1 induced protein the earliest.FSTL1 belongs to Progynon inhibin gene family, has Progynon inhibin spline structure territory and a pair of EF-hand-type structure, but can not combine with activin (Activin).FSTL1 can suppress the growth of tumor cell, and is the target protein of rheumatoid arthritis autoimmune antibody.Although there is Fstl1 mRNA in the nervous system of the known DRG of comprising, the physiological function of FSTL1 in nervous system was still unknown by the people in the past.

The inventor is unexpected the discovery in further investigation, FSTL1 can with sodium-potassium ATP enzyme (Na ⁺, K ⁺-ATPase) combine, and wherein bonded key point is among sodium-potassium ATP enzyme α 1NKA subunit second born of the same parents' outer shroud M3M4 and the 4th the born of the same parents' outer shroud M7M8.And FSTL1 can activate the activity of sodium-potassium ATP enzyme with after sodium-potassium ATP enzyme combines, thereby can be used for treating, alleviating or improvement and na-k-atp enzyme are active reduces or cross low relevant disease or symptom.

Otherwise; Research has also proved the peptide section that derives from na-k-atp enzyme or their homologous sequence; Include but not limited to: the M7M8 peptide section shown in the M3M4 peptide section shown in the SEQ ID NOs:5 and 6, the SEQ ID NOs:7 and 8); Combine with sodium-potassium ATP enzyme through suppressing FSTL1, can reduce the activity of na-k-atp enzyme, thereby can treat, alleviate or improvement and the active raising of na-k-atp enzyme or too high relevant disease or symptom.

Utilize to raise among the present invention or downward modulation FSTL1 treats, alleviates or improvement is relevant with the na-k-atp enzyme activity the disease or the method for symptom, exist in tissue or the cell (for example dorsal root ganglion, heart, lung, kidney etc.) of FSTL1 and na-k-atp enzyme particularly effective at the same time.

The sequence of na-k-atp enzyme α 1NKA subunit is well known in the art, and maybe can confirm through this area conventional method.For example, shown in SEQ ID NO:3 (1023Aa) and SEQ ID NO:4 (1023Aa), their sequence homogeny reaches 96% to the aminoacid sequence of rat and people's na-k-atp enzyme α 1NKA subunit respectively.Crucial born of the same parents' outer shroud in rat and the human sodium-potassium ATP enzyme α 1NKA subunit is as shown in table 1 below:

Table 1. na-k-atp enzyme α 1NKA subunit and crucial born of the same parents' outer shroud

^*Serial number in the 2nd row number is a benchmark with the aminoacid in the aminoacid sequence shown in the SEQ ID NO:3

^*Serial number in the 3rd row number is a benchmark with the aminoacid in the aminoacid sequence shown in the SEQ ID NO:4

The inventor is in further further investigation discovery, and FSTL1 can have regulating action to the sensory information transmittance process from Primary Sensory Neuron and teleneuron secretion.FSTL1 is high expressed in DRG small neuron cell, is transported to afferent nerve endings through little clear vesicle and also can discharges jointly with excitatory transmitter.Electrophysiological recording shows that FSTL1 can be through activating sodium-potassium ATP enzyme (Na ⁺, K ⁺-ATPase) make DRG small neuron cell membrane hyperpolarization.Specificity knocks out the FSTL1 in the DRG neuron, and can cause importing into transportation increases, and increases mammiferous pain sensation sensitivity.Importantly, the expression of nerve injury meeting downward modulation DRG neuron FSTL1, and spinal cord intrathecal injection FSTL1 can alleviate the pain sensation reaction that nerve injury causes.Therefore, the excretory FSTL1 of centripetal fiber tip works to keep normal somesthesia as a kind of important regulatory factor that self suppresses.FSTL1 can effectively alleviate the pain sensation response prompting that nerve injury causes: FSTL1, or the molecule that can activate or raise FSTL1 therapeutic value is arranged in neuropathic pain.

In the present invention, used FSTL1 albumen can be naturally occurring, such as its can be separated or purification from mammal.In addition, described FSTL1 albumen also can be artificial preparation, such as producing reorganization FSTL1 albumen according to the genetic engineering recombinant technique of routine.Preferably, the present invention can adopt the FSTL1 albumen of reorganization.

Any suitable FSTL1 albumen all can be used for the present invention.Described FSTL1 albumen comprises FSTL1 albumen or its bioactive fragment (or be called " active fragment " or " polypeptide of the present invention ") of total length.For example, the proteic aminoacid sequence of described FSTL1 can be substantially the same with the sequence shown in SEQ ID NO:1 or the SEQ ID NO:2.

The proteic aminoacid sequence of FSTL1 that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form is also included among the present invention.FSTL1 albumen or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and said sequence through the aminoacid replacement does not influence its activity or kept the activity of its part.Suitably replacement aminoacid is technology well known in the art, and said technology can be implemented at an easy rate, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, can not change biological activity basically at the inessential area change single amino acids of a peptide species.See Watson etc., " Molecular Biology of The Gene " (" gene molecule biology ", the 4th edition, 1987, Benjamin/Ku Ming publishing company, The Benjamin/Cummings Pub.Co.P224).

The proteic bioactive fragment of any FSTL1 can be applied among the present invention.Here, the implication of the proteic bioactive fragment of FSTL1 is meant that as a peptide species it still can keep the proteic all or part of function of FSTL1 of total length.Generally, described bioactive fragment keeps 50% the proteic activity of total length FSTL1 at least.Under preferred condition, said active fragment can keep proteic 60%, 70%, 80%, 90%, 95%, 99% or 100% the activity of total length FSTL1.

As optimal way of the present invention, the proteic bioactive fragment of described FSTL1 (being also referred to as " polypeptide of the present invention " in the present invention) can include, but is not limited to: (1) has the polypeptide of the basic acid sequence of 158-175 position among 156-173 position among the SEQ ID NO:1 or the SEQ ID NO:2; (2) protein of aminoacid sequence shown in SEQ ID NO:1 or the SEQID NO:2, and wherein lack Progynon inhibin (Follistatin, FS) domain; (3) protein of aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:2, and wherein lack Von Willebrand type C-structure territory (VWC); (4) protein of aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:2, and wherein lack EF hand-type II domain; Or the protein of aminoacid sequence shown in (5) SEQ ID NO:1 or the SEQ IDNO:2, and wherein lack the syndeton territory between EF hand-type I domain and the EF hand-type II domain.Empirical tests, the proteic above-mentioned bioactive fragment of FSTL1 has kept the proteic biological activity of total length FSTL1, or has more excellent biological activity than total length FSTL1 albumen.

In embodiments of the present invention, in SEQ ID NO:1, number be benchmark with the aminoacid in the aminoacid sequence shown in the SEQ ID NO:1, said Progynon inhibin (FS) domain is the 28-96 position, the VWC domain is the 231-285 position; EF hand-type domain is the 142-226 position, and wherein: EF hand-type I domain is the 142-178 position, and EF hand-type syndeton territory is the 179-190 position, and EF hand-type II domain is the 191-226 position.

In embodiments of the present invention, in SEQ ID NO:2, number be benchmark with the aminoacid in the aminoacid sequence shown in the SEQ ID NO:2, said Progynon inhibin (FS) domain is the 30-98 position, the VWC domain is the 233-287 position; EF hand-type domain is the 144-228 position, and wherein: EF hand-type I domain is the 144-180 position, and EF hand-type syndeton territory is the 181-192 position, and EF hand-type II domain is the 193-228 position.

The distribution of each fragment and domain is as shown in table 2 below in the FSTL1 sequence of SEQ ID NO:1 and SEQ ID NO:2:

The distribution of each fragment and domain among the table 2.FSTL1

^*Serial number in the 2nd row number is a benchmark with the aminoacid in the aminoacid sequence shown in the SEQ ID NO:1

^*Serial number in the 3rd row number is a benchmark with the aminoacid in the aminoacid sequence shown in the SEQ ID NO:2

The present invention also can adopt through the FSTL1 albumen of modifying or improveing, such as, can adopt the FSTL1 albumen of modifying or improveing in order to promote its half-life, effectiveness, metabolism and/or proteinic effectiveness.Said can be the proteic conjugate of a kind of FSTL1 through the FSTL1 albumen of modifying or improve, or it can comprise substituted or artificial aminoacid.Said can be to have less common ground with naturally occurring FSTL1 albumen through the FSTL1 albumen of modifying or improve, but also can suppress the mammalian body sensation, and can not bring other harmful effect or toxicity.That is to say that any proteic bioactive version of FSTL1 that do not influence all can be used among the present invention.

Obtained said proteinic sequence in case separate, just can come to obtain in large quantity this protein with recombination method.This normally is cloned into carrier with its encoding gene, changes cell again over to, from the host cell after the propagation, separates obtaining then through conventional method.In addition, for short protein, also can adopt the method for synthetic (as synthetic through Peptide synthesizer) to synthesize relevant sequence, the method for synthetic can obtain needed protein easy and apace.

The present invention has also comprised the isolating nucleic acid of the proteic bioactive fragment of described FSTL1 of encoding, and also can be its complementary strand.The DNA sequence of coding FSTL1 proteic bioactive fragment can the complete sequence synthetic, and also the method for available pcr amplification obtains.After the DNA sequence of the proteic bioactive fragment of described FSTL1 that obtained to encode, it is connected into suitable expression vector, change proper host cell again over to.Through the host cell after the culture transformation, obtain desired protein at last through separation and purification.

The present invention has also comprised the carrier of the nucleic acid molecules that comprises the proteic bioactive fragment of the said FSTL1 of coding.Described carrier also can comprise the expression regulation sequence that links to each other with the series of operations property of said nucleic acid molecules, so that protein expression.Described " operability links to each other " or " operationally being connected in " refer to a kind of like this situation, and promptly the activity of same other part of linear DNA sequence can regulated or control to some part of linear DNA sequence.For example, if the transcribing of promoter control sequence, it is exactly operationally to be connected in coded sequence so.

In addition, the reconstitution cell that contains coding said FSTL1 proteic bioactive fragment nucleotide sequence is also included among the present invention." host cell " comprises prokaryotic cell and eukaryotic cell.Prokaryotic host cell commonly used comprises escherichia coli, bacillus subtilis etc.; For example can be Bacillus coli cells (E.coli), like escherichia coli HMS174 (DE3) or BL21 (DE3).Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell.

Based on the inventor's new discovery, the invention provides the purposes of FSTL1 albumen or its bioactive fragment, be used to prepare the compositions that suppresses the mammalian body sensation; Or be used to screen the material that suppresses the mammalian body sensation.

The inhibitor of FSTL1 albumen or its bioactive fragment

The inhibitor of FSTL1 albumen or its bioactive fragment also is provided among the present invention; These inhibitor can be used to treat, alleviate or improve the disease relevant with this activity, for example renal hypertension, tumor (preferably melanoma, glioblastoma multiforme and nonsmall-cell lung cancer) through the activity that reduces na-k-atp enzyme.

The inhibitor of FSTL1 albumen or its bioactive fragment can be selected from: the antibody of anti-Progynon inhibin appearance albumen 1 or its active fragment; Antisense oligonucleotide, siRNA to Progynon inhibin appearance albumen 1 coded sequence; Derive from the peptide section of na-k-atp enzyme; Or their homologous sequence, include but not limited to: the M7M8 peptide section shown in the M3M4 peptide section shown in the SEQ ID NOs:5 and 6, the SEQ ID NOs:7 and 8.

Those of ordinary skills can obtain or screen said inhibitor through the conventional method in this area in the structure of knowing FSTL1 albumen, its bioactive fragment or its coded sequence or after obtaining said albumen, fragment or coded sequence.

Compositions

The invention provides a kind of compositions, it contains effective dose (like 0.000001-50wt%; Preferable 0.00001-20wt%; Better, described FSTL1 albumen 0.0001-10wt%) or its bioactive fragment, or can activate or raise the molecule of FSTL1 and pharmaceutically acceptable carrier.

Compositions of the present invention can directly be used to improve the na-k-atp enzyme activity, with treatment, alleviation or improvement disease or the symptom relevant with this activity, for example regulates the mammalian body sensation.In addition, also can unite use with other therapeutic agent or adjuvant simultaneously.

Usually, can these materials be formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.

As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and does not have excessive bad side reaction (like toxicity, stimulation and allergy), promptly has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the carrier of therapeutic agent administration, comprises various excipient and diluent.

Compositions of the present invention contains the FSTL1 albumen and the pharmaceutically acceptable carrier of safe and effective amount.This type carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usually pharmaceutical preparation should be complementary with administering mode, and pharmaceutical composition of the present invention can be made into the injection form, for example with normal saline or contain glucose and the aqueous solution of other adjuvant prepares through conventional method.Described pharmaceutical composition should be made under aseptic condition.The dosage of active component is the treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.

The effective dose of FSTL1 albumen of the present invention or its bioactive fragment can change with the order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be confirmed (for example through clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization rate of described FSTL1 albumen or its bioactive fragment, metabolism, half-life etc.; The patient the order of severity, patient's body weight, patient's immune state, the approach of administration etc. of the disease that will treat.Usually, the dosage when FSTL1 albumen of the present invention or its bioactive fragment every day with about 0.00001-50mg/kg the weight of animals (preferable 0.0001-10mg/kg the weight of animals) gives, and can obtain gratifying effect.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.

In addition, a kind of compositions is provided also among the present invention, it contains effective dose (like 0.000001-50wt%; Preferable 0.00001-20wt%; Better, described FSTL1 albumen 0.0001-10wt%) or the inhibitor of its bioactive fragment or its coded sequence, or reduce the molecule of FSTL1 and pharmaceutically acceptable carrier.Compositions of the present invention can directly be used to lower the na-k-atp enzyme activity, with treatment, alleviation or improvement disease or the symptom relevant with this activity, for example improves the sensitivity of mammalian body sensation.In addition, also can unite use with other therapeutic agent or adjuvant simultaneously.

PH in the present composition or carrier etc. can be as indicated above, and can be selected with general knowledge as required by those of ordinary skills.

Usually, the dosage when inhibitor of the present invention every day with about 0.00001-50mg/kg the weight of animals (preferable 0.0001-10mg/kg the weight of animals) gives, and can obtain gratifying effect.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.

Active method of raising/reduction na-k-atp enzyme and administering mode

The invention provides a kind of na-k-atp enzyme activity that improves to treat, to alleviate or to improve the disease relevant or the method for symptom, comprise FSTL1 albumen or its bioactive fragment that gives experimenter's effective dose or the molecule that can activate or raise FSTL1 with this activity.When being used to regulate the mammalian body sensation, preferred FSTL1 albumen or its bioactive fragment that adopts reorganization.

FSTL1 albumen of the present invention or its bioactive fragment or the administering mode that can activate or raise the molecule of FSTL1 have no particular limits, can be whole body or partial.Preferably, FSTL1 albumen of the present invention or its bioactive fragment can give animal through the mode of spinal cord intrathecal injection.

After the proteic purposes of the described FSTL1 of cicada, can adopt several different methods well known in the art that described FSTL1 albumen or its encoding gene or its pharmaceutical composition are delivered medicine to mammal.Preferably, can adopt the means of gene therapy to carry out, such as can be directly with FSTL1 albumen through delivering medicine to the experimenter such as methods such as injections; Perhaps, can will carry FSTL1 expression of gene unit (such as expression vector or virus etc.) through certain approach and be delivered on the target spot, and make it the FSTL1 albumen of expression activity.

As one embodiment of the present invention; Can described FSTL1 albumen directly be delivered medicine to mammal (such as the people); Perhaps; Can the proteic gene of coding FSTL1 be cloned in the appropriate carriers (like conventional protokaryon or carrier for expression of eukaryon or viral vector such as herpesvirus vector or adenovirus vector) through the method for routine, described carrier be imported to express in the proteic cell of said FSTL1, make described cellular expression FSTL1 albumen.Can realize the proteic expression of FSTL1 through an amount of said cell being incorporated into the suitable position of body of mammals.

Method well-known to those having ordinary skill in the art can be used to make up the sequence that contains the FSTL1 protein coding gene and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc.Described DNA sequence can effectively be connected on the suitable promoter in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome binding site and the transcription terminator that translation initiation is used.

Comprise the carrier of above-mentioned suitable gene order and suitable promoter or control sequence, can be used to transform appropriate host cell, so that it can marking protein.Persons skilled in the art all know how to select appropriate carriers, promoter, enhancer and host cell.

Recombiant protein in the above methods can or be secreted into the extracellular at cell inner expression.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) is technological with other various LCs and the combination of these methods.

Aspect opposite; The present invention also provide a kind of reduce the na-k-atp enzyme activity with treatment, alleviate or improve the disease relevant or the method for symptom with this activity, comprise the inhibitor of the FSTL1 albumen that gives experimenter's effective dose or its bioactive fragment or its coded sequence, perhaps can reduce the molecule of FSTL1.The administering mode of these materials has no particular limits, can be whole body or partial.Preferably, FSTL1 albumen of the present invention or its bioactive fragment can give animal through the mode of spinal cord intrathecal injection.

The method of the low active potential material of na-k-atp enzyme is regulated in screening

Getting the described FSTL1 albumen of cicada, can screen expression or the active material of regulating FSTL1 based on this characteristic for after improving the active purposes of na-k-atp enzyme.

Therefore, the method that the present invention provides a kind of screening to can be used for regulating the active potential material of na-k-atp enzyme, described method comprises: candidate substances is contacted with the system of expressing FSTL1; With of the influence of detection candidate substances to FSTL1; If said candidate substances can improve expression or the active or secretion of FSTL1, show that then this candidate substances is to can be used for improving the active potential material of na-k-atp enzyme; If said candidate substances can reduce expression or the active or secretion of FSTL1, show that then this candidate substances is to can be used for reducing the active potential material of na-k-atp enzyme.

In optimal way of the present invention, when screening,, also matched group can be set in order to be easier to observe expression or the active change of FSTL1, described matched group can be the system of not adding the expression FSTL1 of said candidate substances.

Described system includes, but is not limited to: solution system, subcellular fraction system, cell system, organizational framework, organ systems or animal system.Can contain FSTL1 in the described system, be used for adding therein candidate substances, observe the influence of candidate substances FSTL1; Perhaps, can contain the upstream and downstream material that FSTL1 reaches the adjusting path relevant with na-k-atp enzyme in the described system simultaneously, be used for adding therein candidate substances, observe the influence of candidate substances simultaneously for FSTL1 and adjusting path thereof.

As optimal way of the present invention, described method also comprises: the potential material to obtaining carries out further cell experiment and/or animal experiment, with further selection and definite for regulating the active real useful material of na-k-atp enzyme.

On the other hand, the present invention also provide adopt that said screening technique obtains can be used for regulating the active potential material of na-k-atp enzyme.The material that these Preliminary screening go out can constitute a screening storehouse so that people finally can therefrom filter out can be for the expression of regulating FSTL1 and active real useful material.

On the other hand, the present invention also provides a kind of method of screening treatment or lenitive potential material, and it is characterized in that described method comprises: (1) contacts candidate substances with the system of na-k-atp enzyme; (2) detect the influence of candidate substances to na-k-atp enzyme; If said candidate substances can improve the activity of na-k-atp enzyme, show that then this candidate substances is to can be used for treatment or lenitive potential material.

Major advantage of the present invention is:

(1) the present invention has disclosed the relation between FSTL1 and the na-k-atp enzyme activity first, thereby provides a kind of active through regulating na-k-atp enzyme, thereby is used to treat, alleviate or improve the new method and the compositions of disease relevant with this activity or symptom;

(2) the invention provides a kind of brand-new sensory information and transmit regulatory mechanism, having proved FSTL1 first has regulating action to the sensory information transmittance process, thereby provides one to regulate somesthetic new way.

(3) screening for the treatment pain medication provides good approach.Can be with the na-k-atp enzyme activity as the drug screening target spot, the medicine of screening treatment pain.Also can FSTL1 as the drug screening target spot, thereby the expression material of screening and activating FSTL1 is used to prepare the medicine of treating pain.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York, publishing house of cold spring harbor laboratory, New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.

I. material and method

The DRG sample preparation

Get the embryo respectively 14 days, birth back (P) 0,7,14 and 28 day, adult SD rats (available from Chinese Academy of Sciences's Shanghai Experimental Animal Center, male), put to death the anesthesia back, gets waist 4 and waist 5 dorsal root ganglion, use the dry ice quick-freezing, is used for RNA blotting and immunoblotting and tests.

Immunohistochemical assay

Get embryo 12 and 15 days SD rats respectively, soaked 12 hours, directly carry out frozen section with 0.2% picric 4% paraformaldehyde (37 ℃, with the preparation of 0.1mol/L phosphate buffer, pH 6.9) fixative.Get adult SD rats, the anesthesia back pours into normal saline (37 ℃) 50ml earlier through aorta, and then pours into fixative 50ml, pours into the fixative 200ml of 4 ℃ of pre-coolings then immediately, continues 5 minutes.The dabbling amount of liquid of P14 days rat reduces to 1/5 amount.Take out waist 4 and waist 5 spinal cords, in same fixative, fix 90 minutes after 4 ℃, in the phosphate buffer that contains 10% sucrose, soaked 24 hours then.Cut 14 μ m frozen sections.

With an anti-diluent (PBS that contains 1%BSA and 0.3%Triton X-100) dilution first antibody, placed 16-24 hour at 4 ℃.After washing with 0.01mol/LPBS, add with the fluorescently-labeled SA of PBS dilution (1: 100; Jackson ImmunoResearch), puts into 37 ℃ of incubators and hatched 30 minutes, wash the back mounting.Section is gathered pictorial data with laser confocal microscope (Leica SP2).Every rat of statistical data is got 3 sections, every group of 3 rats.

The production of FSTL1-Myc/his fusion rotein and purification

(sequence is referring to the GenBank accession number: NM_024369) insert in the Xho I/Hind III restriction enzyme site of pcDNA3.1Myc/his plasmid (Invitrogen) with rat and human FSTL1 complete encoding sequence for the inventor; Change this recombiant plasmid in the 293T cell (ATCC) over to through calcium phosphate transfection method again; After the DMEM complete medium is hatched 12 hours; Change ISCOVE culture medium (Invitrogen) into; Supernatant was collected once in per 36 hours, and concentrated and purification through HisBind method of purification (Qiagen).

With rat Fstl1cDNA sub-clone in the pcDNA3.1myc/His plasmid.Zones of different through pcr amplification FSTL1 makes up FSTL1 ^{Δ EF}, and it is cloned in the pcDNA3.1myc/His plasmid, the primer sequence is as follows:

FSTL1 ^1-141: 5 '-ATGTGGAAACGCTGGCTGGC-3 ' (forward primer, SEQ ID NO:9)

5 '-ACTGCCTTTAGAGAACCAGC-3 ' (reverse primer, SEQ ID NO:10);

FSTL1 ^249-306: 5 '-TGCGCCCTGGAGGACGAAAC-3 ' (forward primer, SEQ ID NO:11)

5 '-ATCTCTTTGGTGTTCACCT-3 ' (reverse primer, SEQ ID NO:12)

FSTL1 ^E165AStructure adopted the method for two-wheeled PCR.First round PCR is with the zones of different of following primer amplification FSTL1:

5 '-ATGTGGAAACGCTGGCTGGC-3 ' (forward primer, SEQ ID NO:13)

5 '-ATTTCAGGAATGCGCTGGAGTCCAGGTGAGAGT-3 ' (reverse primer, SEQID NO:14);

5 '-GGACTCCAGCGCATTCCTGAAATTCGTGGAGCA-3 ' (forward primer, SEQID NO:15)

5 '-ATCTCTTTGGTGTTCACCT-3 ' (reverse primer, SEQ ID NO:16).

In 1% agarose gel, separate the PCR product and carry out purification, mix the template of taking turns PCR then as second.The full-length cDNA of FSTL1 point mutation increases with following primer, and is cloned in the pcDNA3.1myc/His plasmid.

5 '-ATGTGGAAACGCTGGCTGGC-3 ' (forward primer, SEQ ID NO:17)

5 '-ATCTCTTTGGTGTTCACCT-3 ' (reverse primer, SEQ ID NO:18).

Antibody Preparation

(sequence is referring to the GenBank accession number: NM_024369) insert the pGEX-KG plasmid, obtain corresponding proteins matter through GST expression and purification then with 31 aminoacid of rat and people FSTL1C end and complete encoding sequence.After these two kinds of protein (0.5mg) and complete Freund's adjuvant mixing, be expelled to the subcutaneous of rabbit or mice, inject albumen (0.25mg) and complete Freund's adjuvant mixed liquor after three weeks once more; After fortnight, carry out immunity once more.The serum preparation of getting corresponding rabbit and mice then becomes the FSTL1 antibody in rabbit source and mice source.

Neuron is cultivated

Get the SD rat anesthesia and put to death back exposing spinal cord and neuroganglion; Fast the nerve of neuroganglion both sides is cut off with eye scissors; Take off neuroganglion and be placed in DMEM (Gibco) culture medium that contains digestive enzyme (collagenase 1.0mg/ml, pancreatin 0.4mg/ml and DNA enzyme 0.1mg/ml), put 37 ℃ of water-bath digestion 30 minutes.For the DRG neuron of acute isolation, the culture medium that will contain digestive enzyme with the moderate Pasteur's pipe of thickness is drawn, and with extracellular fluid (ECS:150mM NaCl, 5mM KCl, 1mMMgCl ₂, 2.5mM CaCl ₂, 10mM HEPES and 10mM D-glucose) clean neuroganglion 2-3 time after, blow and beat neuroganglion gently and broken up out until individual cells, the cell kind of breaing up is adherent on coverslip, and cultivation can be put 8-10h room temperature or 4 ℃ in extracellular fluid.Neuron to being used to cultivate cleans digestive enzyme with culture medium, with the cell culture after breaing up DMEM+10%FBS (Gibco) complete medium 48 hours.

COS-7 cell culture and transfection

(sequence is referring to the GenBank accession number: NM_012504) insert pIRES-EGFP2 (Clonteck) with α 3NKA (NM_012506) complete encoding sequence, β 1NKA (NM_013113) complete encoding sequence is inserted pcDNA3.1 (-)-A plasmid (Invitrogen) with rat α 1NKA for the inventor.Through the liposome transfection method α 1NKA and β 1NKA, α 3NKA and β 1NKA recombiant plasmid respectively corotation go in the COS-7 cell (ATCC); After the DMEM complete medium is hatched 36-48 hour, be used for NKA enzymatic activity test kit (bio-engineering research institute is built up in Nanjing) and detect the NKA enzymatic activity.

The neuron immunoblotting stimulates back FSTL1 secretion experiment

Separating the DRG neuron that obtains from back (P) 14 days the rat that is born cultivated 48 hours among the DMEM that contains 10% serum (FBS); Changing the DMEM that contains 1% serum afterwards into cultivated 4 hours again; After in fresh culture, hatching 2 hours, use 55mM KCl and 1 μ M capsaicin (Sigma) to handle neuron 1 hour respectively.Obtain the culture medium of processed group and matched group respectively, precipitate the protein in the culture medium 4 ℃ of methods of placing 10 minutes with 10% trichloroacetic acid, 15, centrifugal 10 minutes of 000g, deposition is dissolved with 100 μ l SDS sample buffers, and carries out boiling water bath 5 minutes.

The neuron of cultivating is used the lysate cracking.Organizing also of rat and mice done same processing at 4 ℃.Carry out the SDS-PAGE electrophoresis after the sample treatment, be transferred on the poly difluoride membranes again, film is with 1 hour (room temperature) of TBST buffer sealing that contains 5% skim milk and 0.1%Tween20.Used the anti-FSTL1 antibody of rabbit (Rb) then respectively on demand (1: 4000; The gst fusion protein immune rabbit gained serum of described in " material and method " part, 31 aminoacid of C-terminal of rat FSTL1 being processed), mouse anti actin antibody is (1: 50000; Chemicon) and mouse anti Myc antibody (1: 4000; Chemicon), under 4 ℃ of conditions, hybridize and spend the night.With the corresponding two anti-hybridization that are connected with horseradish peroxidase, and, make public by the ECL developing liquid developing with after the TBST rinsing.

With the ECL signal intensity of ImageQuant 5.1 mensuration protein hybridizations, the ratio of FSTL1 in the interior and full cell pyrolysis liquid of culture medium calculates the ratio between interior FSTL1 of culture medium and the actin intensity after the computing.Carry out standardization with the corresponding matched group of each experiment.

The calcium record by imaging

Using the fura-2 dyestuff (to calcium sensitive fluorescent dye, Sigma) carries out lucifuge to DRG cell in the culture dish and hatched 30 minutes, take out the cell of hatching then and be positioned on the object stage of fluorescence inverted microscope (OlympusIX50), carry out the calcium imaging.Excite light wavelength to be respectively 340nm and 380nm, obtain radiative fluorescence intensity respectively, calculate the relative value that its ratio obtains calcium concentration.More than test is gone up at TILLvisION imaging system v3.3 (T.I.L.L. company, Germany) and is accomplished.

The spinal cord thin slice is made and record

(the urethane lumbar injection 1.2g/Kg) go laminectomy, exposure lumbosacral enlargement place spinal cord with the animal of anesthesia; Take out lumbosacral enlargement place spinal cord and be positioned in the frozen water mixed liquor of artificial cerebrospinal fluid, the root, root all excises before and after other after keeping a side L5; Peel off the dura mater, arachnoidea and the mantle that cover the spinal cord surface; Vibratome cuts the spinal cord thin slice that has root behind the L5, and the thick 600-650 micron of sheet is positioned over it and is connected with 95%O ₂And 5%CO ₂The artificial cerebrospinal fluid perfusion system in hatch opening entry after 1 hour.

The structure of Fstl1 conditional knock-out mice

The specific Cre recombinase in position can cut the principle of the gene segment between two LoxP sites of reorganization in Using P 1 antibiotics; Utilize pNeotkLoxP carrier (Nanjing University's model animal institute); Made up the conditionality targeting vector of Fstl1, three LoxP sites have been arranged in the targeting vector.

The exon 2 of Fstl1 (F2 includes signal peptide) between a LoxP site, and be used for the later stage at positive and negative sign (neo, tk) that embryonic stem cell screens between another LoxP site.Not I is linearizing restriction enzyme site.Change linearizing targeting vector electricity over to embryonic stem cell subsequently; To change above-mentioned embryonic stem cell with the plasmid of Cre over to then; Targeting vector can be cut into three kinds of situation; Select inventor's conditions needed property targeting vector, be expelled to the embryonic stem cell that contains this conditionality targeting vector then and receive in the stomatoblastula, be transplanted to then in the female Mus body of pseudo-fetus and develop into individuality.The cell (cell of blastaea self and the embryonic stem cell of injection) of two types of separate sources is arranged in this mice body, thereby be called as chimera.Because the totipotency of embryonic stem cell, be expelled to a segmentation cavity to it after, its can with this blastaea each organ-tissue of bud into health together, comprise sexual cell.If embryonic stem cell develops into sexual cell; The genetic modification that obtains through homologous recombination can be imported in the chimeric germ cell line and can pass to the heterozygote mice that the next generation will obtain containing the conditionality targeting vector; With heterozygote mice pairing breeding, just can obtain containing the homozygote mice of conditionality targeting vector then.Use Na subsequently _v1.8-Cre (the Cre recombinase is specific expressed at Na for mice _v1.8 in the male Primary Sensory Neuron, on the minicell, Heidelberg, Germany university provides) breed with the mice pairing that contains the conditionality targeting vector, knock out Na in the Primary Sensory Neuron thereby obtain specificity _v1.8 the mice that FSTL1 expresses on the positive cell.

Behavioristics is observed

Coordination exercise test (Rota-rod test)

With animal place the uniform rotation of Rota-rod Treadmill motion appearance (12 change/adapt to 30s on min) the runner after; Make runner quicken (acceleration is 3 to 30 commentaries on classics in the 5min); The record rat is from beginning to accelerate to the time of falling runner, as the index of the reflection rat motor coordination ability.

Thermostimulation is measured incubation period

It is the transparent organic glass case of the 20cm * 20cm * 25cm of 2mm plate glass that animal is placed a bottom, and is positioned on the shelf that is higher than laboratory table 30cm so that observe.Use the thermostimulation appearance, select 100W halogen delineascope, regulation voltage is to 10V; Regulate the distance between lamp source and the glass plate, making the irradiates light loop diameter that drops on the vola is 5mm, and record the sufficient withdrawal reflex time (s) occurs contracting from beginning to expose to; This is the thermostimulation response latency; Repeated measure 5 times, same position is 10min at interval, and different parts is 5min at interval.Remove measured value for the first time, get back 4 measured values and average, as the thermostimulation response latency and as quantitative target.As shining, in order to avoid cause vola excessive tissue hot injury greater than reactionless then the stopping of 30s.

The mechanical stimulus threshold measurement

Process mechanical stimulus device (von-Frey) fiber of different stimulated intensity with the nylon yarn of different thicknesses and different length.It is on the high shelf of the 30cm of wire gauze that the transparent organic glass case of one 20cm * 20cm * 25cm is positioned over the top, and tested animal is placed case.The hand-held von-Frey fiber of experimenter passes iron wire grid centre, vola, stimulation in rats both sides respectively; Stimulus intensity is ascending; (inferior-minor tick 3～5s), the intensity of foot reflex more than 5 times is decided to be the threshold of reaction of animal to mechanical stimulus with occurring contracting for each intensity repetitious stimulation 10 times.As the foot reflex that contracts not occurring with 588mN von-Frey fiber stimulation in rats vola, then think the painless reaction of rat, mechanical stimulus threshold value＜58.8mN is all in 58.8mN.This is a kind of widely used mechanical stimulus quantitative approach, because its most advanced and sophisticated pressure is constant after nylon fiber is obstructed bending, so can think that each measured value all is constant.

Statistical procedures

All data of this experiment are with mean+SD (mean ± SEM) expression; The SPSS software bag carries out non-ginseng (Mann-Whitney U test) check to the random distribution sample; Paired sample is carried out variance analysis (ANOVA followed by posthoc Fisher ' s PLSD or Sheffe test) and t check, and p＜0.05 is considered to have remarkable statistical significance.

The checking of embodiment 1.FSTL1 selectivity high expressed in the DRG small neuron of growing up

For confirming specificity in the DRG expressed protein, the inventor searches for high expressed in DRG in gene bank, and reaches relatively low gene at its hetero-organization invading the exterior.The inventor found 71 (candidate gene, and through the self-control gene chip detected their express spectras in the adult rat tissue.19 gene Selection property ground high expresseds (expression in DRG is than high more than 3 times or 3 times at the average expression of its hetero-organization) in DRG wherein comprise gene such as CGRP, the P2X of expection ₃Receptor and Na _v1.8 passage.In several genes that specific expressed degree is the highest in detected adult rat DRG, FSTL1 also is one of them (Figure 1A).

Polynucleotide gene chip result shows that Fstl1 mainly expresses at the DRG of adult rat, exceeds about 8 times than other tissue level.And Fstl1 also has high expressed in heart, lung, kidney and the spinal cord of rat.Through RNA blotting (Fig. 1 K) Fstl1mRNA in each tissue is detected further proof, FSTL1 expression in heart, lung, kidney is higher.

The immunoblot experiment (Fig. 1 C) of RNA blotting (Figure 1B) and use FSTL1 specific antibody detects further proof, and FSTL1 is specificity overexpression in adult rat DRG, in spinal cord, also has expressed.It should be noted that in the embryo development procedure that FSTL1 also has expression in Hippocampus and cortex, but the expression in these brain districts disappear (Figure 1A and 1D) after birth.On the contrary, FSTL1 appears in the time of 15 days in the DRG neuron the embryo, peaks in back 28 days and keeps its high expressed to growing up (Fig. 1 D) in birth.

Mainly expression in small neuron of FSTL1 in adult rat DRG (Fig. 1 D, 1H).The DRG small neuron comprises peptidergic neuron and non-peptidergic neuron IB4 (isolectin B4) neuron that contains neuropeptide such as P material and CGRP.The immunofluorescence double labelling shows that the positive small neuron of about 46% FSTL1 expresses the P material, 54% locatees altogether with CGRP and have an appointment, and 45% the FSTL1 positive cell of having an appointment in addition is simultaneously by IB4 labelling (Fig. 1 E, 1G and 1I).In addition, FSTL1 also with label TRPV1 passage, P2X3 receptor and the Na of DRG small neuron cell _v1.8 channel protein coexpressions such as (Fig. 1 F, 1G and 1J).At rat spinal cord, FSTL1 is present in I to II layer (Fig. 1 E), the zone that the positive centripetal fiber of most of just Toplink centripetal fibers and IB4 exists.

This shows that FSTL1 has expression in Toplink and the male small neuron cell space of IB4, and can in its centripetal fiber tip, find.

Embodiment 2.FSTL1 is by special clear vesicle transportation justacrine

The inventor has detected the Subcellular Localization of FSTL1 in cell, finds that FSTL1 is stored by a kind of secretion vesicle of uniqueness.The centripetal fiber tip exists synaptic vesicle and big densified core bubble (LDCV), causes that as stimulation cellular calcium can promote its content of these vesicle secretion when raising.Behind the exocytosis, synaptic vesicle is in the recyclable utilization again of aixs cylinder tip, and big densified core bubble then needs come to be replenished by the cell space transportation.The immunofluorescence double labelling shows, in DRG small neuron cell cell space FSTL1 be associated with non-peptide class vesicle (Fig. 2 A, 1I).Immuno-electron microscope gold labelling shows that FSTL1 is arranged near the little clear vesicle (diameter 30-80nm) (Fig. 2 B) the golgi's network outside after the embedding.

The inventor has also detected the distribution of FSTL1 at the centripetal fiber tip.The method separation that separates subcellular components with SDGC obtains the different vesicle crowds of rat spinal cord dorsal horn; Discovery FSTL1 mainly appears at and contains synaptoporin (synaptoporin; Synpr) among the vesicles crowd, rather than in containing the LDCV component of CGRP (Fig. 2 C).Synaptoporin is a member of synaptophysin (synaptophysin) superfamily, is considered to the ultimate constituent of spinal cord I to II layer afferent nerve fiber tip synaptic vesicle film.The immunofluorescence double labelling shows that most FSTL1 positive particles all are synaptoporin negative (Fig. 2 A), and prompting FSTL1 mainly is present in another kind of and the different vesicles subgroup of synaptoporin.The Electronic Speculum immuno-gold labeling shows, FSTL1 is present in the little clear vesicle (Fig. 2 D) of centripetal fiber tip of back root centripetal fiber and cornu dorsale medullae spinalis I, II layer.What is interesting is that the vesicle that contains FSTL1 also has distribution (Fig. 2 D) in the active region of presynaptic membrane.In sum, FSTL1 is synthetic at cell space, is assembled into little clear vesicle, is transported to then to import the end into and carry out the presynaptic secretion slightly.

With immune labeled comparing stronger in the DRG pericaryon, FSTL1 wants weak many in the dyeing of rat spinal cord centripetal fiber, and in mouse spinal cord basic detect less than immunity positive.But, after the ligation root 24 hours with the blocking-up vesicle forward transportation can cause gather (Fig. 2 E) of centripetal fiber near DRG one end FSTL1, show that there is forward transportation in FSTL1.Therefore, detecting less FSTL1 at spinal cord centripetal fiber tip possibly be because due to the rapid secretion release of its quilt.

The inventor directly detects the secretion of FSTL1 with the rat drg neuron of cultivating.Immunoblot experiment shows that under non-stimulated condition, the horizontal presentative time dependency of FSTL1 raises in the cell culture fluid, and (Fig. 2 F G), shows that there is the spontaneous secretion of FSTL1 in the DRG small neuron.In addition, the high potassium of 55mM stimulates can promote the FSTL1 secretion, and the effect of high potassium is eliminated (Fig. 2 G) under the situation that calcium exists outside acellular.

Can significantly increase the FSTL1 level in the culture fluid with TRPV1 channel agonist capsaicin (Cap) processing, this reaction can be by the blocker capsaizepine of TRPV1 passage (CPZ, Sigma) blocking-up (Fig. 2 J).The high potassium of 55mM stimulate also can promote adult rat spinal cord thin slice and by the secretion of FSTL1 in the rat spinal cord dorsal horn synaptic glomerulus (Fig. 2 H, I).It should be noted that FSTL1 can be divided into CGRP and glutamic acid secretes, and stimulates the rat spinal cord dorsal horn synaptic glomerulus FSTL1 secretion that causes that (Fig. 2 I) only takes place under the condition that extracellular Ca2 exists.

Therefore, FSTL1 is stored in little clear vesicle transportation, and discharges at afferent nerve endings with the mode similar with other neurotransmitter.

Embodiment 3.FSTL1 suppresses the synapse of sensation nervus centripetalis and transmits

The inventor studies the synapse transmission whether FSTL1 can regulate and control nervus centripetalis through on the spinal cord thin slice of adult rat band Dorsal root, carrying out full cell record.

Dyed at the dirty 293T of human embryo kidney (HEK293T) transit cell and can express the plasmid that has Myc and the pulsating rat FSTL1 of His, and the FSTl1 albumen of its synthesis secretion of purification (Fig. 3 E), the structure of expressed FSTL1 is seen Fig. 3 A.

In the spinal cord II layer neuron of about 50% (16/31 neuron) that writes down, perfusion reorganization FSTL1 albumen (60nM or 300nM), the minimizing that the frequency of spontaneous EPSC (sEPSCs) and amplitude are dose dependent (Fig. 3 B).In addition, in about 60% (14/24) spinal cord II layer neuron of record, reorganization FSTL1 albumen can reduce EPSC (eEPSCs) amplitude (Fig. 3 C) that the C fiber brings out.(TTX, 0.5 μ M) writes down miniature EPSCs (mEPSCs) when existing at Fugu ocellatus toxin, finds that FSTL1 albumen can suppress the frequency of mEPSCs, but its amplitude is not had remarkable effect (Fig. 3 D), and prompting FSTL1 discharges glutamic acid has the presynaptic inhibitory action.FSTL1 does not make significant difference to the decay dynamics of sEPSCs and eEPSCs, show FSTL1 to the not directly effect of the character of postsynaptic membrane glutamate receptor passage (Fig. 3 B, C).

The function (Fig. 3 E) that the inventor comes further to have checked FSTL1 with the proteic mutant of multiple FSTL1, (structure is seen Fig. 3 A, E) comprising the FSTL1 mutant protein of calcium binding site (M2) or a pair of EF hand-type structure disappearance loss of function (M1).The inventor finds that the recombiant protein of these FSTL1 sudden changes has lost the inhibitory action (Fig. 3 C) to eEPSC.In addition, the people of purification recombinates Progynon inhibin (Follistatin) to the no effect of centripetal fiber transmission (Fig. 3 F).In sum, external source gives FSTL1 has special inhibitory action to the synapse transmission of spinal cord centripetal fiber.

Also produce inhibitory action for whether the further endogenic FSTL1 of research transmits the synapse of centripetal fiber, the inventor adopt conventional method preparation and purification the antibody of FSTL1 with the excretory FSTL1 of sealing spinal cord thin slice self.Experiment is found; Perfusion FSTL1 antibody (1: 1000) can increase the frequency of sEPSC and amplitude (Fig. 3 B of amplitude and eEPSC; C), and the not effect of the preimmune serum of contrast, the FSTL1 of prompting endogenous excretion under the normal physiological situation promptly has inhibitory action to importing the synapse transmission into.

Embodiment 4.Fstl1 conditional gene knockout mice somesthesia sensitivity increases

After observing the synapse transmission of FSTL1 to the cornu dorsale medullae spinalis nervus centripetalis inhibitory action is arranged, the inventor analyzes FSTL1 possibly participate in regulating normal somesthesia function.The whole body variation that causes for avoiding gene to knock out entirely, inventor's application conditions formula gene knockout method, the FSTL1 in the knock-out mice DRG small neuron observes its effect in elementary sensory information conduction specifically.Have 96.5% and Na in the DRG small neuron of FSTL1 because express _v1.8 the location is the Fstl1 conditionality allele (Fstl1 on basis so use has with loxP altogether ^F) mice and the Na of (Fig. 4 A) _v1.8-Cre BAC transgenic mice strain (SNS-Cre) hybridization can obtain Fstl1 conditional knock out mice (Agarwal etc., Genesis.2004,38:122-129) Fstl1 ^F/F: SNS-Cre (Fstl1 ^-/-).

Fstl1 ^F/F: SNS-Cre (Fstl1 ^-/-) the mice activeness is good, has fertility, significantly deformity do not occur, only be in the DRG neuron, detect expression less than FSTL1 (Fig. 4 B, C).These Fstl1 ^-/-The DRG neuronal quantity of mice and the anatomical structure of size distribution and spinal cord and wild-type mice (Fstl1 ^{+ /+}) similar.And Fstl1 ^-/-Na in spinal cord of mice and the DRG neuron _vSignificant change (Fig. 4 C) does not all take place 1.8 passage, P2X3 receptor, TRPV1 passage, P material and other main ion channel, receptor, neuropeptide and signal transducers are expressed.But, at the spinal cord thin slice the responsive neuronic full cell record of II layer of FSTL1 shown, with Fstl1 ^{+ /+}Mice is compared, Fstl1 ^-/-The increase of the sEPSC frequency that mice 15mM KCl causes will be significantly (Fig. 5 A), showing stimulates the FSTL1 that causes to discharge that (Fig. 2 G is necessary for suppressing strong synapse transmission H).

For detecting the influence that the FSTL1 disappearance is transmitted the body sensation; The inventor carries out outside somatic cell, writing down (according to Li and Chen etc. to wide power territory (WDR) neuron of mouse spinal cord relief angle; Neuroscience; 126 (2001): the method for being put down in writing among the 753-762 writes down), these neurons and Primary Sensory Neuron have synaptic contact, and temperature and mechanical stimulus are all responded.Stimulate the neuronic receptive field of WDR can increase the granting of these neuron action potentials with temperature and mechanical stimulus.What is interesting is that the inventor finds and Fstl1 ^{+ /+}Mice is compared, Fstl1 ^-/-The WDR neuron action potential granting of mouse spinal cord is stronger, and same temperature and mechanical stimulus meeting cause that discharge increases (Fig. 5 B).At Fstl1 ^-/-In the mice, the WDR cell discharge frequency that non-noxious stimulation causes (38 ℃, 7.0 ± 1.5Hz) stimulate with compressing (15.4 ± 1.8Hz) with noxious stimulation at Fstl1 ^{+ /+}The discharge frequency of mice WDR cell induction (45 ℃, 7.9 ± 1.0Hz) with pinch piercing and swash that (16.3 ± 3.9Hz) is similar.In addition, at Fstl1 ^-/-Mouse spinal cord intrathecal injection FSTL1 can change this reaction fully, makes Fstl1 ^-/-The neuronic discharge frequency of mice WDR fades to similar with wild-type mice (Fig. 5 B), proves Fstl1 ^-/-The increase of mice irritability is the direct result of FSTL1 disappearance, rather than owing to developmental defect or other reason.These data show that the DRG small neuron is expressed FSTL1 and kept spinal cord to feel that the loop physiological equilibrium is necessary.

The inventor further detects Fstl1 through behavioristics's experiment ^-/-The pain reaction of mice is to understand the functional meaning of FSTL1 in DRG neuron under the body situation.The inventor finds, Fstl1 in the heat radiation experiment ^-/-Mice significantly shortens (Fig. 5 C) to the response time of thermostimulation, prompting Fstl1 ^-/-Mice is improved the sensitivity of nocuity thermostimulation.In addition, mechanical stimulus device von-Frey fiber shows Fstl1 to the mechanical irritation experiment of the mice rear foot ^-/-Mice also significantly reduces (Fig. 5 C) to the threshold value of mechanical stimulus.Change in these behavioristicss is not because Fstl1 ^-/-There is the motor function defective in mice, because the reaction with wild-type mice similar (Fig. 5 D) of these mices in quickening rotation test (accelerating rotarod test).In addition, mice rear foot injection of formalin is induced inflammatory pain model, Fstl1 ^-/-Mice also has enhanced pain sensation reaction, shows that its time of licking the rear foot of II phase phase of formalin inflammatory reaction significantly increases (Fig. 5 E).In sum, FSTL1 comprises that to normal somesthesia the pain sensation is essential, and in the acute inflammation pain, effect is arranged, and the FSTL1 disappearance can cause the pain sensation responsive.

Behavioristics's experiment shows that at normal adult rat intrathecal injection FSTL1, rat does not significantly change the reaction of nocuity thermostimulation.Yet, give Fstl1 ^-/-Mice intrathecal injection FSTL1 but can fully overturn, and it increases (Fig. 5 F) to sensitivity of thermostimulation.In addition, injection FSTL1 antibody (method preparation described in " material and method " part) also can increase the reaction (Fig. 5 G) of rat to the heat injury sexual stimulus.In sum, FSTL1 down-regulated expression or the excretory FSTL1 of functional block endogenous property all can cause the pain sensation responsive.

Embodiment 5.FSTL1 suppresses the chronic pain that nerve injury causes

Proved FSTL1 that sensory nerve ending discharges for keep normal somesthesia essential after, the inventor thinks further to explore its possibly act in neuropathic pain (being mostly because nerve injury causes).The pathophysiology characteristics of neuropathic pain are the hyperexcitabilities of spinal cord sensation loop.The inventor finds DRG neuron FSTL1 expression decreased behind peripheral nerve injury; Sciatic nerve cuts off the small neuron number that contains Fstl1 mRNA in the rat waist 4-5 sections DRG of back and significantly reduces (Fig. 6 A); And cause the proteic minimizing of FSTL1 in the centripetal fiber (Fig. 6 B, Fig. 6 C) in DRG small neuron cell space and cornu dorsale medullae spinalis.The immunofluorescence double-label experiment finds that its FSTL1 of cell that expresses transcriptional factors 3 (label of DRG neuron axon damage) significantly reduces, and shows that the FSTL1 expression decreased directly occurs in the neuron of damaged.

Cut off neurogenic pain model that (SNI) cause (according to Decosterd at another kind by sciatic nerve branch; I.; And Woolf; C.J. (2000); The method of being put down in writing among the Spared nerve injury:an animal model ofpersistent peripheral neuropathic pain.Pain 87:149-158 is set up this model) in the downward modulation (Fig. 6 D) of DRG neuron FSTL1 appears equally, and intrathecal injection FSTL1 albumen is the elimination of dose dependent and is cut off the unusual machinery pain (Fig. 6 E) that causes by sciatic nerve branch.In sum, the remarkable minimizing of FSTL1 can be imported the mechanism of the disinthibiting participation formation neuropathic pain of transmission through irritability into after the nerve injury.

Embodiment 6.FSTL1 combines α 1 sodium-potassium ATP enzyme (α 1NKA)

The inventor further comes to confirm the receptor of FSTL1 effect.Use the FSTL1-myc that recombinates to hatch the rat Dorsal root ganglion neuron of cultivation, added cross-linking agent (Bis (sulfosuccinimidyl) suberate, the BS of impermeable membrane simultaneously ³), with the myc antibody test to FSTL1 at nearly 150kD place a visible band (Fig. 7 A), and FSTL1-myc is positioned at 37-45kD place (Fig. 7 A) when not adding cross-linking agent, points out the protein binding of a FSTL1-myc and an about 100kD.

In order to confirm this albumen, to BS ³Albumen composition after crosslinked carries out mass spectral analysis, and the peptide section sequence of FSTL1 is arranged in the albumen of being analyzed, and can detect 3-7 peptide section of α 1 sodium-potassium ATP enzyme (α 1NKA) simultaneously, and 15% of α 1NKA aminoacid sequence has been contained in the order-checking of 3 samples.Simultaneously; Detect in the monoclonal antibody of the same sample of handling with α 1NKA; Visible part α 1NKA molecular weight is increased to～145kD (Fig. 7 B) from～110kD, only detects～the single band (Fig. 7 B) at 110kD place with α 3NKA antibody in the same sample, explains that FSTL1 does not combine with α 3NKA.

Immunohistochemical staining is presented at has 51% express alpha 1NKA in the Dorsal root ganglion neuron, wherein 63% is small neuron, and the 36%th, large neuron (Fig. 7 C) has 66% to contain FSTL1 (Fig. 7 C) in the α 1NKA positive cells.The co-immunoprecipitation experiment shows that endogenic FSTL1 can combine α 1NKA (Fig. 7 D) in the rat Dorsal root ganglion.The α 1NKA subunit of corotation zone flag label and FSTL1-myc or FSTL1 (M1)-myc can detect FSTL1-myc in the albumen under the co-precipitation of flag antibody mediated immunity in the COS7 cell, but can not detect FSTL1 (M1)-myc (Fig. 7 E).

Scatchard analysis (Current Protocols in Protein Science (2000) A.3H.1-A.3H.55) shows ¹²⁵I-FSTL1 dose dependent ground combines the COS7 cell (Kd=43nM) (Fig. 7 F) of coexpression α 1NKA subunit and β 1 subunit.Therefore, FSTL1 can combine α 1NKA.

The inventor compares born of the same parents' outer shroud of α 1NKA subunit and α 3NKA subunit in order to confirm in α 1NKA subunit and the interactional critical sites of FSTL1, and having found has different amino acid (Fig. 7 G) at second and the 4th born of the same parents' outer shroud.The sequence alignment of Fig. 7 G shows that the 314th (E) in the α 1NKA subunit maybe be relevant with the effect of FSTL1 with the 889th (T) aminoacid.In the COS7 cell of expressing FSTL1-myc and α 1NKA subunit, co-immunoprecipitation is tested to be presented in the albumen that the co-precipitation of flag antibody mediated immunity gets off and is had FSTL1-myc, at coexpression FSTL1-myc and α 1NKA subunit ^E314G-flag or α 1NKA subunit ^T889N-flag or α 1NKA subunit ^{E314G, T889N}In the cell of-flag, the FSTL1-myc that exists in the albumen that the co-precipitation of flag antibody mediated immunity is got off significantly descends, and proves that the 314th (E) in the α 1NKA subunit is relevant with the effect of FSTL1 really with the 889th (T) aminoacid.Simultaneously, FSTL1-myc does not combine with α 3NKA subunit, but with α 3NKA subunit ^{G304E, N879T}-flag combines (Fig. 7 G), further proves the 314th (E) and the importance of the 889th (T) aminoacid in α 1NKA and FSTL1 combination in the α 1NKA subunit.Therefore, in the α 1NKA subunit among second born of the same parents' outer shroud M3M4 among the 314th amino acids E and the 4th the born of the same parents' outer shroud M7M8 the 889th amino acids T be the pivotal role site of α 1NKA subunit and FSTL1.

Embodiment 7.FSTL1 activates α 1 sodium-potassium ATP enzyme (α 1NKA)

NKA is a kind of memebrane protein that cell membrane potential is changed through inhomogeneous transhipment sodium ion and potassium ion.In the Dorsal root ganglion neuron of cultivating, also can detect FSTL1 and be the activity (Fig. 8 A) that dose dependent ground raises NKA.β 1 subunit of corotation α 1NKA-flag and band myc label in the COS7 cell can detect the activity (Fig. 8 A) of FSTL1 dose dependent ground rise NKA, then can not (Fig. 8 A) during corotation α 3NKA-flag.

FSTL1 is at corotation α 1NKA subunit ^E314G-flag or α 1NKA subunit ^T889N-flag or α 1NKA subunit ^{E314G, T889N}Can not increase the activity of NKA in the COS7 cell of-flag and β 1 subunit, FSTL1 ^E165ACan not improve the active alpha (Fig. 8 B) of NKA in the COS7 cell of coexpression α 1NKA subunit and β 1 subunit.COS7 is complete, and cell record is observed; In the cell of corotation α 1NKA-flag and β 1-myc subunit FSTL1 cause the film hyperpolarization (～8mV); Significantly reduce cell membrane potential (Fig. 8 C), transmembrane potential is not influenced (Fig. 8 C) at the cell FSTL1 of corotation α 3NKA-flag and β 1-myc subunit.β 1 subunit and α 1NKA subunit corotation are realized that to FSTL1 strong and stable function is important, in the cell of independent transfection α 1NKA subunit FSTL1 can only cause moment and low-level film hyperpolarization (～2mV).Therefore, FSTL1 can activate α 1NKA specifically, thereby causes the cell hyperpolarization.These results are also illustrated in 314 (E) and 889 (T) aminoacid on the α 1NKA subunit, and 165 (E) aminoacid on FSTL1 is important to the function of FSTL1 and α 1NKA.

In the COS7 cell of coexpression α 1NKA subunit and β 1 subunit, preincubate M3M4 (EC ₅₀=3.6 μ M) or M7M8 (EC ₅₀=2.9 μ M) peptide section (synthetic by Ji Tai company) can make ¹²⁵The combination rate of I-FSTL1 significantly descends, and is illustrated in the calmodulin binding domain CaM that M3M4 and M7M8 peptide section in the α 1NKA subunit contain FSTL1, and relatively, M1M2 peptide section can not reduce said combination rate (Fig. 8 D).Preincubate M3M4 or M7M8 peptide section can weaken the NKA increased activity effect (Fig. 8 E) that FSTL1 causes, more confirm the importance of M3M4 or M7M8 peptide section.In addition, these results represent that M3M4 or M7M8 or similar peptide section can be used as the inhibitor of Progynon inhibin appearance albumen 1, reduce the combination of FSTL1 and NKA because these peptide sections can combine with FSTL1.

Adult rat Dorsal root ganglion small neuron (diameter is 15～30 microns) to acute isolation is done full cell record; FSTL1 (30nM) reduces the frequency of neuron action potential; And induce the film hyperpolarization (; This influence was reversed when specific inhibitor ouabain (the OB 100 μ M) time that gives NKA simultaneously, and the effect of the film hyperpolarization that FSTL1 causes can be overturn by ouabain (Fig. 8 F), also can be by the 2nd born of the same parents' outer shroud M3M4 peptide section institute's antagonism (Fig. 8 F) of preincubate α 1NKA.Therefore, α 1NKA is the receptor of FSTL1.

In cornu dorsale medullae spinalis; SABC shows that α 1NKA mainly is distributed in the nerve fiber of cornu dorsale medullae spinalis I-V layer (Fig. 8 G); These nerve fibers that contain α 1NKA mainly are primary afferent terminations; Because research in the past and present research do not find that all dorsal horn neurons has the mRNA of α 1 subunit, provides the foundation for FSTL1 presynaptic generation effect like this.The full cell record of spinal cord brain sheet of band Dorsal root shows; FSTL1 reduces the frequency of sEPSC; This influence is reversed (Fig. 8 H) when giving the specific inhibitor ouabain (OB 100 μ M) of NKA simultaneously, the 2nd born of the same parents' outer shroud M3M4 peptide section of preincubate α 1NKA can obviously strengthen the frequency (Fig. 8 I) of sEPSC.The full cell record demonstration of spinal cord brain sheet II layer neuron M3M4 peptide section is hatched the eEPSC amplitude (Fig. 8 J) that the stimulation of C fiber causes that increases.Therefore, excretory FSTL1 regulates and imports that to transmit be through activating α 1NKA into.

The proteic functional area research of embodiment 8.FSTL1

The inventor further comes to confirm to produce in the FSTL1 albumen zone of function; Noxious stimulation capsaicin (Capsaicin) can cause that the capsaicin receptor VR1 on the small neuron in the Primary Sensory Neuron is open; Cause flow of calcium ions, can detect this process with the calcium method for imaging.When adding capsaicin, add each mutant protein of FSTL1 (the concentration 60nM that adds capsaicin 1 μ M and mutant protein), detect the influence of flowing in the calcium that each mutant protein causes capsaicin.

Experimental result shows (Fig. 9): the FSTL1 full-length molecule can significantly suppress the inductive flow of calcium ions of capsaicin; The FSTL1 albumen of disappearance FS domain, VWC domain does not influence its inhibitory action to the inductive flow of calcium ions of capsaicin; Be illustrated among the FSTL1; FS domain and VWC domain are not participated in the inhibitory action of FSTL1 to the inductive flow of calcium ions of capsaicin; Relatively, disappearance EFI+EF hand-type connection+EFII or the 165th amino acids be mutated into alanine the FSTL1 proteins lose to the inhibitory action of the inductive flow of calcium ions of capsaicin, show that the 165th amino acids is important to this function of FSTL1.In fact, the synthetic polypeptide (SP76) of 156-173 amino acids sequence just has the similar function of FSTL1 (being described below) in this domain.

Further on the COS7 cell of stable transfection α 1NKA and β 1NKA subunit, detect α 1NKA enzymatic activity; The FSTL1 total length can significantly activate the activity of α 1NKA enzyme, and the synthetic polypeptide (SP76) of 156-173 amino acids sequence also can significantly activate the activity of α 1NKA enzyme among the EF chiral structure territory I.

These results show; FS in FSTL 1 albumen and VWC fragment are not participated in the inhibitory action of FSTL1 to the inductive flow of calcium ions of capsaicin; EFI+EF hand-type connection+EFII fragment is most important to the inhibitory action of the inductive flow of calcium ions of capsaicin to FSTL1, and the 165th amino acids among the EFII is most important to the inhibitory action of 1 pair of inductive flow of calcium ions of capsaicin of FSTL.156-173 amino acids sequence possibly be the minimum function fragment of FSTL1 among the FSTL1 of rat.

This of rat section 156-173 amino acids sequence (SEQ ID NO:1) is equivalent to people's 158-175 amino acids sequence (SEQ ID NO:2).The 159th (H) amino acids that can find out rat from rat and people's sequence alignment is made into 161 (R) in people's sequence.This is substituting of conservative.In this area, have general Chang Shizhe all to know, these substitute (for example: Ala, Leu, Ile substitute mutually) with other similar conservative not too can influence proteic activity.So the present invention includes alternate this function fragment of conservative.

Embodiment 9. screening of medicaments

Express male small neuron cell as the cell model that is used to screen with FSTL1.

Testing group: the culture of the above-mentioned cell of handling with candidate substances;

Matched group: the culture of the above-mentioned cell of handling without candidate substances.

If compare with matched group; Significantly rising (for example more than 30%) of the proteic expression of FSTL1 in the testing group, activity, amount or secretion; Explain that then this candidate substances is potential na-k-atp enzyme agonist; Can be used for treatment or alleviation and the relevant disease of the active reduction of na-k-atp enzyme, for example reduce mammalian body sensation, treatment or alleviate familial hemiplegia type migraine, diabetes or serious symptom depression etc. through improving the na-k-atp enzyme activity.

Otherwise; If the proteic expression of the FSTL1 in the testing group, activity, amount or secretion significantly descend (for example more than 30%); Explain that then this candidate substances is potential na-k-atp enzyme inhibitor; Can be used for treatment or the alleviation disease relevant, for example be used to increase mammalian body sensation, treatment or alleviate melanoma or other growth of tumor and generation with the na-k-atp enzyme hyperactivity through reducing the na-k-atp enzyme activity.

The inventor finds in further investigation, FSTL1 can with sodium-potassium ATP enzyme (Na ⁺, K ⁺-ATPase) combine, and wherein bonded key point is among sodium-potassium ATP enzyme α 1NKA subunit second born of the same parents' outer shroud M3M4 and the 4th the born of the same parents' outer shroud M7M8.And FSTL1 can activate the activity of sodium-potassium ATP enzyme with after sodium-potassium ATP enzyme combines, thus prompting FSTL or its promoter can be used for treats, alleviates or improvement and na-k-atp enzyme are active reduces or cross and hang down relevant disease or symptom.Otherwise; Research has also proved through the active of downward modulation FSTL1 or has suppressed FSTL1 and combined with sodium-potassium ATP enzyme; Can reduce the activity of na-k-atp enzyme, thereby the inhibitor of prompting FSTL1 can be used for treating, alleviating or improvement improves or too high relevant disease or symptom with na-k-atp enzyme is active.

The inventor is for FSTL1 and somesthetic discovering, endogenous FSTL1 is somesthetic a kind of modified, and the reduction of FSTL1 helps the generation of the pain sensation.Those skilled in the art know that the excitatory synapse transmission can be regulated by excretory inhibitive factor in the relay cell of cornu dorsale medullae spinalis and the descending inhibitory system.It is that the inhibition that transmits of the somesthesia of a peripheral nerve endings autocrine is modified that the inventor's research discloses FSTL1.FSTL1 mainly is expressed on the minicell of DRG, and is transported to the afferent nerve endings of cornu dorsale medullae spinalis through little clear vesicle, and FSTL1 can discharge altogether with excitatoty neurotransmitter and carry out inhibitory action, to keep normal somatosensory information transmission.Because the FSTL1 selective expression is on the minicell of DRG, so the autocrine effect of FSTL1 is that a kind of exceptional function of the minicell of DRG can be kept somesthetic steady-state adjustment.

FSTL1 belongs to SPARC family, has Progynon inhibin spline structure territory (FS domain) and EF hand-type domain.Existing research shows that the Progynon inhibin domain of FSTL1 is not participated in the inhibitory action that FSTL1 induces generation, and this also is consistent with the result that the Progynon inhibin does not produce direct repression.FSTL1 and Dan Baijutang (agrin) all belong to Progynon inhibin gene subfamily, and different with the function of Progynon inhibin.Dan Baijutang is vital for the postsynaptic differentiation, and it is through suppressing Na ⁺/ K ⁺-ATP enzyme alpha 3 subunit strengthens the irritability reaction of cortical neuron.The inventor discovers the FSTL1 high expressed on the DRG minicell, and through Na on the activated membrane ⁺/ K ⁺-ATP enzyme suppresses cell excitement property.

Matrix metalloproteinase (MMP) and PGE2 are considered to participate in the destruction of joint process in the rheumatoid joint.Fstl1 can reduce the generation (.IntImmunol.2003Jan such as Mimori T of MMP and PGE2 in the synovial fluid; 15 (1): 71-7).In the rheumatoid arthritis disease model of mice, Fstl1 and the little peptide that derives from Fstl1 155-176 amino acids can releasing arthritis diseases (.ArthritisRheum.2004Feb such as Ozaki S; 50 (2): 660-8.).Fstl1 also is considered to an excretory muscle protein or cytokine, and in ischemia injury, Fstl1 can improve inner skin cell function (.J Biol Chem.2008Nov 21 such as Walsh K; 283 (47): 32802-11.), directly stimulate vascularization and the death that suppresses the cardiovascular cell, therefore, Fstl1 possibly can be used to treat ischemic heart disease, and (.Circ such as Walsh K J.2009Jan; 73 (1): 13-8.; .Circulation.2008Jun 17 such as Walsh K; 117 (24): 3099-108).

Based on above result of study; FSTL1 is synthesized and is sorted in the little clear vesicle in the minicell cell space of DRG; The nerve ending that these vesicles that contain FSTL1 are transported to cornu dorsale medullae spinalis discharges, and its releasing pattern is similar with big dense vesicle with traditional synaptic vesicle.Under normal physiological condition, neuronic excitement causes FSTL1 to discharge to be increased, thereby makes the teleneuron hyperpolarization, and then suppresses presynaptic excitatory transmitter and modified release.On the contrary, feel that active minimizing causes FSTL1 to discharge and reduces, thereby make teleneuron go levelization, and then presynaptic excitatory transmitter of facilitation and modified release.Yet under pathological conditions,, cause the FSTL1 persistence to reduce, thereby cause the hypersensitivity felt like peripheral nerve injury or arthritis.

Knock out the FSTL1 in the neuron or give the nocuity hypersensitive phenomenon explanation that the antibody of FSTL1 causes; The hyperexcitability of the function of FSTL1 in synapse is transmitted and teleneuron and pain are quick, and relevant (removal of inhibit function that it should be noted that neurotransmission can cause the symptom of chronic pain clinically: hyperpathia and touch and bring out bitterly.This area has known that the inhibition system in spinal cord after nerve injury is upset, and shows as the minimizing of Mu opiate receptor in the primary afferent fiber and losing of GABA ability spinal neuron.Having proposed the FSTL1 minimizing among the present invention is a key factor of neuropathic pain, and these find that prompting FSTL1 has the good curing effect to neuropathic pain.

All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims

1. Progynon inhibin appearance albumen 1 is in the purposes of preparation in the compositions, and said compositions is used for treating, alleviating or improvement and relevant disease or the symptom of na-k-atp enzyme activity through improving the na-k-atp enzyme activity.

2. purposes as claimed in claim 1 is characterized in that, said disease or symptom are selected from: pain, familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung or pulmonary edema.

3. purposes as claimed in claim 1 is characterized in that, said compositions is used for treatment, alleviates or improve pain.

4. purposes as claimed in claim 3 is characterized in that, said pain is body source property pain.

5. purposes as claimed in claim 4 is characterized in that, said body source property pain is selected from: neuropathic pain or nociceptive pain.

6. purposes as claimed in claim 5 is characterized in that, said neurogenic is selected from bitterly: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.

7. purposes as claimed in claim 5; It is characterized in that said neuropathic pain is the pain that causes of damage or pathological change by maincenter or peripheral nervous system that is selected from down group: lumbago and backache, neuralgia, fibromyalgia, neuralgia that diabetes are relevant, neuralgia that multiple sclerosis is relevant, PHN and with the related neurogenic neuralgia of HIV.

8. purposes as claimed in claim 5 is characterized in that, said nociceptive pain is selected from: body nociceptive pain or internal organs nociceptive pain.

9. purposes as claimed in claim 8 is characterized in that, said nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.

10. purposes as claimed in claim 1 is characterized in that, said Progynon inhibin appearance albumen 1 is:

(a) protein of aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:2;

(c) replacement, disappearance or the interpolation of aminoacid sequence in (a) through 1-20 amino acid residue formed, and it is active by (a) deutero-protein to have a raising na-k-atp enzyme.

11. purposes as claimed in claim 10 is characterized in that, said (c): replacement, disappearance or the interpolation of aminoacid sequence in (a) through 1-10 amino acid residue formed, and it is active by (a) deutero-protein to have a raising na-k-atp enzyme.

12. purposes as claimed in claim 10 is characterized in that, said (c): replacement, disappearance or the interpolation of aminoacid sequence in (a) through 1-5 amino acid residue formed, and it is active by (a) deutero-protein to have a raising na-k-atp enzyme.

13. purposes as claimed in claim 10 is characterized in that, said (b) is selected from:

(1) has the polypeptide of 156-173 aminoacid sequence among the SEQ ID NO:1;

(2) has 158-175 amino acids polypeptide of sequence among the SEQ ID NO:2;

(3) by the sequential amino acid deletion Progynon inhibin domain of SEQ ID NO:1 or SEQ ID NO:2, i.e. FS domain, formed polypeptide;

(4) by the sequential amino acid deletion Von Willebrand type C-structure territory of SEQ ID NO:1 or SEQ ID NO:2, i.e. VWC domain, formed polypeptide;

14. a separated polypeptide, said polypeptide is selected from:

15. isolating polynucleotide is characterized in that, the described polypeptide of described polynucleotide encoding claim 14.

16. a carrier is characterized in that, it contains the described polynucleotide of claim 15.

17. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 16; Or be integrated with the described polynucleotide of claim 15 in its genome.

18. the method for preparing of the described polypeptide of claim 14 is characterized in that, this method comprises:

(a) be fit to cultivate the described host cell of claim 17 under the condition of expressing;

(b) from culture, isolate the described polypeptide of claim 14.

19. one kind through improving the na-k-atp enzyme activity is treated, alleviated or improvement is relevant with the na-k-atp enzyme activity the disease or the pharmaceutical composition of symptom, it contains:

(a) protein of the described polypeptide of the claim 14 of safe and effective amount, SEQ ID NO:1 or SEQ ID NO:2; And

(b) pharmaceutically acceptable carrier.

20. pharmaceutical composition as claimed in claim 19 is characterized in that, said disease or symptom are selected from: familial hemiplegia type migraine, diabetes, depression, digitalism, injury of lung, pulmonary edema or pain.

21. pharmaceutical composition as claimed in claim 20 is characterized in that, described pain is body source property pain.

22. pharmaceutical composition as claimed in claim 21 is characterized in that, said body source property pain is selected from: nociceptive pain or neuropathic pain.

23. pharmaceutical composition as claimed in claim 22 is characterized in that, described neurogenic is selected from bitterly: dorsal root ganglion neuropathic pain, trigeminal ganglion neuropathic pain or sciatica.

24. pharmaceutical composition as claimed in claim 22; It is characterized in that said neuropathic pain is the pain that causes of damage or pathological change by maincenter or peripheral nervous system that is selected from down group: lumbago and backache, neuralgia, fibromyalgia, neuralgia that diabetes are relevant, neuralgia that multiple sclerosis is relevant, PHN or with the related neurogenic neuralgia of HIV.

25. pharmaceutical composition as claimed in claim 22 is characterized in that, said nociceptive pain is selected from: body nociceptor property pain or internal organs nociceptor property pain.

26. pharmaceutical composition as claimed in claim 25 is characterized in that, said nociceptive pain is selected from: cancer pain, arthritis pain, postoperative pain or with the related nociceptive pain of HIV.

27. the protein shown in protein shown in the described polypeptide of claim 14, the SEQ ID NO:1 and/or the SEQ ID NO:2 is used for the purposes through the pharmaceutical composition that improves the na-k-atp enzyme activity is treated, alleviated or improvement is relevant with the na-k-atp enzyme activity disease or symptom in preparation.

28. the described polypeptide of claim 14 is used for treating the purposes of the pharmaceutical composition of rheumatoid arthritis and/or ischemic heart desease in preparation.

29. a screening is used for treating, alleviate or improve the method for the potential material of disease relevant with this activity or symptom through improving the na-k-atp enzyme activity, it is characterized in that described method comprises:

30. a method of screening treatment or lenitive potential material is characterized in that described method comprises:

(1) candidate substances is contacted with the system of na-k-atp enzyme;

(2) detect the influence of candidate substances to na-k-atp enzyme;

31. the inhibitor of Progynon inhibin appearance albumen 1 preparation through reduce the na-k-atp enzyme activity and treat, alleviate or the compositions of disease that improvement is relevant with the na-k-atp enzyme activity or symptom in purposes.

32. purposes as claimed in claim 31 is characterized in that, said disease or symptom are selected from: renal hypertension or tumor.

33. purposes as claimed in claim 32 is characterized in that, said tumor is selected from: melanoma, glioblastoma multiforme or nonsmall-cell lung cancer.

34. purposes as claimed in claim 31 is characterized in that, said inhibitor is selected from: the antibody of anti-Progynon inhibin appearance albumen 1 or its active fragment; Antisense oligonucleotide or siRNA to Progynon inhibin appearance albumen 1 coded sequence; Or derive from the peptide section of na-k-atp enzyme.

35. purposes as claimed in claim 31 is characterized in that, said inhibitor is selected from: