CN101935673B - siRNA for inhibiting mouse gamma -GABA transfer protein subtype 1 - Google Patents
siRNA for inhibiting mouse gamma -GABA transfer protein subtype 1 Download PDFInfo
- Publication number
- CN101935673B CN101935673B CN 200910054351 CN200910054351A CN101935673B CN 101935673 B CN101935673 B CN 101935673B CN 200910054351 CN200910054351 CN 200910054351 CN 200910054351 A CN200910054351 A CN 200910054351A CN 101935673 B CN101935673 B CN 101935673B
- Authority
- CN
- China
- Prior art keywords
- gat
- liver
- mouse
- albumen
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The invention provides application of a coding sequence of gamma-amino butyric acid transport protein subtype 1 (GAT-1) or gamma-amino butyric acid transport protein subtype 1 in preparing a medicament for promoting liver regeneration. The invention also relates to a method for preparing or screening a substance for promoting liver regeneration based on GAT-1 genes or GAT-1 protein.
Description
Technical field
The present invention relates to express the promotion liver regeneration by regulating GAT-1.
Background technology
Human and mammiferous liver has very strong regenerative power behind physics, chemistry, infection or ischemia injury.Under certain conditions (as surgery because of tumour or transplantation cut-out liver or lobe of the liver; Transplant little donor liver to a big acceptor; Cause liver cell destruction etc. because of chemicals, virus etc.), the volume of liver changes, and touches liver regeneration.Since 20th century, soaring one of disease of serious threat human health that becomes gradually of the sickness rate of hepatic diseases.The various liver problem sufferer of China has nearly 400,000 people to die from hepatic diseases just in rising trend every year.At present, the hepatotomy operation is widely used in treating multiple hepatic diseases by many hospitals.Behind the part hepatotomy, liver is faced with the compensatory problem of residue liver function.If the residue liver function then can cause liver regeneration and rehabilitation gradually can be compensatory.Otherwise, then can move towards acute hepatic failure, cause death.Studies show that people to liver regeneration induces the survival rate that may improve the acute hepatic failure patient that hepatectomy causes and shortens its rehabilitation duration.How to improve effectively and to promote that liver regeneration, liver function recovery become present problem demanding prompt solution.
The most frequently used animal model of research liver regeneration is partially hepatectomized (the about 70% hepatotomy) model of mouse or rat.Within the short several minutes behind the hepatotomy, hepatocellular activation namely is touched.In this course, the various kinds of cell factor, somatomedin, neuroendocrine regulatory factor etc. all participate in regulating kinase whose activation, the activation of transcription factor and the recovery of hepatocellular propagation and liver function.The index of estimating liver regeneration has: the variation of liver weight, liver cell mitotic index etc.
γ-An Jidingsuan (GABA) is as important inhibitory nerve mediator, keeps the normal function of central nervous system with the mutual balance of excitatory neurotransmitter glutamate.Be released into the GABA part of synaptic cleft and the GABA receptors bind that is positioned at postsynaptic membrane by neurone, most of GABA then is transported in the cell by the gaba transporter of presynaptic membrane.GAT-1 is as one of main transporter of inhibitory nerve mediator, found by scientists the earliest, think that at present it mainly is expressed in olfactory bulb, neural cortex, cerebellum, superior colliculus, matrix nuclear, and mainly be distributed in presynaptic membrane end, neurone and spongiocyte, the GABA of low concentration plays an important role in the body for keeping.There are some researches prove that at present GABA can the negative regulation liver regeneration, but whether participating in this process for GAT-1 it be unclear that.
Summary of the invention
The present invention utilizes the animal model of liver regeneration, has proved obviously rise in 24 hours after being expressed in liver and cutting of GAT-1.Utilize GAT-1 genetic flaw mouse, find that first GAT-1 disappearance back Mouse Liver regenerative power obviously descends, and has confirmed that GAT-1 plays a significant role in the liver regeneration pathophysiological mechanism.Studies show that further utilize the RNAi technology to disturb after GAT-1 expresses in the mouse liver, the Mouse Liver regenerative power obviously weakens.
Therefore, the invention provides γ-An Jidingsuan translocator hypotype 1 or its encoding sequence purposes in the material that preparation promotion liver regeneration is used.
In a preferred embodiment, described material is the expression vector that contains described encoding sequence.
In another preferred embodiment, described material is the agonist of γ-An Jidingsuan translocator hypotype 1.
In other preferred embodiment, the medicament of described material for being used for promoting that liver regeneration is used.In a preferred embodiment, described medicament contains the expression plasmid that supplies γ-An Jidingsuan translocator hypotype 1, its encoding sequence or contain described encoding sequence of the present invention.In other preferred embodiment, described medicament contains the agonist of γ-An Jidingsuan translocator hypotype 1.
The invention provides the purposes of agonist in the medicine that preparation promotion liver regeneration is used of γ-An Jidingsuan translocator hypotype 1.
In a preferred embodiment, the agonist of described γ-An Jidingsuan translocator hypotype 1 is selected from (but being not limited to): oestrogenic hormon, proteolytic enzyme C agonist, inhibitor of phospholipase enzymes.
In other preferred embodiment, described medicament contains the agonist of described γ-An Jidingsuan translocator hypotype 1 of the amount of effective promotion liver regeneration.In other embodiments, described medicament also contains pharmaceutically acceptable carrier and/or vehicle.
The invention provides the purposes of expression vector in the medicine that preparation promotion liver regeneration is used of the encoding sequence that contains γ-An Jidingsuan translocator hypotype 1.
The invention provides the method that a kind of screening is used for the potential material of promotion liver regeneration, described method comprises:
(1) candidate substances is contacted with the system of expressing γ-An Jidingsuan translocator hypotype 1;
(2) detect candidate substances to the influence of γ-An Jidingsuan translocator hypotype 1;
If described candidate substances can improve expression, activity or the secretion of γ-An Jidingsuan translocator hypotype 1, show that then this candidate substances is the potential material that can be used for promoting liver regeneration.
In a preferred embodiment, described step (1) comprising: in test group, candidate substances is joined in the system of constructive expression or inducible expression's γ-An Jidingsuan translocator hypotype 1; And/or
Step (2) comprising: detect expression or the activity of γ-An Jidingsuan translocator hypotype 1 in the system of test group, and with control group relatively, wherein said control group is the system of not adding the expression γ-An Jidingsuan translocator hypotype 1 of described candidate substances;
If the expression of γ-An Jidingsuan translocator hypotype 1 in the test group, activity or secretion are higher than control group statistically, just show that this candidate substances is the potential material that can be used for promoting liver regeneration.
In a preferred embodiment, described system is selected from: cell system, ubcellular system, solution system, organizational framework, organ systems or animal system.
In a preferred embodiment, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, further to select from candidate substances and to determine for promoting the useful composition of liver regeneration.
Description of drawings
Different time points after Fig. 1 is presented at liver and cuts, the expression of GAT-1 in liver.C57BL/6 mouse (each time point 3-4 only) carried out liver resection (about 70% hepatotomy) by giving mouse at the 0th day, touch the residue liver regeneration.Mouse is put to death in after liver cuts the 0th hour, 12 hours, 24 hours and 48 hours, and the liver that takes out mouse extracts RNA.Detect the expression level of GAT-1 by the method for real-time quantitative PCR.Shown in data represent 3 times the test the result.
After Fig. 2 shows that liver cuts, GAT-1-/-obviously decline of mouse liver regeneration.Control group (wild-type) and experimental group (GAT-1-/-), every group of 9-10 mouse carried out liver resection (about 70% hepatotomy) by giving mouse at the 0th day, to touch the residue liver regeneration.After liver cuts the 24th hour, 48 hours and 72 hours, weighing mouse body weight is put to death mouse respectively, and the liver that takes out mouse also claims its weight.Calculate liver and the body weight ratio of every mouse according to body weight * 100 of mouse liver weight/mouse.Shown in data represent 3 times the test the result.
Fig. 3 shows that mouse liver regeneration obviously descends by after the RNAi technology interference GAT-1 expression.Control group and experimental group (GAT-1 siRNA), every group of 3-4 mouse at the 0th day, given 0.9% physiological saline or the GAT-1 siRNA of mouse tail vein injection 2ml.After 48 hours, implement liver resection (about 70% hepatotomy) to mouse, touch Mouse Liver regeneration.Liver cut back 48 hours, put to death mouse, calculated liver and the body weight ratio of every mouse according to the described method of Fig. 2.
Fig. 4 shows and to utilize Protocols in Molecular Biology to raise after GAT-1 expresses in the liver that mouse liver regeneration obviously increases.Control group and experimental group (GAT-1 plasmid), every group of 3-4 mouse at the 0th day, given the empty plasmid of mouse tail vein injection 2ml or the expression plasmid of GAT-1.After 48 hours, implement liver resection (about 70% hepatotomy) to mouse, touch Mouse Liver regeneration.Liver cut back 48 hours, put to death mouse, calculated liver and the body weight ratio of every mouse according to the described method of Fig. 2.
Fig. 5 shows the GAT-1 plasmid map.
Embodiment
γ-An Jidingsuan (GABA) is as important inhibitory nerve mediator, keeps the normal function of central nervous system with the mutual balance of excitatory neurotransmitter glutamate.Be released into the GABA part of synaptic cleft and the GABA receptors bind that is positioned at postsynaptic membrane by neurone, most of GABA then is transported in the cell by the gaba transporter of presynaptic membrane.γ-An Jidingsuan translocator hypotype 1 (GAT-1) is as one of main transporter of inhibitory nerve mediator, found by scientists the earliest, think that at present it mainly is expressed in olfactory bulb, neural cortex, cerebellum, superior colliculus, matrix nuclear, and mainly be distributed in presynaptic membrane end, neurone and spongiocyte, the GABA of low concentration plays an important role in the body for keeping.
The present invention utilizes GAT-1 genetically deficient animal, finds that first GAT-1 disappearance back Mouse Liver regenerative power obviously descends, and has confirmed that GAT-1 plays a significant role in the liver regeneration pathophysiological mechanism.Studies show that further utilize the RNAi technology to disturb after GAT-1 expresses in the mouse liver, the Mouse Liver regenerative power obviously weakens.
Therefore, one aspect of the present invention relates to the method that promotes the object liver regeneration, and this method comprises this object of promotion GAT-1 expression of gene.
The GenBank accession number of the nucleotide sequence of GAT-1 gene is NM_178703, and the GenBank accession number of aminoacid sequence is NP_848818.
Described object comprises various Mammalss, for example people, various performing animal, pet etc.
Promote the method for GAT-1 genetic expression to comprise and adopt biotechnology to go into to contain this GAT-1 expression carrier to the object transit cell, this expression vector can be expressed GAT-1 albumen in described object cell.Perhaps, promote GAT-1 expression of gene in the cell by the agonist that gives GAT-1 genetic expression.
The present invention relates to the method that promotes the object liver regeneration on the other hand, and this method comprises the agonist that GAT-1 albumen or GAT-1 are provided to this object.
Used GAT-1 albumen can be naturally occurring, such as its can be separated or purifying from Mammals.In addition, described GAT-1 albumen also can be artificial preparation, such as producing reorganization GAT-1 albumen according to the genetically engineered recombinant technology of routine.Preferably, the present invention can adopt the GAT-1 albumen of reorganization.
Any suitable GAT-1 albumen all can be used for the present invention.Described GAT-1 albumen comprises GAT-1 albumen or its bioactive fragment of total length.Preferably, the aminoacid sequence of described GAT-1 albumen can with the GenBank accession number: the sequence shown in the NP_848818 is substantially the same.
The aminoacid sequence of the GAT-1 albumen that passes through replacement, disappearance or the interpolation of one or more (for example several) amino-acid residue and form is also included among the present invention.GAT-1 albumen or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and described sequence of replacing through amino acid does not influence its activity or kept the activity of its part.Suitably replacing amino acid is technology well known in the art, and described technology can be implemented and guarantee not change the biological activity of gained molecule at an easy rate.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, the 4th edition, 1987, TheBenjamin/Cummings Pub.Co.P224.
Particularly, amino acid is generally divided into four classes: (1) acidity---aspartic acid and L-glutamic acid; (2) alkalescence---Methionin, arginine, Histidine; (3) nonpolar---L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane; (4) uncharged polarity---glycine, l-asparagine, glutamine, halfcystine, Serine, Threonine, tyrosine.Sometimes phenylalanine, tryptophane and tyrosine are classified as aromatic amino acid.For example, have reason to predict: replace leucine, replace aspartic acid, replace Threonine with Serine with L-glutamic acid with Isoleucine or Xie Ansuan separately, perhaps with the similar conservative amino acid of aminoacid replacement relevant on the structure, such substituting will can not have material impact to biological activity.For example, GAT-1 albumen of the present invention can comprise up to about 5-10 conservative or not conservative aminoacid replacement, even up to about 15-25 conservative or not conservative aminoacid replacement, or any integer between the 2-25, as long as the required function of this molecule is still kept complete.Those skilled in the art can easily measure the zone that can tolerate change in the molecule (s) of interest in conjunction with Hopp/Woods well known in the art and Kyte-Doolittle graphic representation.
The bioactive fragment of any GAT-1 albumen can be applied among the present invention.Here, the implication of the bioactive fragment of GAT-1 albumen refers to that as a peptide species it still can keep all or part of function of the GAT-1 albumen of total length.Generally, described bioactive fragment keeps the activity of 50% total length GAT-1 albumen at least.Under preferred condition, described active fragments can keep 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length GAT-1 albumen.
The present invention also can adopt GAT-1 albumen modified or improvement, such as, can adopt the GAT-1 albumen of being modified or improveing for the effectiveness that promotes its transformation period, validity, metabolism and/or albumen.Described can be a kind of conjugate of GAT-1 albumen through the GAT-1 albumen of modifying or improve, or it can comprise substituted or artificial amino acid.Described can be to have less common ground with naturally occurring GAT-1 albumen through the GAT-1 albumen of modifying or improve, but also can promote liver regeneration, and can not bring other detrimentally affect or toxicity.That is to say that any bioactive version that does not influence GAT-1 albumen all can be used among the present invention.
Aminoacid sequence according to GAT-1 albumen can draw its corresponding nucleotide coding sequence easily.Preferably, the nucleotide sequence of described GAT-1 albumen can with the GenBank accession number: the sequence shown in the NM_178703 is substantially the same.
Therefore, protein of the present invention comprises: (1) protein shown in GenBank accession number NP_848818; (2) in the aminoacid sequence shown in the GenBank accession number NP_848818, through the protein of being derived by GenBank accession number NP_848818 that replaces, lacks or add one or several amino acid and have the activity of albumen shown in the GenBank accession number NP_848818.
Can adopt prepared in various methods protein of the present invention.Usually with recombinant methods protein of the present invention.The polynucleotide that can prepare code book invention protein with the standard molecular biology method.For example, with recombination method can obtain the to encode polynucleotide sequence of above-mentioned molecule, for example by from expressing cell screening cDNA and the genomic library of this gene, or by this gene of deriving from the known carrier that comprises this gene.Also can synthesize but not clone and prepare interested gene.The codon of available suitable particular sequence digests this molecule.To assemble complete sequence with the overlapping oligonucleotide of standard method preparation then, and be fitted in the complete encoding sequence.Referring to as Edge (1981) Nature 292:756; Nambair etc. (1984) Science223:1299; With (1984) J.Biol.Chem.259:6311 such as Jay.
Therefore, can obtain concrete nucleotide sequence from the carrier that carries required sequence, or with various oligonucleotide synthesis methods known in the art, as site-directed mutagenesis and polymerase chain reaction (PCR), synthetic wholly or in part.Referring to as Sambrook, " molecular cloning: laboratory manual " (Molecular Cloning:aLaboratory Manual), second edition, 1989.Specifically, the method that obtains the nucleotide sequence of the required sequence of coding is the overlapping synthetic oligonucleotide with the annealing supplementary set of conventional automatic polynucleotide synthesizer preparation, connect with suitable dna ligase then, and the nucleotide sequence that connects with the PVR amplification.Referring to as (1991) Proc.Natl.Acad.Sci.USA 88:4084-4088 such as Jayaraman.In addition, also can use the synthetic (Jones etc. of oligonucleotide orientation in the present invention, the enzymatic that the oligonucleotide directed mutagenesis (Riechmann etc., (1988) Science 239:1534-1536 such as (1988) Nature 332:323-327 and Verhoeyen) in Nucleotide zone is arranged (1986) Nature 54:75-82), earlier and carry out with the T4DNA polysaccharase is mended the oligonucleotide synthetic (Queen etc. (1989) Proc.Natl.Acad.Sci.USA86:10029-10033) of flat rubber belting breach.
In case the preparation or separated encoding sequence, just these sequence clones can be gone in any suitable carriers or the replicon.To those skilled in the art, various cloning vectors are known, and the screening of suitable cloning vector is the selection problem.Suitable carriers comprises (but being not to be limited to): plasmid, phage, transposon, clay, karyomit(e) or when reproducible virus when suitable controlling elements is combined.
Then cloned sequence is placed under the control of suitable controlling elements, this depends on for the system of expressing.Therefore, encoding sequence can be placed promotor, ribosome bind site (being used for bacterial expression) and randomly under the control of operon, thereby by suitable transformant interested dna sequence dna be transcribed among the RNA.This encoding sequence can comprise or not comprise signal peptide or leader sequence (can be removed at the translation post-treatment by the host subsequently).Referring to as U.S. Patent No. 4,431,739; 4,425,437; 4,338,397.
Except control sequence, can add the adjusting sequence, thus can be with respect to the expression of the growth regulating sequence of host cell.It is well known by persons skilled in the art regulating sequence, and the example comprises that those can cause replying the adjusting sequence that chemistry or physical stimulation (comprising the existence of regulating compound) start or close genetic expression.The regulatory element that also can have other type in the carrier.For example, can use enhancer element to increase the expression level of construction.Example comprises SV40 early gene enhanser (Dijkema etc. (1985) EMBOJ.4:761); The enhancers/promoters (Gorman etc. (1982) Proc.Natl.Acad.Sci.USA 79:6777) of deriving from the sarcoma viral long terminal repeat of Rous (LTR); With the element of deriving from people CMV (Boshart etc., (1985) Cell 41:521), as the element (U.S. Patent No. 5,688,688) that comprises in the CMV intron A sequence.Also can be included in potential energy and the strong promoter of replication orgin, one or more selectable markers of self-replicating in the proper host cell, one or more restriction site, high copy number amount in the expression cassette.
Construction of expression vector, make concrete encoding sequence be arranged in this and have the carrier that is fit to regulate sequence, the position of the encoding sequence relevant with control sequence and orientation make encoding sequence transcribe (that is this encoding sequence of rna polymerase transcribe that, is incorporated into dna molecular in the control sequence) under " control " of control sequence.May need the sequence of coding molecules of interest is modified to realize this purpose.For example, may need in some cases to modify this sequence, thereby make it be connected in the control sequence that is fit to orientation, namely keep frame.Before being inserted into carrier, control sequence and other are regulated sequence may be connected in encoding sequence.Perhaps, encoding sequence directly can be cloned in the expression vector that has comprised control sequence and suitable restriction site.
By the one or more Nucleotide in sequence, insertion sequence and/or alternative this sequence of lacking the interested polypeptide of part coding, can be for the preparation of mutant or the analogue of the albumen of analyzing of the present invention.The method of modified nucleotide sequence is well-known to those skilled in the art as directed mutagenesis etc.Referring to as Sambrook etc., above-mentioned; Kunkel, T.A. (1985) Proc.Natl.Acad.Sci.USA (1985) 82:448; Geisselsoder etc. (1987) BioiTechniques 5:786; Zoller and Smith (1983) Methods Enzymol.100:468; Dalbie-McFarland etc. (1982) Proc.Natl.Acad.SciUSA 79:6409.
This molecule can be expressed in various systems, comprises insect well known in the art, Mammals, bacterium, virus and yeast expression system.
For example, insect cell expression system such as rhabdovirus system are well known by persons skilled in the art, are described in as Summers and Smith Texas Aguricultural Experiment Station BulletinNo.1555 (1987).The material and the method that are used for baculovirus/insect cell expression system all can kit form be buied, Invitrogen especially, San Diego CA (" MaxBac " test kit).Similarly, bacterium and mammalian cell expression system also are well known in the art, and its description is seen as Sambrook etc., as above.Yeast expression system also is well known in the art, and the Butterworths as " yeast genetic engineering " (YeastGenetic Engineering) (editor such as Barr, 1989), London are seen in its description.
Many host cells that are fit to for said system also are known.For example, mammal cell line is known in the art, it comprises the clone of the immortalization that can obtain from American type culture collection (ATCC), as (but being not to be limited to) Chinese hamster ovary cell (CHO), Hela cell, young hamster kidney (BHK) cell, monkey-kidney cells (COS), HEKC, human liver cell cancer cells (as Hep G2), Madin-Darby ox kidney (" MDBK ") cell etc.Similarly, in expression constructs of the present invention, also can use host bacterium, as intestinal bacteria, Bacillus subtilus and suis.Available yeast host also in the present invention is particularly including yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), maltose candiyeast (Candidamaltosa), Candida albicans (Candida albicans), multiple-shaped nuohan inferior yeast (Hansenulapolymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), season is Meng Shi pichia spp (Pichia guillerimondii) also, pichia pastoris phaff (Pichia pastoris), grain wine fragmentation sugar yeast (Schizosaccharomycespombe) and yarrowia lipolytica.The insect cell that can use with baculovirus expression system is particularly including Aedes aegypti (Aedes aegypti), Autographa californica multicapsid nucleopolyhedrosisvirus (Autographacalifornica), silkworm (Bombyx mori), black-tailed fruit flies (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and cabbage looper (Trichoplusia ni).
Send the method for passing with range gene well known in the art, the stable additive type element that the nucleic acid molecule that comprises interested nucleotide sequence stably can be integrated in the host cell gene group or in proper host cell is kept.Referring to as U.S. Patent No. 5,399,346.
According to selected expression system and host, under the condition of marking protein, cultivation prepares this molecule with aforesaid expression vector transformed host cells.Isolate expressed protein and purifying from host cell then.If expression system with protein secreting in substratum, then directly from the substratum purified product.If not what secrete, then separate from cell pyrolysis liquid.The culture condition that is fit to and the selection of recovery method all are within those skilled in the art's ability.
Therefore, the present invention also comprises the nucleotide sequence of code book invention GAT-1 protein and contains the expression vector of this nucleotide sequence, and uses this class nucleotide sequence or its expression vector to promote liver regeneration.
The purposes of expression vector in the material that preparation promotion liver regeneration is used that the invention still further relates to described GAT-1 albumen, its encoding sequence and contain described encoding sequence.Described preparation also comprises based on described GAT-1 albumen, its encoding sequence and contains the expression vector of described encoding sequence and screen the material that can promote liver regeneration.
Therefore, more on the one hand, the invention provides the method that a kind of screening is used for the potential material of promotion liver regeneration, this method comprises:
(1) candidate substances is contacted with the system of expressing γ-An Jidingsuan translocator hypotype 1;
(2) detect candidate substances to the influence of γ-An Jidingsuan translocator hypotype 1;
If described candidate substances can improve expression, activity or the secretion of γ-An Jidingsuan translocator hypotype 1, show that then this candidate substances is the potential material that can be used for promoting liver regeneration.
In optimal way of the present invention, when screening, in order to be easier to observe expression or the active change of GAT-1, also control group can be set, described control group can be the system of not adding the expression GAT-1 of described candidate substances.
The system of described expression GAT-1 for example can be cell (or cell culture) system, and described cell can be the cell of endogenous expression GAT-1; It maybe can be the cell of recombinant expressed GAT-1.The system of described expression GAT-1 can also be (but being not limited to) ubcellular system, solution system, organizational framework, organ systems or animal system (as animal model) etc.
As a kind of optimal way of the present invention, described system is the cell system of expressing GAT-1.Preferably, the immunocyte of expression GAT-1 can derive from following 3 kinds of preparation methods:
1. derive from the mononuclearcell of normal mammalian
Get normal mammalian mononuclearcell (lymphoglandula, peripheral blood, spleen) process, for example after the plain stimulation of (but being not limited to) anti-CD3 and anti-CD28 or phorbol ester and ion enzyme, this cell can be expressed GAT-1 albumen.With the cell model of this kind cell as the medicine that is used for screening promotion liver regeneration.
2. the T lymphocyte series is selected from (but being not limited to): OVA-specificity T cell line/lineage, MOG-specificity T cell line/lineage, MBP-specificity T cell line/lineage, Jurkat clone, YAC-1 clone, human T lymphocyte system (H9), Hut-102 etc.
After getting the plain stimulation of T lymphocyte series process antigen or anti-CD3 and anti-CD28 or phorbol ester and ion enzyme, this cell can be expressed GAT-1 albumen.With the cell model of this kind cell as the medicine that is used for screening promotion liver regeneration.
3. derive from behind the antigen immune or the mammiferous mononuclearcell of morbid state
Get behind the antigen immune or the mammiferous mononuclearcell (peripheral blood, lymphoglandula, spleen, lesions position etc.) of morbid state after stimulation oversaturation (antigen, anti-CD3 and anti-CD28, phorbol ester and ion enzyme element) activation, this cell can be expressed GAT-1 albumen.With the cell model of this kind cell as the medicine that is used for the promotion liver regeneration.
As another kind of optimal way of the present invention, described system is the animal model system.Preferably, can adopt and have pathogenic antigen or pathogenic immunocyte prepares pathogenic animal model.Described animal includes but not limited to mouse, rat, cavy, rabbit etc.Induction method can be as follows: initiatively induce pathogenic through antigen immune; Induce pathogenic through passive transfer pathogenic (but being not limited to) T cell or B cell or immune factor (as antibody etc.).Described animal model also can be the animal model of idiopathic morbidity.
As optimal way of the present invention, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, with further selection and definite for promoting the real useful material of liver regeneration.
The present invention has no particular limits for the detection method of expression, activity, amount or the secretion situation of GAT-1 albumen.Can adopt conventional protein quantification or half-quantitative detection technology, for example (but being not limited to): SDS-PAGE method, Western-Blot method, ELISA etc.
Therefore, the present invention also provides the potential material that can be used for promoting liver regeneration that adopts described screening method to obtain.The material that these preliminary screening go out can constitute a screening storehouse so that people finally can therefrom filter out can be for promoting the real useful material of liver regeneration, thereby be used for clinical.
Therefore, further aspect of the present invention also comprises the agonist of GAT-1 and promotes purposes aspect the medicine that liver regeneration uses in preparation.
As used herein, the agonist of described GAT-1 comprises promotor, goes up adjustment etc.The material of transcribing and translating of the activity of any GAT-1 of raising albumen, the stability of keeping GAT-1 albumen, the expression that promotes GAT-1 albumen, the secretion that promotes GAT-1 albumen, prolongation GAT-1 albumen effective acting time or promotion GAT-1 all can be used for the present invention, as the active substance that can be used for promoting liver regeneration.
As optimal way of the present invention, the agonist of described GAT-1 includes, but is not limited to: oestrogenic hormon, proteolytic enzyme C agonist (phorbol ester, (-)-Indolactam V etc.), inhibitor of phospholipase enzymes (cyclocyto enzyme A, Okadaic Acid etc.).
As optimal way of the present invention, the agonist of described GAT-1 albumen includes, but is not limited to: expression vector or the expression constructs that can express (the preferred mistake expressed) GAT-1 after changing cell over to.Usually, described expression vector comprises a box gene, and the gene that described box gene contains the GAT-1 that encodes reaches the continuous expression regulation sequence of operability with it.Described " operability links to each other " or " operationally being connected in " refer to a kind of like this situation, and namely the activity of same linear DNA sequence other parts can be regulated or control to some part of linear DNA sequence.For example, if the transcribing of promotor control sequence, it is exactly operationally to be connected in encoding sequence so.
Among the present invention, the GAT-1 polynucleotide sequence can be inserted in the recombinant expression vector.As long as can copy in host and stablize, any plasmid and carrier may be used to the present invention.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
As mentioned before, method well-known to those having ordinary skill in the art can be used for make up the dna sequence dna that contains GAT-1 and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Conversion carrier also comprises ribosome bind site and the transcription terminator that translation initiation is used.
The present invention also provides a kind of composition, and it contains significant quantity (as 0.000001-50wt%; Preferable 0.00001-20wt%; Better, 0.0001-10wt%) described GAT-1 albumen or its agonist, and pharmaceutically acceptable carrier.
Composition of the present invention can be directly used in the promotion liver regeneration.In addition, also can unite use with other therapeutical agent or assistant agent simultaneously.
Usually, these materials can be formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, preferably, pH is about 6-8.
As used herein, term " contains " the various compositions of expression and can be applied to together in mixture of the present invention or the composition.Therefore, term " mainly by ... form " and " by ... composition " be included in during term " contains ".As used herein, term " significant quantity " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and does not have excessive bad side reaction (as toxicity, stimulation and transformation reactions), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.
Composition of the present invention contains GAT-1 albumen and the pharmaceutically acceptable carrier of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, 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 physiological saline or contain glucose and the aqueous solution of other assistant agents is prepared by ordinary method.Described pharmaceutical composition should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity.Pharmaceutical preparation of the present invention also can be made into sustained release preparation.
The significant quantity of GAT-1 albumen of the present invention or its agonist can change with severity of the pattern of administration and pending liver injury etc.The selection of preferred significant quantity can be determined (for example by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio of described GAT-1 albumen or its agonist, metabolism, transformation period etc.; The severity of the disease that the patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Usually, the dosage when GAT-1 albumen of the present invention or its agonist every day with about 0.00001mg-50mg/kg the weight of animals (preferable 0.0001mg-10mg/kg the weight of animals) gives, and can obtain gratifying effect.For example, by an urgent demand for the treatment of situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
As optimal way of the present invention, described agonist is oestrogenic hormon, when every day during with the administration of 0.1-800 μ g/kg the weight of animals, has effect preferably; Preferably every day is with the administration of 0.5-300 μ g/kg the weight of animals; More preferably every day is with the administration of 1-200 μ g/kg the weight of animals.
The present invention also provides a kind of method that promotes liver regeneration, comprises the GAT-1 albumen or its agonist that give experimenter's significant quantity.
The administering mode of GAT-1 albumen of the present invention or its agonist has no particular limits, can be whole body or local.For example, GAT-1 albumen of the present invention or its agonist can give animal, comparatively preferably intravenous injection by the mode of abdominal injection, intravenous injection, oral, subcutaneous injection, intradermal injection etc.
After the purposes of the described GAT-1 albumen of cicada, can adopt several different methods well known in the art that described GAT-1 albumen or its encoding gene or its pharmaceutical composition are delivered medicine to Mammals.Preferably, can adopt the means of gene therapy to carry out, such as can be directly with GAT-1 albumen by delivering medicine to the experimenter such as methods such as injections; Perhaps, can will carry GAT-1 expression of gene unit (such as expression vector or virus etc.) by certain approach and be delivered on the target spot, and make it the GAT-1 albumen of expression activity.
As one embodiment of the present invention, described GAT-1 albumen directly can be delivered medicine to Mammals (such as the people), perhaps, the gene of coding GAT-1 albumen can be cloned in the appropriate carriers (as conventional protokaryon or carrier for expression of eukaryon or virus vector such as herpesvirus vector or adenovirus carrier) by the method for routine, described carrier is imported in the cell that can express described GAT-1 albumen, make described cell expressing GAT-1 albumen.Can realize the expression of GAT-1 albumen by an amount of described cell being incorporated into the suitable position of body of mammals.
The administering mode of the agonist of GAT-1 albumen depends primarily on type and the characteristic of described agonist, and this is that those skilled in the art can assess.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the 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:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Experiment material therefor and reagent among the embodiment:
1. animal: C57BL/6 mouse, GAT-1-/-construction process of mouse is with reference to Cai YQ etc., Journalof Neuroscience Research, 2006,84:255-67.
2. reagent: GAT-1 and β-actin primer synthesizes the Bioisystech Co., Ltd in Shanghai Ying Jun; RNA extraction agent box, RNA reverse test kit and purchase Invitrogen; SYBR Green PCR master mix is available from Applied Biosystems; Synthesize in Shanghai JiMa pharmacy Technology Co., Ltd from GAT-1 siRNA.
3. testing method: real-time quantitative PCR
1) extraction of RNA and reverse transcription
1. the extraction of RNA
Use the Qiagen Rneasy Mini Kit of company, operation to specifications is summarized as follows:
The EP that 350 μ l cell pyrolysis liquids are housed from-80 ℃ of taking-ups manages, and waits for its thawing
↓
Add equal-volume 350 μ l 70% ethanol, put upside down mixing for several times
↓
All be transferred to Rneasy mini colume, be placed on the 2ml collection tube
↓
＞=8000g (＞=10000 rev/mins), centrifugal about 15 seconds of normal temperature removes to collect liquid in pipe (if sample surpasses 700 μ l, repeating once again)
↓
Add 350 μ l RW1 solution,＞=8000g (＞=10000 rev/mins) often mixed centrifugal about 15 seconds, removed to collect liquid in pipe, replaced new 2ml collection tube
↓
Add 80 μ l DNA enzymes (as far as possible directly being added on the film), room temperature effect 15 minutes
↓
Add 350 μ l RW1 solution,＞=8000g (＞=10000 rev/mins), centrifugal about 15 seconds of normal temperature removes to collect liquid in pipe, replaces new 2ml collection tube
↓
Add 500 μ l RPE solution (working concentration),＞=8000g (＞=10000 rev/mins), centrifugal about 15 seconds of normal temperature removes to collect liquid in pipe
↓
Add 500 μ l RPE solution (working concentration),＞=8000g (＞=10000 rev/mins), centrifugal about 2 minutes of normal temperature removes to collect liquid in pipe
↓
Rneasy mini colume is placed on the new 1.5ml collection tube
↓
Add 30-50 μ l RNAse-free water,＞=8000g (＞=10000 rev/mins), centrifugal about 1 minute of normal temperature is collected liquid in pipe, surveys the OD value
↓
If RNA＞30 μ g repeats to add 30-50 μ l RNAse-free water,＞=8000g (＞=10000 rev/mins), centrifugal about 1 minute of normal temperature is collected liquid in pipe, surveys the OD value
↓
The uv-spectrophotometric instrument detects RNA concentration
↓
Packing and mark are in-80 ℃ of preservations
2. reverse transcription
Use the Sensiscript RT Kit of Qiagen company, operation to specifications is summarized as follows: (primer of synthetic cDNA is the random hexamer of Nucleotide)
| Component | Volume/reaction (μ l) |
| Master mix | |
| 10* damping fluid RT | 2.0 |
| DNTP mixture (5.0mM) | 2.0 |
| Random primer (0.15 μ g/ml) | 1.0 |
| Rnase inhibitor 10U/ μ l | 1.0 |
| The Sensiscript ThermoScript II | 1.0 |
| No RNase water | In right amount |
| Template ribonucleic acid (＜50 μ g) | In right amount |
| Total amount | 20μl |
Operation is centrifugal behind the mixing on ice, carries out following reaction:
A) 37 ℃ 60 minutes or 2 hours
B) 93 ℃ 5 minutes
C)4℃
Take out reaction tubes, centrifugal, preserve cDNA for-20 ℃.
3. fluorescence real-time quantitative PCR
A) design of primers
The expression of GAT-1 mRNA detects by fluorescence real-time quantitative PCR (Real-time PCR).In this process, we select β-Actin to come total quantitative difference of RNA in each sample of balance as internal control gene, do not add the negative contrast of template simultaneously.The design of primers of gene is used Prime Express 2.0 softwares of AppliedBiosystems, and sequence is as shown in the table:
| Gene | Primer sequence |
| β-actin | Forward, 5 ' TGTCCACCTTCCAGCAGATGT 3 ' (SEQ ID NO:1) is reverse, 5 ' AGCTCAGTAACAGTCCGCCTAGA 3 ' (SEQ ID NO:2) |
| GAT-1 | Forward, 5 '-CAAGCCCAAAACCCTGGTAGT-3 (SEQ ID NO:3) is reverse, 5 '-CCACGCAGGACATGAGGAA-3 ' (SEQ ID NO:4) |
[0141]B) experimental procedure
Each reacts with 4.85 μ l cDNA (cDNA sample and water are in the uniform mixture of 1: 4 ratio), add 5 μ l SYBR Green PCR Master Mix, add 0.15 μ l primer (mixture of upstream and downstream primer, concentration are 5 μ M) again, namely reacting final volume is 10 μ l.Sample is added behind the 384 hole PCR Sptting plates centrifugal, sticks sealing membrane then onboard, and as far as possible will around seal tightly, again plate is put into the PCR instrument, configure reaction process, beginning PCR reaction.Being collected among the ABI 7900 SequenceDetection System of data carried out.Reaction process is as follows:
4. experimental procedure:
1) GAT-1-/-mouse partially hepatectomized experiment
After mouse is anaesthetized through 2% vetanarcol, be put on the operation plate, behind the unhairing, cut skin, cut off peritonaeum, fully expose the abdominal cavity, isolate the right upper lobe of liver, upper left leaf and lower-left leaf (account for altogether mouse liver volume 70%) gently with the cotton swab of adhesional wetting, connect bundle with No. 5 lines respectively after, cut away the liver of ligation, peritoneal suture and skin, and give mouse back injection 4ml 0.9% physiological saline.After 1-2 hour, mouse revives.After liver cuts 24 hours, 48 hours and 72 hours take by weighing the weight of mouse body weight and residue liver, calculate the weight/mouse body weight of residue liver.
2) siRNA disturbs the expression of GAT-1 in the liver
The sense strand of employed siRNA is CAGUGUAUUUCAUGUUUGUTT (SEQ ID NO:5); Antisense strand is: ACAAACAUGAAAUACACUGTT (SEQ ID NO:6).
The siRNA of 5OD is dissolved in 0.9% physiological saline of 2ml, squeezes into fast in mouse (body weight the is the 20-22 gram) body by the i.v injection in second at 4-10.After 48 hours, give mouse and implement the part liver and cut operation. after mouse revived 48 hours, take by weighing the weight of mouse body weight and residue liver, calculate the weight/mouse body weight of residue liver, to estimate the influence of disturbing after the expression of GAT-1 in liver liver regeneration.
3) plasmid transfection raises liver regeneration situation after the expression of GAT-1 in the liver
The plasmid of following construction expression GAT-1.(forward primer: AGCTGAGAGGTTGCAGGCGA (SEQ ID NO:7) and reverse primer CCAAGGCGGGCAGAAAAGT (SEQ ID NO:8)) does pcr amplification to the cDNA of spinal cord with outer primer, be that template uses inner primer (forward primer: GGCGaagcttATGGCGACTGACAACAGCAAGG (SEQ ID NO:9) and reverse primer GCGCgaattcCTAGATGTAGGCCTCCTTGCTG (SEQ ID NO:10), lowercase is represented HindIII restriction enzyme site and ECORI restriction enzyme site respectively) to be PCR again with the PCR product.PCR product post precipitation is used HindIII and ECORI double digestion PCR product and plasmid.After enzyme is cut, carry out electrophoresis.Reclaim the PCR enzyme and cut product and plasmid enzyme restriction product.Cut product and plasmid enzyme restriction product with T4 ligase enzyme ligase enzyme.In the further experiment, will connect product and transform DH5a intestinal bacteria competence, converted product coating Amp resistance LB flat board behind the picking clone, shakes bacterium and little upgrading grain.Behind the plasmid electrophoresis, carry out enzyme and cut.Get the plasmid that enzyme cuts out about 1800bp and send order-checking, after order-checking is correct, shakes bacterium and do plasmid and take out greatly, to obtain the plasmid (as shown in Figure 5) of a large amount of expression GAT-1.
The plasmid of the expression GAT-1 of the above-mentioned structure of 50 μ g is dissolved in 0.9% physiological saline of 2ml, squeezes into fast in mouse (body weight the is the 20-22 gram) body by the i.v injection in second at 4-10.After 48 hours, give mouse enforcement part liver and cut operation.After mouse revived 48 hours, take by weighing the weight of mouse body weight and residue liver, calculate the weight/mouse body weight of residue liver, to estimate the influence of raising after the expression of GAT-1 in liver liver regeneration.
4) the GAT-1 agonist is for the promoter action of liver regeneration
Control group (not giving the GAT-1 agonist estrogens) and experimental group (giving the GAT-1 agonist estrogens), every group of 3-4 mouse.Carry out liver partly excise before or give injected in mice oestrogenic hormon simultaneously, cut back 48 hours execution mouse until liver, take by weighing the weight of mouse body weight and residue liver, calculate the weight/mouse body weight of residue liver, to estimate behind the activation GAT-1 influence to liver regeneration.Give estrogenic dosage and be made as 25mg/kg, 50mg/kg, 100mg/kg.
5. result
(1) C57BL/6 mouse (each time point 3-4 only) carried out liver resection (about 70% hepatotomy) by giving mouse at the 0th day, touched the residue liver regeneration.Mouse is put to death in after liver cuts the 0th hour, 12 hours, 24 hours and 48 hours, and the liver that takes out mouse extracts RNA.Detect the expression level of GAT-1 by the method for PCR in real time.The result is presented among Fig. 1.Shown in data represent 3 times the test the result.
(2) control group (wild-type) and experimental group (GAT-1-/-), every group of 9-10 mouse carried out liver resection (about 70% hepatotomy) by giving mouse at the 0th day, to touch the residue liver regeneration.After liver cuts the 24th hour, 48 hours and 72 hours, weighing mouse body weight is put to death mouse respectively, and the liver that takes out mouse also claims its weight.Calculate liver and the body weight ratio of every mouse according to body weight * 100 of mouse liver weight/mouse.Shown in data represent 3 times the test the result.The result is presented among Fig. 2.This figure demonstration, after liver cuts, GAT-1-/-obviously decline of mouse liver regeneration.
(3) control group and experimental group (GAT-1 siRNA), every group of 3-4 mouse at the 0th day, given 0.9% physiological saline or the GAT-1 siRNA of mouse tail vein injection 2ml.After 48 hours, implement liver resection (about 70% hepatotomy) to mouse, touch Mouse Liver regeneration.Liver cut back 48 hours, put to death mouse, calculated liver and the body weight ratio of every mouse according to the described method of aforementioned (2) part.The result is presented among Fig. 3.This figure shows that after expressing by RNAi technology interference GAT-1, mouse liver regeneration obviously descends.
(4) control group and experimental group (GAT-1 expression plasmid), every group of 3-4 mouse at the 0th day, given empty plasmid or the GAT-1 expression plasmid of mouse tail vein injection 2ml.After 48 hours, implement liver resection (about 70% hepatotomy) to mouse, touch Mouse Liver regeneration.Liver cut back 48 hours, put to death mouse, calculated liver and the body weight ratio of every mouse according to the described method of aforementioned (2) part.Found that, induce GAT-1 to express by plasmid transfection after, (Fig. 4) obviously raised in mouse liver regeneration.
(5) control group (not giving the GAT-1 agonist estrogens) and experimental group (giving the GAT-1 agonist estrogens) mouse cut liver and to be condemned to death in back 48 hours.Calculate liver and the body weight ratio of every mouse according to the described method of aforementioned (2) part.Found that the application of GAT-1 agonist can promote the mouse liver regenerative power significantly.
Embodiment 2: drug screening
1. cell levels screening
The first step: express the preparation of GAT-1 immunocyte.The immunocyte of expressing GAT-1 prepares by the following method: ordinary method is obtained the mononuclearcell of mouse lymph knot, after stimulating 2-12 hour with the ion enzyme of the phorbol ester of 20ng/ml concentration and 0.5 μ mol/l concentration is plain, detect this cell through Real time PCR or Westernblot and can express GAT-1 albumen.With the cell model of this kind cell as the medicine that is used for screening promotion liver regeneration.
Second step: drug screening:
Test group: the culture of the above-mentioned cell of handling with candidate substances;
Control group: the culture of the above-mentioned cell of handling without candidate substances.
Appropriate time after processing adopts ordinary method to measure expression, activity, amount or the secretion situation of the GAT-1 albumen of described cell.If compare with control group, the expression of the GAT-1 albumen in the test group, activity, amount or secretion are significantly risen (statistical analysis P＞0.05% has significant difference), illustrate that then this candidate substances is the material of potential promotion liver regeneration.
Adopt the agonist estrogens of this method test GAT-1 albumen.The result shows, compares with control group, gives the expression amount that oestrogenic hormon can significantly improve GAT-1 albumen in the cell.
2. animal level screening
With normal mouse as animal model.
Test group: give (also can give by filling stomach, intravenous injection, intramuscular injection, hypodermic mode) mouse with the candidate substances abdominal injection;
Control group: give mouse without candidate substances.
Appropriate time after processing adopts ordinary method to measure expression, activity, amount or the secretion situation of GAT-1 albumen in the mouse liver.If compare with control group, the expression of the GAT-1 albumen in the test group, activity, amount or secretion are significantly risen (statistical analysis P＞0.05% has significant difference), illustrate that then this candidate substances is the material of potential promotion liver regeneration.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉express the promotion liver regeneration by regulating GAT1
<130>091489
<160>10
<170>PatentIn version 3.3
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>1
tgtccacctt ccagcagatg t 21
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>2
agctcagtaa cagtccgcct aga 23
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
caagcccaaa accctggtag t 21
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
ccacgcagga catgaggaa 19
<210>5
<211>21
<212>RNA
＜213〉artificial sequence
<220>
＜223〉sense strand of siRNA
<220>
<221>misc_feature
<222>(20)..(20)
＜223〉n is T
<220>
<221>misc_feature
<222>(21)..(21)
＜223〉n is T
<400>5
caguguauuu cauguuugun n 21
<210>6
<211>21
<212>RNA
＜213〉artificial sequence
<220>
＜223〉antisense strand of siRNA
<220>
<221>misc_feature
<222>(20)..(20)
＜223〉n is T
<220>
<221>misc_feature
<222>(21)..(21)
＜223〉n is T
<400>6
acaaacauga aauacacugn n 21
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>7
agctgagagg ttgcaggcga 20
<210>8
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>8
ccaaggcggg cagaaaagt 19
<210>9
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>9
ggcgaagctt atggcgactg acaacagcaa gg 32
<210>10
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>10
gcgcgaattc ctagatgtag gcctccttgc tg 32
Claims (1)
1. be used for suppressing the siRNA of mouse γ-An Jidingsuan translocator hypotype 1, its sense strand is CAGUGUAUUUCAUGUUUGUTT (SEQ ID NO:5), and antisense strand is ACAAACAUGAAAUACACUGTT (SEQ ID NO:6).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910054351 CN101935673B (en) | 2009-07-03 | 2009-07-03 | siRNA for inhibiting mouse gamma -GABA transfer protein subtype 1 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910054351 CN101935673B (en) | 2009-07-03 | 2009-07-03 | siRNA for inhibiting mouse gamma -GABA transfer protein subtype 1 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101935673A CN101935673A (en) | 2011-01-05 |
| CN101935673B true CN101935673B (en) | 2013-08-21 |
Family
ID=43389242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910054351 Expired - Fee Related CN101935673B (en) | 2009-07-03 | 2009-07-03 | siRNA for inhibiting mouse gamma -GABA transfer protein subtype 1 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101935673B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974465B (en) * | 2016-10-20 | 2022-11-29 | 上海南方模式生物科技股份有限公司 | Slc6a13 gene and application of protein thereof |
| CN106754950A (en) * | 2017-02-21 | 2017-05-31 | 河南师范大学 | A kind of c9orf116 genes for adjusting liver regeneration and its siRNA disturbance target points and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002020753A2 (en) * | 2000-09-01 | 2002-03-14 | Lexicon Genetics Incorporated | Human gaba transporter protein and polynucleotides encoding the same |
| AU2006217543A1 (en) * | 2005-02-28 | 2006-08-31 | Integragen | Human autism susceptibility genes encoding a neurotransmitter transporter and uses thereof |
-
2009
- 2009-07-03 CN CN 200910054351 patent/CN101935673B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002020753A2 (en) * | 2000-09-01 | 2002-03-14 | Lexicon Genetics Incorporated | Human gaba transporter protein and polynucleotides encoding the same |
| AU2006217543A1 (en) * | 2005-02-28 | 2006-08-31 | Integragen | Human autism susceptibility genes encoding a neurotransmitter transporter and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| Minuk GY.γ-氨基丁酸与肝脏.《国外医学（消化系疾病分册）》.1993,第13卷(第4期), * |
| Olaf Dransfeld 等.Oligonucleotide micrarray analysis of differential transporter regulation in the regenerating rat liver.《Liver international》.2005,第25卷(第6期),1243-1258. * |
| 姜杰.小鼠γ-氨基丁酸转运蛋白亚型1（GAT1）与疼痛.《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》.2005,(第1期),A006-25. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101935673A (en) | 2011-01-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101420977B (en) | NOGO receptor antagonist | |
| CN102321158B (en) | Polypeptide for preventing cell DNA synthesis and inhibiting cell proliferation and application | |
| CN101511181A (en) | Proteins, nucleic acids encoding the same and associated methods of use | |
| CN101854949A (en) | Methods and compositions for modulating t cells | |
| CN103415615B (en) | Amplifying candidate stem cell | |
| CN109526229A (en) | Screen prevention or the therapeutic agent of the prevention of the disease as caused by interleukin-6, interleukin-13, TNF, G-CSF, CXCL1, CXCL2 or CXCL5 or the method for therapeutic agent and the disease as caused by interleukin-6, interleukin-13, TNF, G-CSF, CXCL1, CXCL2 or CXCL5 | |
| CN107557337B (en) | anti-ROR1 safe chimeric antigen receptor modified immune cell and application thereof | |
| US20220112498A1 (en) | Methods for diagnosing and treating metastatic cancer | |
| BR112020014803A2 (en) | MODIFICATION OF GENOMIC LOCIES RELATED TO THE IMMUNE SYSTEM USING CRISPR NICKASES PAIRED RIBONUCLEOPROTEINS | |
| US20110184045A1 (en) | Silencng and rig-i activation by dual function oligonucleotides | |
| CN105079792A (en) | Application of IL-17 in improving mesenchymal stem cell immunity inhibition function | |
| CN110257491B (en) | Method for transplanting and tracking mouse ovarian stem cells | |
| CN101935673B (en) | siRNA for inhibiting mouse gamma -GABA transfer protein subtype 1 | |
| CN111035648A (en) | Application of long-chain non-coding RNA GAS5 in preparation of medicines for promoting nerve regeneration and repairing nerve injury | |
| CN103339507A (en) | Recombinant Fc-fusion protein of the fifth fibronectin type iii domain of DCC | |
| Perry et al. | Injected human umbilical cord-derived mesenchymal stromal cells do not appear to elicit an inflammatory response in a murine model of osteoarthritis | |
| CN101899443B (en) | Method for regulating and controlling FAF1 (Fas-associated Factor-1) genes, composition and application thereof | |
| CN110170051A (en) | Application of the KLF12 albumen in preparation treatment non-small cell lung cancer drug | |
| CN106139163B (en) | It is a kind of inhibit osteoarthritis microRNA and its application | |
| CN103223176B (en) | miR-24 and application of inhibitor of miR-24 | |
| CN107227362A (en) | A kind of gene related to liver cancer and its application | |
| KR101433794B1 (en) | Composition comprising inhibitors of Progranulin for the prevention or treatment of osteoporosis | |
| US20220105122A1 (en) | Therapeutic and prophylactic agent for glioma, brain tumor malignancy marker, brain tumor prognostic marker, method for determining malignancy and prognosis of brain tumor and antibody inhibiting tumor proliferation | |
| CN108707603B (en) | ESCRT-III core subunit Snf7 double-stranded RNA and preparation method and application thereof | |
| KR20220041934A (en) | Therapeutic immune cells with improved function and method for preparing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: 200031 Yueyang Road, Shanghai, No. 319, No. Patentee after: Shanghai Institute of nutrition and health, Chinese Academy of Sciences Address before: 200031 No. 320, Yueyang Road, Shanghai Patentee before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES |
|
| CP03 | Change of name, title or address | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130821 Termination date: 20200703 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |