CN109771667A - The purposes of Spy1 gene and its expression albumen in treatment amyotrophic lateral sclerosis - Google Patents

Abstract

The invention discloses the purposes of Spy1 gene and its expression albumen in treatment amyotrophic lateral sclerosis.The present invention by experiment in vivo confirm in SOD1G93A ALS mouse the expression of Spy1 be decreased obviously and with the occurrence and development of disease it is closely related, Spy1 gene is overexpressed by gene regulation in neuronal cell, it can be by inhibiting DNA damage acknowledgement mechanism to promote the survival of neuron, the high above-mentioned Spy1 gene of expression of prompt can treat amyotrophic lateral sclerosis, the invention also discloses a kind of for treating the polymer nano granules of amyotrophic lateral sclerosis, by the brain of administration targeting subject and the motor neuron of spinal cord, for treating amyotrophic lateral sclerosis.The present invention provides new technological means for the prevention and treatment of amyotrophic lateral sclerosis.

Description

The purposes of Spy1 gene and its expression albumen in treatment amyotrophic lateral sclerosis

Technical field

The present invention relates to a kind of methods of gene therapy amyotrophic lateral sclerosis, in particular to Spy1 gene and its expression egg The white purposes in treatment amyotrophic lateral sclerosis, the invention belongs to neural field of biotechnology.

Background technique

Amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS) is also known as " amyotrophic lateral sclerosis disease " one The fatefulue neurodegenerative disease of kind.Mainly involve upper and lower motor neuron, show as the myasthenia and atrophy of progressive, Mean survival time (MST) 3-5.Newest epidemiology statistics show that the illness rate of ALS is about annual every 5/100000ths.About 20% familial ALS and SOD1 gene mutation are closely related.At present research think, the pathogenesis of ALS mainly with RNA generation It is thin to thank disorder, protein homeostasis exception, mitochondria dysfunction, oxidative stress, toxicity of excitatory amino acid, DNA damage, colloid Born of the same parents' lesion and intracellular caryoplasm transport disorders etc. are related.Current research expects the year two thousand forty, and global ALS patient will be in 2015 Increase by 69% on the basis of (222801 people).

Although ALS produces increasing threat to the health of the mankind, and brings to family and society heavy Burden, but up to the present still without control disease effective means.First oral drugs Riluzole of FDA approval (Riluzole), expensive, and be only capable of extending patient vitals about 3-4 months；The most newly approved free radical of FDA in 2017 is clear Except drug Edaravone (Edaravone), there are still disputes for early clinic research, and the Edaravone of research prompt at present seems not Suitable for all types of ALS patients.Since RNA or protein mediated toxicity are the most common pathogenic mechanisms of ALS, pass through The RNA or albumen of the means reduction toxicity of gene therapy simultaneously play the role of protecting motor neuron by controlling gene expression Have become the forward position studied at present.Although the gene therapy of the mankind remains at initial stage, a series of early clinics Test has confirmed that the safety and validity of this treatment means.The gene therapy research of ALS is based primarily upon at present Antisense oligonucleotides (ASOs), adenovirus (AAV)/slow virus (LV) carrier and mescenchymal stem cell slow virus carrier system. The current research of ASOs targeting SOD1 has come into I phase clinical stage, research shows that SOD1ALS patient gives 3mg intrathecal ISIS333611 does not show toxicity and serious adverse reaction；However discovery mutation is tested to the I phase of ISIS33611 The content of SOD1 albumen is not reduced, and the Phase I clinical trial of the SOD1 ASO (BIIB067 or IONIS-SOD1Rx) improved It is currently under conceptual phase.ASOs is used in the neuron of ALS/FTD patient iPSC (derivable multipotential stem cell) induction The C9ORF72 of targeting amplification reduces the aggregation of toxicity RNA and the toxicity of glutamic acid.It recent studies have shown that, give C9ORF72 ALS mouse intracerebroventricular injection ASOs targeting C9ORF72 repeat amplification protcol segment can reduce RNA aggregation and dipeptides repetitive proteins simultaneously Improve behaviouristics performance relevant to aging.1 clinical trial phase of the ASOs for giving targeting C9ORF72 intrathecal at present (NCT03626012) it is in and raises subject's stage.It is current using adenovirus/slow virus as the gene therapy of carrier in ALS Mainly based on zooscopy.The research of multiple research groups confirms to subtract by striking using AAV/LV vector administration shRNA The life cycle of SOD1 extension ALS G93A rat model.It is intramuscular to give AAV/LV be carrier neurotrophic factor (GDNF, IGF-1 and VEGF), the morbidity of ALS SOD1-G93A rat can be delayed, improve its behavior and motor function and extend existence Phase, and the administration mode by being directly targeted central nervous system, as intracerebroventricular injection, injection through vertebral canal or small injection of brain are delivered These also play partial rescue disease phenotype using virus as the neurotrophic factor of carrier and extend the work of ALS surviving rats phase With.Experiment in vitro expresses antioxidant genes using LV as carrier in cell can reduce the horizontal cell that promotes of oxidative stress Survival.The forelimb function that hUPF1 gene therapy can improve rat is given by carrier of AAV in ALS TDP43 mouse model. In addition, mescenchymal stem cell therapeutic modality is also by the common concern of researcher, research shows that moving in SOD1-G93A rat The human mesenchymal stem cell with LV carrier expression GDNF is planted, it being capable of skeletal muscle, motor neuron to ALS SOD1-G93A rat Neuroprotection is played with neuromuscular junction, and increases survival.Another research is using LV carrier system in human mesenchyme It is overexpressed VEGF and GDNF in stem cell, is able to extend the life cycle of SOD1-G93A ALS rat, maintains ventricornu movement The function of neuron and neuromuscular junction.In addition to this, in motor neuron disease gene therapy have been achieved with it is breakthrough Progress, a kind of antisense oligonucleotides drug --- Spinraza has been obtained for the approval of FDA at present and withers for treating ridge flesh Contracting disease (SMA).

To sum up, although it is receive more and more attention in the research of field of gene at present, gene therapy Still suffer from many challenges: the obstruction of invalid administration mode and blood-brain barrier has seriously affected therapeutic effect, such as antisense widow Nucleotide cannot blood-brain barrier (BBB) and can only by intrathecal injection by way of be administered；Still lack targeting nervous centralis The administration mode of system privileged site or cell subsets；And production for clinical a large amount of viral vectors need it is expensive at This, furthermore viral vectors can not only activate the immune system of host, moreover it is possible to which the later period that has an impact of inducing immunological memory answers again With the curative effect of viral vectors.

Nano-carrier (nano-vectors) is a kind of safe and efficient, ideal novel gene treatment of Recent study Carrier, it is that target gene is contained in interparticle or is adsorbed in surface, utilizes nanoparticle using nanoparticle as carrier Target gene is transferred to target cell interior by the properties such as small-size effect, specific surface effect, interfacial effect, discharges target gene, To reach the final purpose of gene therapy.Compared with viral vectors, nano-carrier is micro- by the way that Plasmid DNA is compressed into nanoscale Grain, not only can be to avoid nucleolysis, and can efficiently mediated dna transfect to intracellular, while also avoiding using disease The disadvantages of poisonous carrier bring cytotoxicity, immunogenicity and potential oncogenicity, it is often more important that, it is repaired using different surfaces Decorations, nano-carrier can effectively determine trend, positioning transmitting and the high efficient expression of foreign gene in vivo, to show excellent Targeting more.Therefore it is low will largely to improve central nervous system administration bioavilability for the appearance of nano-carrier And it can not be by the situation of blood-brain barrier (BBB) etc., up to the present, although in Alzheimer disease (AD) He Pajin There has been the research much based on nano-carrier drug delivery system in gloomy disease field (PD), but the research of ALS therapy field still very It is limited.Earlier studies have shown that by being covalently attached the medicine combined Riluzole and carbon nanotube (CNTs) without influencing Riluzole Object activity, it is also free of toxic effects to cell.It is negatively charged that 88nm is made with solid lipid nano-particles (SLNs) package Riluzole Compound brain tissue targeting more higher than common Riluzole and lower non-is shown by intraperitoneal injection approach administration Specific biological distribution.Experiment in vivo in ALS SOD1G93A mouse by the administration mode of intracerebroventricular injection, will be more with rouge The liposome of sugar-modified package anti-inflammatory drug (minocycline) is directly targeted the TLR4 receptor of microglia, increases drug Uptake ratio and slow down the progress of disease.It recent studies have shown that, calcium phosphate lipidic nanoparticles formula package SOD1 ASO exists It is injected under microscope in zebra fish body, successfully detects nano particle in its brain tissue, spinal cord and blood, it was demonstrated that phosphoric acid Calcium lipidic nanoparticles are a kind of safe and effective genophores, improve ASO to motor neuron administration route.To have anti- The cerium oxide nanoparticles of oxidation characteristic are injected in ALS SOD1G93A mouse by tail vein injection approach, and it is small to extend ALS The mean survival time (MST) of mouse.For the correlative study of nano-gene carrier targeted therapy ALS, there is not been reported at present.

Many researchs are with rabies virus glycoprotein (rabies virus glycoprotein, RVG) as targeting brain recently The ligand of tissue is simultaneously modified on the surface of transmitting siRNA carrier, such as trimethylene chitosan, mannitol-polyethyleneimine, Excretion body, and siRNA is transmitted in brain tissue by way of systemic injection.However, only transfer gene passes through blood brain Barrier be it is inadequate, there are many cell subsets in central nervous system, therefore in order to ensure the safety of gene delivery and effectively Property, it would be desirable to it is capable of the nanometer transfer gene system of targeted neuronal.One research group recent studies have shown that, bacteriophage Show that l peptide TGNYKALHPHNG (TGN) can make PEG-PLGA (polyethylene glycol-polylactic acid glycolic acid copolymer) nanometer Grain facilitates penetration of blood-brain barrier, therefore constructs novel Brain targeting drug delivery system, after which contains polypeptide drugs It can protect it not degraded by vivo environment, improve stability, be a kind of effective, hypotoxicity Brain targeting drug delivery system. On the basis of another research group is studied herein, the reversed isomer C GN peptide (d-CGNHPHLAKYNGT) of TGN is synthesized, and is demonstrate,proved Real CGN ratio TGN shows higher brain targeting.And the research group constructs a kind of difunctional nano complex application Blood-brain barrier targeting ligand-CGN and the dual modification of neuron targeting ligand-Tet1.Tet1 (HLNILSTLWKYR), passes through body The peptide of outer phage display identification, in conjunction with GT1B gangliosides sphingomyelins highly expressed on neuron.Polyethylene glycol- Polymethylacrylic acid N, N- dimethylaminoethyl (PEG-PDMAEMA) has hypotoxicity, and high buffer capacity and high transfection efficiency are One ideal nano-carrier, therefore target gene is packed by core of this PEG-PDMAEMA by the study group, then to pass through blood brain Barrier and the dual modification of targeted neuronal ligand, by CGN-PEG-PDMAEMA and Tet1-PEG-PDMAEMA according to weight ratio 1:1 Mixing, shows good stability in blood, does not lead to haemolysis.Nano gene complex can be mediated by clathrin Endocytosis and micro- full drink phenomenon enter neuron, and small recessed protein mediated endocytosis enters in brain capillary at it It is played an important role in chrotoplast.This nano-particles reinforcement body can successfully escape the dissolution of lysosome, be directly entered mind In cytoplasm through member, effective transfection of gene is realized.

Many epidemiological study reports, the disease incidence of neurodegenerative disease decreases in tumor patient, newest to grind Study carefully and show that many genes in tumour and neurodegenerative disease, albumen and signal path regulation obstacle show opposite situation, Which results in our great interest, inventor is had found by early-stage study, the closely related former cancer with tumour growth Gene Spy1 is expressed in ALS SOD1G93A mouse spinal cord Motoneurons and is decreased obviously, and with the generation of ALS and hair Open up closely related, there is not been reported for the relationship between this proto-oncogene and ALS.

Spy1 is that xenopus cell cycle-regulatory gene corresponds to gene in the mankind, is in Speedy/Ringo family A member.The family member contains Speedy/Ringo box structural domain, is cell cycle dependent kinases (cyclin Dependent kinases, CDKs) activation factor.Spy1 is proved in kinds of tumors close with tumor development Correlation, also some researches show that Spy1 also plays an important role in the development and damage of nervous system.

Summary of the invention

The purpose of the present invention is to provide the new use of Spy1 gene and its expression albumen in treatment amyotrophic lateral sclerosis On the way.

In order to achieve the above object, present invention employs following technological means:

Amyotrophia funiculus lateralis is treated in preparation firstly, the invention proposes cell cycle-regulatory gene Spy1 and its expression albumen Harden the purposes in drug.

Wherein, the nucleotide sequence of the cell cycle-regulatory gene Spy1 is as shown in SEQ ID NO.1.

Wherein, it is preferred that the drug is used for by inhibiting DNA damage response pathway and increasing the survival of neuron Improve amyotrophic lateral sclerosis caused by being mutated by SOD1G93A.

Wherein, it is preferred that the drug is the nano particle being prepared by nano-carrier combination Spy1 gene, coagulates Glue or emulsion, the nano particle is including but not limited to polymer nano granules, lipidic nanoparticles, carbon nano-particle, metal Nano particle.

Further, the invention also provides the synergist of cell cycle-regulatory gene Spy1 and its expression albumen to prepare Treat the purposes in amyotrophic lateral sclerosis drug.

Wherein, it is preferred that the drug is used for by inhibiting DNA damage response pathway and increasing the survival of neuron Improve amyotrophic lateral sclerosis caused by being mutated by SOD1G93A.

Wherein, it is preferred that the synergist be cell cycle-regulatory gene Spy1 and its express albumen agonist, on Adjustment or stabilizer.

Wherein, it is preferred that the synergist refers to any activity that Spy1 gene or albumen can be improved, improves Spy1 The stability of gene or albumen, up-regulation Spy1 gene or albumen expression, increase Spy1 gene or albumen effective acting time Substance, the synergist are compound, chemical small molecule or biomolecule.

Wherein, it is preferred that the biomolecule can be DNA or RNA, or regulation Spy1 gene or protein expression Viral product.

Further, the invention also provides a kind of for treating the polymer nano granules of amyotrophic lateral sclerosis, It is prepared by the following method to obtain:

(1) diblock copolymer Mal-PEG-PDMAEMA is synthesized；

(2) by the diblock copolymer Mal-PEG-PDMAEMA of synthesis and sulfydryl CGN peptide or Tet1 peptide in phosphate-buffered The ratio of 1:1 and at room temperature effect 4 hours in molar ratio in liquid, reaction solution is dialysed with the bag filter of MWCO5ka, dialysis Liquid is distilled water, CGN-PEG-PDMAEMA and Tet1-PEG-PDMAEMA product is obtained after freeze-drying；

(3) CGN-PEG-PDMAEMA and Tet1-PEG-PDMAEMA is dissolved respectively in DEPC water, then with Spy1 Gene is mixed in the ratio that N/P is 1-10:1, is vortexed 30 seconds, then cultivates obtain the polymer after twenty minutes at room temperature Nano particle.

Purposes of the polymer nano granules in preparation treatment amyotrophic lateral sclerosis drug, wherein it is preferred, The polymer nano granules by inhibit DNA damage response pathway and increase neuron survival, for improve by Amyotrophic lateral sclerosis caused by SOD1G93A is mutated.

Administration mode includes telocoele, intrathecal injection, intravenous injection and nasal-cavity administration.Subject include mammal and People.

It is proposed of the invention provides new technological means for the prevention and treatment of amyotrophic lateral sclerosis.

Detailed description of the invention

Fig. 1 is that hSOD1G93A transgenic mice and wild-type mice anterior horn motor neurons Spy1 immunohistochemistry contaminate Color and quantitative analysis results；

Wherein: (A) immunohistochemistry shows the Motoneurons Spy1 table compared with wild-type mice in ALS mouse Up to decline.Low-power field: scale=100 μm, high power field: scale=50 μm；(B) qRT-PCR detection hSOD1G93A turns base Because Spy1 mRNA level in-site decreases compared with wild-type mice in mouse；(C, D) Western blotting shows Spy1 Protein expression level also significantly reduces in hSOD1G93A transgenic mice.

Fig. 2 is that the expression of Spy1 is declined in stable transfection mSOD1 group in NSC34 cell line；

Wherein: (A) fluorescence intensity of Spy1 (red) in mSOD1 cell line is decreased obviously, scale=25 μm； (B,C) Western blotting is analyzed and quantified: the protein expression of Spy1 also decreased significantly in mSOD1 cell line；(D)qRT- The mRNA level in-site of PCR detection display Spy1 is also declined in mSOD1 cell line.* P < 0.001 P < 0.05, * * *；

Fig. 3 is expression of the DNA damage response pathway associated molecules in NSC34, pLV, wtSOD1 and mSOD1 cell line；

Wherein: the expression of the prompt of (A) Western blotting result p-ATM, p-ATR, p-Chk1, p-Chk2 and p53 There is significant increase compared with other three groups in mSOD1 group；(B) quantitative analysis of A result；(C) qRT-PCR shows p53mRNA Level increased in mSOD1 group, P < 0.001 * P < 0.05, * * P < 0.01, * * *；(D) quantitative analysis shows that 8-OHdG exists MSOD1 group is horizontal to be increased, and (E) immunofluorescence dyeing shows that 8-OHdG (red) fluorescence intensity increases；

Fig. 4 is that Spy1 negative regulation DNA damage response pathway is lowered in mSOD1 cell line；

Wherein: simultaneously quantitative result shows p-ATM, p-ATR, p- after silencing Spy1 for (A, B) Western blotting detection The protein level of Chk1, p-Chk2 and p53 increased；(C) qRT-PCR detects Spy1 and p53 mRNA expression；(D) Silencing Spy1 flow cytometer showed detects Apoptosis situation in mSOD1 cell line, and apoptosis rate increases by 25% after showing silencing Spy1； (E) quantitative analysis of D result.* P < 0.001 P < 0.05, * * P < 0.01, * * *；

Fig. 5 is the activity for being overexpressed Spy1 and increasing mSOD1 cell line；

Wherein: (A) transfects the Spy1 of mCherry label in mSOD1 cell line.48 hours standby Western The expression of blotting detection Spy1 and CDK2.(B) Western blotting is overexpressed Spy1 as the result is shown leads to p-ATM, P-ATR, p-Chk1, p-Chk2 and p53 protein expression level are remarkably decreased.(C) quantitative analysis DNA damage response access is related Level of the albumen after Spy1 overexpression.P < 0.001 * P < 0.01, * * *；(D) mtt assay detects cell activity, G93A mutation SOD1 causes the activity of NSC34 cell to reduce, and such case can partially be over-expressed Spy1 and be reversed.* P < 0.01, * * * P<0.001；(E) expression of qRT-PCR method detection Spy1 and p53 mRNA；

Fig. 6 is that the hSOD 1G93A mutain of high concentration causes the expression of Spy1 to be lowered.

Wherein: (A) detects Spy1, the protein expression level of SOD1 and β-actin using Western blotting； (B) Using the activity of mtt assay detection cell, SOD1G93A is mutated the activity for reducing NSC34 cell.* P < 0.05, * * * P < 0.001；(C, D) quantitative analysis transfects Spy1 and SOD1 protein expression level after hSOD1G93A plasmid.Spy1 protein expression water It is flat to be decreased obviously.The mRNA level in-site application of * P < 0.01, * * * P < 0.001. (E) Spy1 after transfecting hSOD1G93A plasmid QRT-PCR detection, the mRNA level of Spy1 is declined as the result is shown.*P<0.05,**P<0.01,***P<0.001.

Specific embodiment

It elaborates with reference to the accompanying drawing to specific embodiment provided by the invention.Those skilled in the art It is contemplated that a variety of modifications and adaptations carried out, without changing the spirit or scope of the present invention.This modifications and adaptations include Within the scope of the invention.Following embodiments are not limit the invention in any way.

Embodiment 1

One, method and material

1, experimental animal and reagent

1.1 transgenosis C57BL/6J hSOD1G93A genotype positive mice 6, C57BL/6J wild-type mice 6, Half male and half female is purchased from U.S. Jackson Laboratory company, breeds in Harbin Veterinary Medicine Inst., China Academy of Agriculture Amplification and identified for genes.

1.2 NSC34 cell lines are purchased from Canada CEDARLANE Corporation, save in liquid nitrogen.

PLenti viral-NSC34 cell line, wtSOD1-NSC34 cell line and hSOD1G93ANSC34 stable transfection Cell line is constructed by Guangzhou Ai You biology Co., Ltd, is screened and is stablized passage, cultivate in DMEM culture medium.Primary neuron Cell is derived from tire mouse cortical neuron, Neurobasal culture medium culture.

1.3 main agents

Thermo Forma company, the U.S. protein electrophoresis Marker

The green skies company in Western and IP protein lysate Beijing

The green skies company in SDS lysate Beijing

The green skies company in the Beijing PMSF

The green skies company in Lodding buffer (5 ×) Beijing

SIGMA-ALDRICH company, the U.S. bovine serum albumin(BSA) (BSA)

Company, Anti- β-actin antibody mouse anti-mouse how anti-Beijing Zhong Shan Golden Bridge

Anti-Spy1 antibody rabbit anti-mouse how anti-U.S. Novus company

Anti-CDK2 antibody mouse anti-mouse Wuhan doctor's moral company

Cell Signaling company, the Anti-ATM antibody U.S.

Cell Signaling company, the Anti-p-ATM antibody U.S.

Cell Signaling company, the Anti-ATR antibody U.S.

Cell Signaling company, the Anti-p-ATR antibody U.S.

Cell Signaling company, the Anti-Chk1 antibody U.S.

Cell Signaling company, the Anti-p-Chk1 antibody U.S.

Cell Signaling company, the Anti-Chk2 antibody U.S.

Cell Signaling company, the Anti-p-Chk2 antibody U.S.

Abcam company, the Anti-p53 antibody U.S.

Santa Cruz company, the Anti-SOD1 antibody U.S.

Anti-MAP2 antibody Beijing Bo Aosen company

Santa Cruz company, the Anti-8-OHdG antibody rabbit anti-mouse U.S.

The green skies company in rabbit igg Beijing

The green skies company in the Beijing mouse IgG

IRDye800 couples Rockland company, the goat anti-mouse IgG U.S.

IRDye800 couples Rockland company, the goat anti-rabbit igg U.S.

SIGMA-ALDRICH company, the DAPI dyeing liquor U.S.

Santa Cruz company, the U.S. Protein A/G PLUS-Agarose

Invitrogen company, Trizol (total serum IgE extraction agent) U.S.

One-step method fluorescent dye PCR quantification kit Dalian treasured biotech firm

Abnova company, the PE-Anexin V/7-AAD apoptosis detection kit U.S.

Invitrogen company, the U.S. Lipofectamine2000

Invitrogen company, the U.S. Opti-MEM

MTT powder Shanghai Hua Shun bioengineering Co., Ltd

SIGMA-ALDRICH company, the puromycin U.S.

Spy1 siRNA synthesizes Shanghai Ji Ma company

MCherry-Spy1-pEZ-M95 plasmid synthesizes Guangzhou reactivation genome company

EGFP-SOD1G93A-pcDNA3.1 plasmid synthesizes Guangzhou Ai You Biotechnology Co., Ltd

QRT-PCR matches primer

NQBB company, fetal calf serum (FBS) Australia

Invitrogen company, the U.S. DMEM

Invitrogen company, the U.S. Neurobasal Medium (1X)

Invitrogen company, the B-27 additive U.S.

Invitrogen company, the L-Glutamine U.S.

1.4 primer

QRT-PCR matches primer

Mouse Spy1 (forward): 5 '-AAG GGA CCA ACT CTG GGA CA-3 '

Mouse Spy1 (reverse): 5 '-ACT GTG ATG CAC AGA CCG CT-3 '

MSOD1 (forward): 5 '-GGG AAG CTG TTG TCC CAA G-3 '

MSOD1 (reverse): 5 '-CAA GGG GAG GTA AAA GAG AGC-3 '

Mouse p53 (forward): 5 '-TAC CAC CAT CCA CTA CAA G-3 '

Mouse p53 (reverse): 5 '-AAA CAC GAA CCT CAA AGC-3 '

Mouse β-actin (forward): 5 '-CCA GCC TTC CTT GGG TAT-3 '

Mouse β-actin (reverse): 5 '-TGC TGG AAG GTG GAC AGT GAG-3 '

The mating primer of transgenic mice genotype identification (U.S. Jackson Lab)

Target gene SOD1 (forward): CAT CAG CCC TAA TCC ATC TGA

Target gene SOD1 (reverse): CGC GAC TAA CAA TCA AAG TGA

Internal reference IL-2 (forward): CTA GGC CAC AGA ATT GAA AGA TCT

Internal reference IL-2 (reverse): GTA GGT GGA AAT TCT AGC ATC ATC C

Spy1 siRNA synthesizes (Shanghai Ji Ma company)

Spy1 siRNA (si-Spy1) (forward): 5 '-CCA GAG GGC CUU GUC UAA UTT-3 '

Spy1 siRNA (si-Spy1) (reverse): 5 '-AUU AGA CAA GGC CCU CUG GTT-3 '

Spy1siRNA negative control (si-NC) (forward): 5 '-UUC UCC GAA CGU GUC ACG UTT-3′

Spy1 siRNA negative control (si-NC) (reverse): 5 '-ACG UGA CAC GUU CGG AGA ATT-3′

5%BSA confining liquid, 10% chloraldurate, 4% paraformaldehyde solution, 0.1mol/L phosphate buffer, DAB examination Agent box, deionized-distilled water, penicillin, streptomysin etc. are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture.

1.5 solution are prepared

0.1 M PBS (pH=7.4): high pressure steam sterilization packing saves after filtering.

4% paraformaldehyde fixer: paraformaldehyde 4g is dissolved in 0.1mol/L PBS100ml, and it is molten to be heated up to 60 DEG C of stirrings Solution, 4 DEG C of preservations.

75% ethyl alcohol: dehydrated alcohol and DEPC water are prepared according to 1:3, are put in -20 DEG C and are saved backup.

The preparation of penicillin, Streptomycin Solution: 0.48g/ bottles of penicillin (i.e. 800,000 units) is dissolved in the double steamings of 4ml sterilizing Water.1g/ bottles of streptomysin (i.e. 1,000,000 units) is dissolved in 5ml sterilizing distilled water.Two kinds of solution concentrations are every milliliter 200,000 single Position.5 bottles of penicillin solutions and 4 bottles of Streptomycin Solutions are mixed, that is, it is mixed to can reach every milliliter of each 100,000 units of Penicillin, streptomysin Close solution.

10% cell culture fluid is prepared: 450ml is added in 50ml fetal calf serum Fetal Bovine Serum (FBS) In Dulbecco ' s modified Eagle ' s medium (DMEM), prepared penicillin, Streptomycin Solution are added 5ml, 4 DEG C of packing save.

Primary neuronal culture liquid is prepared: the addition 10ml B27 additive in Neurobasal Medium (1X), then plus Enter to prepare penicillin, Streptomycin Solution 5ml, 4 DEG C of packing save.

Solution used in Western blot is prepared

Electrophoresis liquid: being dissolved in distilled water 500ml for Tris-base6g, glycine 36g, SDS5g, is 5 × mother liquor, uses When need to add distilled water dilute five times.

Film washing liquid (TBST): Tris-base6g, sodium chloride 20g are dissolved in 500ml distilled water, are configured to 5 × mother Liquid, by 100ml mother liquor when use, 0.5ml tween, 0.5ml cryosel acid dissolution is configured to 500ml TBST in 400ml distilled water Solution, 4 DEG C of preservations.

Confining liquid: 5% skimmed milk power confining liquid or 5%BSA confining liquid, 4 DEG C of preservations are prepared with the TBST dissolution prepared.

2, method

The grouping of 2.1 NSC34 cell experiments

NSC34 cell culture, by constructing plasmid, slow-virus transfection plasmid screens simultaneously stable transfection, obtains passage PLenti viral-NSC34 cell line, wild type SOD1-NSC34 cell line and hSOD1G93A-NSC34 stable transfection are thin Born of the same parents system.Experiment is divided into four groups, is respectively designated as blank control group (NSC34 group), empty viral group (pLV group), wild type group (wtSOD1 group) and anomaly group (mSOD1 group).

The grouping of 2.2ALS mouse experiment

Animal is divided into two groups: one groups for ALS transgenic mice group (hSOD1G93A-Positive mouse group), and one group is open country Raw control group (hSOD1G93A-Negative mouse group), every group each 5.After behavior evaluation, myeloid tissue is taken, through 4% poly Formaldehyde, which impregnates, to be fixed, and paraffin embedding, slice carries out immunohistochemical staining and immunofluorescence dyeing.

2.3 NSC34 cell line cultures

The recovery of NSC34 cell line

Water-bath is preheated to 37 DEG C, by cryopreservation tube after being taken out in liquid nitrogen container, water-bath is quickly placed in, should be noted and freeze The sealing orifice of pipe, ceaselessly shakes, and after liquid in pipe melts, liquid in cryopreservation tube is sucked out, is put into containing cell culture In the centrifuge tube of liquid, it is centrifuged 5min (750rpm/min), discards supernatant.It rejoins cell culture fluid and is made into cell suspension, After cell suspension piping and druming uniformly, it is added in culture bottle, is put into cell incubator and cultivates, cell culture fluid is replaced after 24.

The culture and passage of NSC34 cell

The superclean bench for being passed through ultraviolet light irradiation using 75% alcohol wipe, is lighted alcolhol burner, is ready to use Disinfection after empty culture bottle, take out preheated cell culture fluid and PBS solution.Cell bottle is taken out from cell incubator, After the injection of 75% alcohol, alcolhol burner light-burned bottle collar extension after sterilization, bottle cap is opened, and burn bottle cap internal orifice, outwells bottle Interior old culture solution is cleaned once with the PBS of preheating, and after outwelling PBS, the cell culture fluid of preheating is added, and cell is blown from bottle wall Get off, piping and druming to uniformly after, cell suspension is dispensed into three new culture bottles, often it is bottled in add 4ml cell culture fluid. Using the light-burned bottleneck of alcolhol burner, and culture bottle cap, bottleneck is screwed, culture bottle is put into and continues to cultivate in cell incubator.

Slow-virus transfection constructs stable transfection, screening cell line

Inventor's previous experiments have successfully constructed pLenti viral-NSC34 cell line, the wild type of stable transfection SOD1-NSC34 cell line and hSOD1G93A-NSC34 cell line after being screened by puromycin (200 μ g/ml), carry out Subsequent experimental.

NSC34 cell freezes

Cell bottle is taken out from cell incubator, discards supernatant, and new cell culture fluid is added, it will be adherent using dropper Cell is gently blown and beaten to cell suspension, is put into centrifuge tube, and 5 minutes (750rpm/min) is centrifuged, and discards supernatant, is added and is prepared In the good cells frozen storing liquid containing 10%DMSO, cryopreservation tube is put into gradient into cryopreservation tube by packing after cell piping and druming uniformly In the freezing storing box of cooling, -80 DEG C of short-term preservations save in rear dislocation liquid nitrogen container for a long time overnight.

The culture of 2.4mSOD1 cell line and siRNA interference

Adherent mSOD1 cell is washed using the cell culture fluid of antibiotic-free and is resuspended by first 24 hours of transfection, every hole 2 ×10⁵Cell is added in 6 orifice plates and is cultivated, and next day is transfected.Firstly, preparing transfection reagent: operating, use to specifications Opti-MEM dilutes Spy1 siRNA and Lipofectamine2000, is placed at room temperature for 5 minutes.Above two transfection reagent is mixed It closes, room temperature and gently vibration are placed 25 minutes, are added in cell plates after forming transfection reagent and Spy1 siRNA mixture, 6 is small When after replacement cell liquid be the cell culture fluid containing 10% serum.Cell RNA is extracted after 48 hours, is detected using qRT-PCR dry Disturb efficiency.

The conversion of 2.5 plasmids, amplification, extracting

MCherry-Spy1-pEZ-M95 plasmid is purchased from Guangzhou reactivation genome company, EGFP-SOD1G93A-pcDNA3.1 Plasmid experimental group is purchased from Guangzhou Ai You Biotechnology Co., Ltd early period.

2.5.1 the conversion of plasmid

By DH5 α competence ice bath to dissolving, it is separately added into above-mentioned plasmid, pipettor mixes gently, and common ice bath 30 divides Clock is then rapidly added 900 μ l non-resistant LB culture mediums, is put into through heat shock (42 degrees Celsius heat shock 90 seconds, ice bath 3 minutes) In shaken cultivation case, 37 DEG C of oscillations (220rpm) shake bacterium 45 minutes.4500rpm is centrifuged 5 minutes, discards supernatant liquid, remaining 100 microlitres, gently thallus is blown afloat, be spread evenly across on the LB agar plate containing amicillin resistance (Amp+), stands 5 points Clock.37 DEG C of incubators are incubated overnight.Then, single full independent bacterium colony is chosen to be placed in equipped with 5ml LB liquid medium (Amp +) test tube in, 37 DEG C of oscillations (220rpm) shake bacterium, are incubated overnight.

2.5.2 the extraction of plasmid

Referring to Omega company E.Z.N.A.It measures and extracts in Endo-Free Plasmid Midi Kit endotoxin-free plasmid Kit specification extracts plasmid.

2.6 plasmids transiently transfect

Plasmid transient transfection is transfected using Lipofectamine2000 reagent.First 24 hours of transfection, mSOD1 cell Or NSC34 cell is with every hole 1 × 10⁶A cell kind is in 6 orifice plates.MSOD1 cell transient transfection contains the plasmid of Spy1 gene, MCherry-Spy1-pEZ-M95 plasmid is diluted in 200 μ L Opti-MEM with every 2 μ g of hole, meanwhile, use 200 μ L Opti- MEM dilutes Lipofectamine2000 reagent (every 4 μ L of hole), is stored at room temperature 5 minutes.Then, above two reagent is mixed Lipofectamine2000/DNA compound is formed, is stored at room temperature 20 minutes, tissue culture plate is added, cultivates 6 in incubator After hour, replacement culture solution is the cell culture fluid containing 10% serum, continues culture 42 hours.NSC34 cell gradient instantaneously turns Contaminate the plasmid containing SOD1G93A, EGFP-SOD1G93A-pcDNA3.1 plasmid with every hole 0 μ g, 1 μ g, 2 μ g, 3 μ g, 4 μ g's Concentration is transfected using Lipofectamine2000, and fluorescence microscope detection is carried out after transfection 48 hours and is carried out subsequent Experiment.

The detection of 2.7 apoptosis rates

(1) by mSOD1 cell with every hole 5 × 10⁵The amount kind of cell grows into 60%~70% to cell in six orifice plates When, Spy1 interference sequence is transfected, it is 10% serum cell culture fluid that cell liquid is replaced after 6 hours, continues culture 42 hours, is carried out Annexin-PE/7-AAD apoptosis kit detects early apoptosis；

(2) cell is resuspended using new culture solution, and 4 DEG C of 750rpm are centrifuged 5min, collects cell；

(3) PBS being pre-chilled with 4 DEG C is washed cell 2 times, and 750rpm is centrifuged 5min, collects cell；

(4) it prepares 1 × Binding Buffer: using 4 times of 4 × Binding of dilution Buffer of deionized water；

(5) 250 microlitres of 1 × Binding Buffer is added, cell, adjuster concentration 1 × 10 is resuspended⁶Cell/ml；

(6) it takes 100 microlitres of cell suspensions in 5ml streaming pipe, Annexin V and 10 microlitres that 5 microlitres of PE are marked is added 7-AAD is mixed gently；

(7) it is protected from light, reacts at room temperature 15min；

(8) 400 microlitres of 1 × Binding Buffer is added, mixes, flow cytometry analysis；

(9) fluorescence compensation adjustment: using the normal cell handled without apoptosis induction, fluorescence compensation is carried out as control and is adjusted It saves away spectra overlapping and sets the position of cross door；

(10) Annexin V-PE is fluorescent red-orange, and excitation wavelength Ex=488nm, launch wavelength is Em=578nm, Annexin V-PE can be in conjunction with the cell membrane of apoptosis early stage cell, and 7-AAD is red fluorescence, excitation wavelength Ex=546nm, Launch wavelength is Em=647nm, and 7-AAD cannot be through normal cell or the complete cell membrane of viable apoptotic cell, but can be with Penetrate non-viable apoptotic cell or non-viable non-apoptotic cell and in conjunction with the DNA in it；

(11) flow cytometry analysis, using Annexin V-PE as abscissa, 7-AAD is ordinate.Living cells: Annexin V-PE feminine gender/7-AAD is negative；Viable apoptotic cell: the Annexin V-PE positive/7-AAD is negative；Late apoptic is thin Born of the same parents and non-viable non-apoptotic cell: the Annexin V-PE positive/7-AAD is positive.

2.8 MTT detection

The preparation of MTT solution: being added 2ml solvent for 10mg MTT powder and mix, and is configured to the MTT that concentration is 5mg/ml Solution is protected from light preparation.

MTT solution is added: at corresponding time point, the attached cell in 96 orifice plates being detected, it is micro- that every hole is added 20 Rise MTT solution, 5%CO2,37 degrees Celsius cultivate 4 hours, terminate culture, carefully draw and discard culture solution.

Colour generation: 150 microlitres of dimethyl sulfoxides are added in every hole, slight oscillatory 10 minutes, dissolve crystal sufficiently.

Colorimetric: 96 orifice plates are put into microplate reader, select 540nm wavelength, measure each hole absorbance value, record result.Simultaneously Zeroing hole and corresponding control wells are set

2.9 tire mouse Primary cortical neurons cultures

2.9.1 poly-D-lysine is coated with

Poly-D-lysine coating buffer is appropriate, instills in the culture plate for preparing developing approach, is coated with 30 minutes, blots and dried in the air Night, secondary daily PBS cleaning twice, are dried spare.

2.9.2 Primary cortical neurons culture

It takes tire murine brain: C57BL/6J wild females mouse and hSOD1G93A transgenic male mice is mated, Female mice yin bolt is observed, pregnant 18-21 days to female mice, after putting to death pregnant mouse, 75% alcohol soaking disinfection 1 minute is placed in sterile training It supports in ware, is placed on ice.In in super-clean bench, taking out embryo on ice, dissection, separation brain tissue, while leave and take portion of tissue into Row subsequent gene type identifies (being detailed in trangenic mice genotype identification part).

Separate cerebral cortex: removal pia mater, blood vessel take cerebral cortex to be placed in a small amount of DMEM, with eye scissors by cortex Fragment is cut into repeatedly.

Digestion: being added 2mg/ml papain and appropriate DNA enzymatic, and 37 degrees Celsius digest 25~30 minutes, every five minutes It slightly shakes once, equivalent complete medium is taken to stop digestion.

Cell suspension is transferred in 15ml centrifuge tube, gently blows and beats 15~20 times, uses 200 mesh sterilizing stainless steel gauzes Filtering.The cell suspension centrifugation that will acquire, 800rpm 3 minutes, take precipitating, complete medium are added, blow and beat into cell suspension. Take the cell suspensions drop of 10 microlitres of mixings in carrying out cell count in cell counting board.Cell density is adjusted using complete medium It is 4~5 × 10⁶/ ml is seeded in the culture plate that poly-D-lysine was coated with, and inoculum density is 4~5 × 10⁵/cm², added Full culture medium is to minimum volume of culture.The cell of inoculation is placed in 37 degrees Celsius of incubators and is cultivated.Microscope after 6~8 hours After lower observation cell is adherent, replacement cell culture fluid is neuronal culture (Neurobasal A+B27+L- glutamine).With 2~3 days half amounts change liquid afterwards, cultivate to one week or so, after carrying out neuronal specificity immunofluorescence dyeing assay approval, then carry out Subsequent experimental.

2.10 immunohistochemical staining

(1) 6 μ m-thick of paraffin section, gradient dewaxing, dehydration of alcohol.

(2) 3%H2O2, room temperature 10 minutes, 0.1MPBS flushing 5 minutes, in triplicate.

(3) dropwise addition 5%BSA confining liquid, room temperature 20 minutes.

(4) diluted 4 DEG C of primary antibody Spy1 (1:500) overnight incubations are added dropwise.0.1MPBS flushing 5 minutes, in triplicate.

(5) biotinylation is added dropwise and marks secondary antibody, 37 DEG C are incubated for 20 minutes.0.1MPBS flushing 5 minutes, in triplicate.

(6) horseradish enzyme is added dropwise and marks strepto- avidin, 37 DEG C are incubated for 20 minutes.0.1MPBS flushing 5 minutes, in triplicate.

(7) DAB color developing agent color development at room temperature is used.

(8) haematoxylin is redyed, mounting.

(9) microscopically observation acquires image.

2.11 immunohistofluorescence's double staining

(1) 6 μ m-thick of paraffin section, gradient dewaxing, dehydration of alcohol.

(2) 3%H2O2, room temperature 10 minutes, 0.1MPBS flushing 5 minutes, in triplicate.

(3) dropwise addition 5%BSA confining liquid, room temperature 10 minutes.

(4) primary antibody is added to be incubated for, it is anti-using primary antibody diluted rabbit-anti Spy1 antibody (1:200) and the anti-MAP2 of goat Body (1:200) is added on sample after mixing.4 DEG C overnight.

(5) slice is rinsed 10 minutes with 0.1MPBS, in triplicate, fluorescent marker secondary antibody, Alexa is added 488 Mark anti-rabbit IgG (1:200), Alexa594 labels anti goat igg (1:200), mix, and are incubated at room temperature 2 hours.

(6) slice is rinsed 10 minutes with 0.1MPBS, in triplicate, uses anti-fluorescent quenching mountant mounting.

(7) microscopically observation acquires image.

2.12 cellular immunofluorescence detects

(1) corresponding growth time point cell is taken, is washed three times with 0.1M PBS, 10 minutes every time.

(2) 4% paraformaldehyde room temperature fixes 30 minutes.

(3) it is washed three times with 0.1M PBS, 10 minutes every time.

(4) it uses 0.1%Triton X-100 permeable membrane 2 minutes.

(5) it is washed three times with 0.1M PBS, 10 minutes every time.

(6) 5%BSA room temperature is closed 30 minutes.

(7) add corresponding primary antibody, 4 DEG C overnight.

(8) it is washed three times with 0.1M PBS, 10 minutes every time.

(9) add corresponding fluorescence secondary antibody, close 30 minutes.

(10) it is washed three times with 0.1M PBS, 10 minutes every time.

(11) it is dyed 3 minutes with DAPI.

(12) it is washed three times with 0.1M PBS, 10 minutes every time.

(13) microscopically observation acquires image.

2.13 enzyme-linked immunosorbent assay detect 8-OhdG (8-OHdG)

2.13.1 reagent prepares

(1) using preceding that all kit samples are slowly balanced to room temperature

(2) this preparation of standard: being added standard dilutions 1ml for every bottle of standard items, is stored at room temperature 10 minutes after covering, together When gently shake and (avoid blistering), concentration 6000pg/ml, prepare 5 dilution standard product EP pipe, each EP Guan Zhongjia Enter the standard dilutions of 600 μ L, successively three times are diluted to 6000pg/ml, 2000pg/ml, 666.67pg/ml, 222.22pg/ml, 74.07pg/ml.Standard dilutions (0pg/ml) are directly as blank well.

(3) detect solution A and detection solution B: detection solution A and detection solution B before use, centrifugal treating when few, with Make the liquid deposition of tube wall or bottle cap to tube bottom.It is filled respectively with detecting diluent A or B with the dilution proportion of 1:100 before use Divide and mixes.

(4) dense cleaning solution: the dense cleaning solution of 20ml is diluted to 600ml with 580ml distilled water or deionized water, carries out 30 times Dilution.

(5) substrate solution: the tmb substrate of volume needed for being drawn with the suction pipette head of sterilizing makes into another clean solution With.

2.13.2 operating procedure

(1) it is loaded: setting gauge orifice, sample to be tested hole, blank well respectively.If 5 hole of gauge orifice, it is different that 50 μ L are added with this The standard items of concentration.Blank well adds 50 μ L standard dilutions, and 50 μ L of Yu Kongjia sample to be tested, every hole adds detection molten immediately after 50 μ L of liquid A working solution, gently vibrates, and mixes, pays attention to that bubble is avoided to generate, and ELISA Plate adds overlay film, and 37 DEG C incubate 1 hour.

(2) liquid in hole is discarded, every hole is washed with the cleaning solution of 300 μ L, impregnates 1~2 minute, enzyme is patted on blotting paper Target removes all liq in hole.It repeats board-washing 3 times, after last time is washed, draws or pour out remaining washing buffer, ELISA Plate is tipped upside down on blotting paper, the liquid remained in hole is all blotted.

(3) every hole adds detection 100 μ L of solution B working solution, in addition overlay film, 37 DEG C are incubated 30 minutes.

(4) liquid in hole is discarded, is dried, board-washing 5 times.

(5) every hole adds 90 μ L of substrate solution, and ELISA Plate adds overlay film, and 37 DEG C are protected from light colour developing, and the reaction time 20 minutes or so, There is an apparent gradient blue in 3 holes behind gauge orifice, when preceding 3 gradient pores are unobvious, can terminate.

(6) every hole adds 50 μ L of stop bath, terminates reaction, blue switchs to yellow at this time.The addition sequence of terminate liquid should use up It measures identical as the addition sequence of substrate solution.

(7) after ensuring ELISA Plate bottom without bubble-free in water droplet and hole, immediately with microplate reader in each hole of 450nm wavelength measurement Optical density (OD value).

2.14 qRT-PCR is detected

2.14.1 NSC34 cell and ALS mouse spinal cord total tissue RNA are extracted

(1) cell in six orifice plates is cleaned one time using PBS, abandons PBS, then 1ml Trizol, piping and druming is added in every hole It after cracking uniformly, is added in 1.5ml EP pipe, vibrates 30 seconds.Tissue homogenate: the mortar after taking out autoclave sterilization is cut 50mg~100mg frozen tissue is placed in mortar, pours into liquid nitrogen, is ground.It is added in every 50mg~100mg homogenised tissue sample The above homogenate sample is transferred in 1.5ml EP pipe, places 5 minutes at room temperature by 1ml Trizol.

(2) 200 microlitres of chloroforms are added in every pipe, after overturning oscillation mixing, are placed in and are stored at room temperature 3 minutes, at 4 DEG C, 13000rpm is centrifuged 15 minutes.

(3) upper layer colourless aqueous phase is drawn, (about 400 microlitres) are moved into another EP pipe.

(4) it is added in new EP pipe 400 microlitres of isopropanols (supernatant liquid drawn with previous step is isometric), -20 It DEG C places 30 minutes or more.

(5) 13000rpm at 4 DEG C is centrifuged 15 minutes.

(6) liquid is outwelled, the 75% ethyl alcohol 1ml prepared in advance using DEPC water is added, carries out washing precipitating, overturns vibration It swings.

(7) 10000rpm at 4 DEG C is centrifuged 10 minutes.

(8) liquid is outwelled, by the back-off of EP pipe on filter paper, draws raffinate, in being air-dried at room temperature about 10 minutes.

(9) 50 microlitres of DEPC water dissolution precipitatings are added, leaves and takes appropriate amount of fluid and carries out survey concentration, integrality identification.

(10) after dispensing RNA, -80 DEG C of preservations are put in.

RNA concentration calculation (μ g/ml)=[OD260-OD310] × 40 × extension rate

The detection of RNA integrality: denaturing formaldehyde agarose electrophoresis determines extract RNA integrality and DNA pollution situation.

2.14.2 qRT-PCR is detected

RNA reverse transcription synthetic DNA (Qrt-PCR one-step method)

In advance by test needed for reagent thaw on ice, storeed instrument and superclean bench, in advance open PCR to preheat, It is formulated as follows the reaction system of table 1:

Table 1

The reaction system prepared is added in eight connecting leg of PCR, lightly after of short duration centrifugation, is put into Roche quantitative PCR instrument Middle progress reverse transcription and amplified reaction.

Response procedures are as follows:

Hold

42 DEG C 5 minutes

95 DEG C 10 seconds

Pattern2: quantitative reaction

Cycle:40

95 DEG C 5 minutes

60 DEG C 30 seconds

Pattern3: dissociation

Amplification curve is recorded after reaction, reads CT value.

2.15 Western blot detection

2.15.1 the extraction of monolayer adherence total protein of cell

Culture solution in culture bottle is outwelled, cell is cleaned three times using the PBS of pre-cooling, discards PBS.Rapidly will on ice Attached cell, which scrapes, to be collected into 1.5ml EP pipe, of short duration centrifugation, discards PBS, and every bottle of cell is added 100 μ l and contains PMSF's Western and IP lysate, piping and druming uniformly, are cracked 30 minutes on ice, were vortexed 10 seconds every 10 minutes.Cell cracking is filled After point, the 13000rpm at 4 DEG C, 20 minutes.Supernatant is taken, is total protein.2 μ l detectable concentrations are taken, all samples are adjusted to phase Same concentration is added 5 × loading buffer of four times of volumes, boils, packing.It is put into -80 DEG C of refrigerators to save, avoids freezing repeatedly Melt.

2.15.2 SDS-PAGE electrophoresis

(1) after being aligned glass plate, it is put into clamping in folder, prepares encapsulating

(2) prepare 10% separation gel, be added after TEMED after mixing immediately can encapsulating, then on glue plus one layer of water, liquid It is honored as a queen, it is solid to accelerate gelling.When forming a defiber between Dang Shui and glue, water is blotted with blotting paper.

(3) the concentration glue for preparing 5%, mixes encapsulating after TEMED is added immediately, and comb is inserted into concentration glue.

(4) prepare loading after electrophoresis liquid being added.

(5) electrophoresis to bromophenol blue to glue bottom terminates electrophoresis, carries out transferring film.

2.15.3 transferring film

(1) 6 filter paper and 1 nitrocellulose filter are impregnated 10 minutes in transfer liquid in advance.

(2) three layers of filter paper, a nitrocellulose filter, gel, three layers of filter paper sandwich.It is removed using glass bar In bubble.

(3) sandwich product is put into transfer instrument, film is in anode, and glue is in cathode.Under the conditions of electric current 50mA, transferring film 1.5 hour.

(4) nitrocellulose filter transferred is unloaded, whether observation Marker is transferred on film from glue completely, is placed in Room temperature rocks incubation 1 hour in confining liquid.

After nitrocellulose filter is taken out in confining liquid, after TBST cleaning three times, 10 minutes every time.

Nitrocellulose filter is placed in diluted primary antibody antibody-solutions, 4 DEG C overnight.A specific antiantibody is pressed in advance Be diluted according to ratio: Spy1 (1:500), CDK2 (1:400), ATM (1:1000), p-ATM (1:1000), ATR (1:500), p-ATR(1:500)、Chk1(1:500)、p-Chk1(1:500)、Chk2(1:1000)、 p-Chk2(1:1000)、p53(1: 500)、SOD1(1:200)、β-actin(1:1000)。

After nitrocellulose filter is taken out, on room temperature shaker three times using TBST cleaning, 10 minutes every time, by washed film It is placed in diluted fluorescence two corresponding anti-solution and (is prepared according to 1:4000), 37 DEG C of shaking tables are incubated for 1 hour.

After film is washed film using TBST, sweeps film and obtain band image.

2.16 co-immunoprecipitation experiment

(1) well-grown mSOD1 cell culture to degrees of fusion is reached 70% or so, instantaneously turned in mSOD1 cell Spy1 plasmid is contaminated, after continuing culture 48 hours.Culture solution is abandoned, three times using pre-cooling PBS rinsing, abandons net PBS.

(2) the IP cell pyrolysis liquid of pre-cooling is added, cracks 5 minutes, is scraped cell from culture dish on ice using cell scraper Get off, moves in EP pipe.

(3) continue to slowly shake 30 minutes on ice, crack it sufficiently.It is centrifuged 10 minutes at 4 DEG C with 13000rpm, It takes supernatant to be placed in new EP pipe, is used for co-immunoprecipitation experiment.

(4) prepare Protein A/G Agarose, wash twice of pearl using PBS, being then configured to concentration using PBS is 50% mixed liquor.

(5) the Protein A/G Agarose of 100 μ l is added in every 1ml total protein, is slowly rocked 4 hours in 4 DEG C, with Non-specific foreign protein is removed, background is reduced.

(6) in 4 DEG C, 12000rpm is centrifuged 15 minutes, supernatant is transferred in new EP pipe, is discarded precipitating.

(7) protein concentration is measured using BCA method, total protein is then diluted to 1mg/ml using PBS, it is anti-that Spy1 is added Body and Protein A/G Agarose do immunoprecipitation, and CDK2 carries out Western Blot detection.It is added simultaneously first with antibody The IgG in source is linked up as control.Antibody antigen mixture is slowly shaken, overnight in 4 DEG C.

(8) 1ml IP cell pyrolysis liquid is added, repeats five times with 300rpm centrifugation 5 minutes in 4 DEG C.

(9) suitable 5 × sample-loading buffer is taken to hang Protein A/G Agarose-antigen antibody complex, gently It mixes.

(10) 100 DEG C of loading sample are boiled 10 minutes, with dissociate Protein A/G Agarose, antibody, antigen, 13000rpm is centrifuged 5 minutes, and supernatant is taken to carry out electrophoresis.

The breeding and identification of 2.17 ALS transgenic models mouse

2.17.1 the breeding breeding and raising of ALS transgenic models mouse

It is bred according to Propogation and culture specified in model mouse specification, the C57BL/6J hSODG93A of purchase is turned Gene masculine semizygote hero mouse mates with C57BL/6J feminine gender female mice.Mouse is raised in the sterilizing incubator that constant temperature and humidity is ventilated It supports, it is small that a C57BL/6J hSODG93A transgene-positive hemizygous hero mouse with two C57BL/6J feminine gender female mices is mated 48 When more than, female mice is taken out into Dan Fangyi cage, whether observation female mice is pregnant, and after positive hero mouse sky cage, continues next round and mates Mating.When newborn mice to four week old, extracts its DNA and carry out identified for genes, and every mouse marks.

2.17.2 ALS transgenic models mouse progeny genotypes are identified

Extracting genome DNA (carries out) according to Tiangeng genome DNA extracting reagent kit specification

(1) the tail point tissue of 30 days clip 0.5cm, is placed in EP pipe after mouse is born, it is thoroughly crushed, at room temperature 12000rpm is centrifuged 1 minute abandoning supernatant, and 200 μ l buffer GA are added, and sufficiently oscillation mixes.

(2) 20 μ l Proteinase Ks are added, mix well.60 DEG C of water-baths or it is dry set, until histocyte suspension is completely dissolved, If EP inside pipe wall has droplet that should carry out brief centrifugation.

(3) the GB buffer of 200 μ l is added, is sufficiently mixed by inversion and acutely oscillation for a moment, is set 10 minutes in 75 DEG C of heat, Solution appearance is answered clear at this time, and the droplet of EP inside pipe wall is removed with of short duration centrifugation.

(4) it takes 100% ethyl alcohol, 200 μ l to be added, is sufficiently mixed by inversion, and acutely oscillation a moment, if cap wall has droplet It should carry out brief centrifugation.

(5) adsorption column CB3 is put into collecting pipe, gained mixed liquor is all sucked out and is added in adsorption column, room temperature 13000rpm is centrifuged 1 minute, takes out waste liquid in CB3 reject pipe, then CB3 is put back to.

(6) the GD buffer of 500 μ l is added dropwise into adsorption column CB3, room temperature 13000rpm is centrifuged 60 seconds, takes out CB3 reject Waste liquid in pipe, then CB3 is put back to.

(7) the PW rinsing liquid of 600 μ l is added dropwise into adsorption column CB3, room temperature 13000rpm is centrifuged 60 seconds, takes out CB3 reject Waste liquid in pipe, then CB3 is put back to.

(8) last action is repeated.

(9) it is centrifuged again, room temperature 13000rpm is centrifuged 2 minutes, takes out waste liquid in CB3 reject pipe.CB3 room temperature is put Several minutes are set to thoroughly drying.

(10) adsorption column CB3 is put into clean 1.5ml EP pipe, the TE elution of 100 μ l is added, stands about 5 at room temperature Minute, with dissolving DNA, then room temperature 13000rpm is centrifuged 2 minutes, and solution is collected into EP pipe.

(11) gained DNA solution is dispensed into freezen protective, and leaves and takes 5 μ l detection concentration of specimens.

PCR reaction

(1) on ice, by following reagents, according to table 2, proportion carries out preparing PCR reaction system accordingly；

Table 2

(2) PCR reaction condition is set:

95 DEG C initial denaturation 5 minutes, 95 DEG C be denaturalized 45 seconds, 58 DEG C anneal 30 seconds, 72 DEG C extension, 30 circulation, 72 DEG C extension 5 minutes.

(3) PCR instrument will be put into prepared reaction system addition PCR tubule to carry out amplification reaction.

PCR product identification

(1) Ago-Gel is prepared, the tbe buffer liquid (0.5 ×) of 20ml is added in 0.4g agarose, is added in micro-wave oven and adds Heat about 5 minutes to being completely melt.

(2) final concentration of 2% Ago-Gel liquid is taken out into cooling a moment from micro-wave oven.

(3) it picks a small amount of Ethidium Bromide (0.5mg/ml) with pipette tips to be immersed in gel liquid, oscillation shakes up.

(4) gel mixed liquor is poured into stick mixing tank, is plugged comb, be cooled to room temperature solidification.

(5) sample comb is extracted, gel is removed and is put into Horizontal electrophoresis tank, immerse in electrophoresis liquid.

(6) 10 μ l of loading carries out electrophoresis, and 120V electrophoresis to band is located at the 2/3 of gel, and it is full-automatic solidifying to take out glue merging The scanning of glue Image analysis system.

2.18 statistical analysis

All data are all made of mean ± standard deviation (± SD), are counted using software systems.It adopts cell experiment part With the variance analysis of single factor test, compares two-by-two and statistical procedures are carried out to corresponding data, compare and use between Animal Experimental grouping ANOVA analysis and independent samples t test.Think with statistical significant difference P < 0.05.

2.19 synthesis PDMAEMA derivatives

PEG-PDMAEMA and Mal-PEG-PDMAEMA (MPEG-PDMAEMA) diblock copolymer are according to document before Synthesis (Y.Qian, Y.Zha, B.Feng, Z.Pang, B.Zhang, X.Sun, J.Ren, C. Zhang, X.Shao, Q.Zhang, X.Jiang,PEGylated poly(2-(dimethylamino)ethyl methacrylate)/DNA polyplex micelles decorated with phage-displayed TGN peptide for brain-targeted gene delivery,Biomaterials.34(2013)2117-2129.).Mal-PEG-PDMAEMA carrier and sulfydryl CGN peptide (d- CGNHPHLAKYNGT) or Tet1 peptide (HLNILSTLWKYR) (PBS, pH7.0) in phosphate buffer presses 1:1 (mol/mol) Ratio and effect 4 hours at room temperature.Reaction solution is dialysed with distilled water (MWCO5ka), obtains CGN-PEG- after freeze-drying PDMAEMA and Tet1-PEG-PDMAEMA product.

2.20 preparing polymer nano granules

PDMAEMA derivative, including MPEG-PDMAEMA (M), CGN-PEG-PDMAEMA (C), Tet1-PEG- PDMAEMA (CT, 1:1, w/w) is dissolved respectively in DEPC water, then from Spy1 in different N/P ratios (1:1,2:1,5:1 or It 10:1) mixes, is first vortexed 30 seconds, then cultivate obtain after twenty minutes at room temperature.The granular size of polymer nano granules and Zeta current potential application dynamic light scattering technique (DLS) is measured in N/P=10.The form application projection electricity of nano gene complex Sub- microscope (TEM) is observed.TEM preparation of samples is by drawing a drop complex solution in the grid copper of a standard On grid and spontaneously dry it in air.These grids are with phosphotungstic acid (2%, w/v) negative staining and dry before detection.Macromolecule The ability of polymer concentration Spy1 is assessed by agarose gel electrophoresis, and is detected with ultra violet illumination in 302nm and used fluorine Chemical imaging system is taken pictures.

3, experimental result

3.1 Spy1 are in hSOD1G93A transgenosis ALS mouse model motor neuron and hSOD1G93A stable transfection Expression quantity reduces in neuronal cell model

In order to study Spy1 expression in ALS animal model and cell model, we select hSOD1G93A to turn DNA murine is ALS animal model, and hSOD1G93A stable transfection NSC34 cell line is ALS cell model, passes through immuning tissue The methods of the double dyes of chemical staining, immunofluorescence, Western Blotting and quantitative fluorescent PCR detection Spy1 expression. Firstly, identifying hSOD1G93A positive transgenic mouse by PCR method, hSOD1G93A transgenic mice will be carried and trained It supports, the same time is brood wild type negative mice observes mouse growth developmental state, scores its function as control group, After 130 day age of mouse, general anaesthesia mouse is fixed, saline infusions, and paraformaldehyde perfusion fixation takes mouse ridge Myeloid tissue is fixed.Specimens paraffin embedding slices carry out immunohistochemical staining, as a result as shown in Figure 1, as the result is shown in the terminal phase In the ventricornu of hSOD1G93A transgenic mice, Spy1 is in sepia in neuron plasma, with wild type negative mice phase Compare, Spy1 positive staining significantly reduces in hSOD1G93A transgenic mice Motoneurons.In order to further clarify In single motor neuron, Spy1 expression, using Image Pro Plus6.0 software to sun in single neural neuron Property dyeing analyzed, with integral optical density value (intergrated optical density, IOD) as measure Spy1 table Index up to intensity carries out statistical analysis.This is the results show that the ventricornu in hSOD1G93A transgenic mice moves mind Through in member, Spy1 expression quantity is significantly reduced compared with wild-type mice.In order to further clarify Spy1 in hSOD1G93A transgenic mice Expression in anterior horn motor neurons marks neuron using MAP-2 dyeing by the double dyeing methods of immunofluorescence Note takes on a red color fluorescence, and Spy1 positive staining is in green fluorescence, Spy1 positive staining mainly in dynamoneure endochylema, In hSOD1G93A transgenic mice Motoneurons Spy1 positive staining (i.e. green fluorescence) intensity significantly lower than control Group fluorescence intensity (Figure 1A).Using western blotting and real-time fluorescence quantitative PCR (qRT-PCR) method, detection Spy1 expression in hSOD1G93A transgenic mice myeloid tissue.Compare from protein level, hSOD1G93A transgenosis is small Spy1 protein content is significantly lower than wild type negative mice in mouse, has statistical significance (Fig. 1 C, D).It is carried out from mRNA level in-site Compare, hSOD1G93A transgenic mice myeloid tissue Spy1 mRNA expression significantly reduces, and has system compared with the control group Meter learns meaning (Figure 1B).

In order to further confirm, the cortical neuron that we extract tire mouse carries out originally culture, while carrying out base to tire mouse Because type is identified.When primary neuronal culture was to the 7th day, neuron purity is identified using MAP-2 fluorescent staining first, is then carried out Subsequent experimental.Using immunofluorescence dyeing method, in the cortical neuron of hSOD1G93A transgenic positive mouse, Spy1 green Fluorescent staining intensity significantly reduces compared with wild type negative mice.Finally we use hSOD1G93A-NSC34 cell line again It is verified, Spy1 expression is compared by immunofluorescence dyeing, western blotting and qRT-PCR method.Knot Fruit as shown in Fig. 2, by immunofluorescence dyeing as it can be seen that compared with NSC34 cell line, pLV cell line, wtSOD1 cell line, Spy1 fluorescence intensity obviously weakens (Fig. 2A) in mSOD1 cell line.Compare from protein level, Spy1 albumen in mSOD1 cell line Content is significantly lower than control group, and has statistical significance (Fig. 2 C, D).Compare from mRNA level in-site, Spy1 mRNA exists MSOD1 cell line expression is significantly lower than control group, and has statistical significance (Fig. 2 B).

There are core DNA damage and DNA damages in 3.2 hSOD1G93A stable transfection NSC34 cell line ALS cell models Reaction is in state of activation

There are DNA damages in previous research discovery ALS pathologic process, it may be possible to due to superoxide dismutase -1 Caused by (superoxide dismutase-1, SOD1) dysfunction, in order to further inquire into hSOD1G93A in DNA damage Effect detects the content of 8-OHdG and DNA damage in hSOD1G93A stable transfection NSC34 cell line and reacts access correlation egg White expression.In hSOD1G93A transgenosis ALS mouse model, there are oxidative stress DNA damage, 8- for previous research discovery OHdG is that researching DNA damages most sensitive one of index.Research finds 8-OHdG in hSOD1G93A transgenosis ALS mouse mould Type myeloid tissue is compared with wild type negative mice in high expression.Therefore, this research is seen first by immunofluorescence dyeing method The expression for examining 8-OHdG in hSOD1G93A stable transfection NSC34 cell line, in mSOD1 cell line, 8-OHdG is positive Dyeing quantity it is more and concentrate be distributed in around nucleus, be in high fluorescent, and NSC34 cell line, pLV cell line, It is in low fluorescence intensity in wtSOD1 cell line.Increase to further clarify 8-OHdG in mSOD1 cell line content, passes through enzyme Linked immunosorbent adsorption test method detects 8-OHdG contents level in four kinds of cell line cell lysates.As a result as shown in figure 3, result is aobvious Show, in four kinds of cell lines, mSOD1 cell line 8-OHdG concentration obviously increases compared with control group, and has statistical significance.In synthesis Two kinds of experimental results are stated, there are core DNA oxidations in hSOD1G93A stable transfection NSC34 cell line ALS neuron models for prompt Stress damage.Extract the change situation that cell protein carries out Western Blotting detection DNA damage reaction pathway protein.Knot Fruit shows, compared with NSC34 cell, pLV cell, wtSOD1 cell, phosphorylated protein p-ATM, p-ATR in mSOD1 cell line, P-Chk1, p-Chk2 and p53 expression quantity obviously increase, and have statistical significance, rather than phosphorylated protein ATM, ATR, Chk2, Chk1 are without significant change (Fig. 3 A, B).Compare from mRNA level in-site, qRT-PCR shows p53 mRNA level in-site in mSOD1 Group increased, and have statistical significance (Fig. 3 C).Quantitative analysis shows that 8-OHdG increases (Fig. 3 D) in mSOD1 group level, Immunofluorescence dyeing shows that 8-OHdG (red) fluorescence intensity increases (Fig. 3 E).Therefore in hSOD1G93A stable transfection NSC34 cell It is in ALS cell model, DNA damage reaction is active.

3.3 knockout Spy1 lead to the reduction of mSOD1 cell vigor, and further DNA damage can be activated to react, and promote to wither It dies

In order to study the effect between Spy1 and neuronal activity, the RNA interference sequence that specificity is directed to Spy1 is chosen SiRNA-Spy1 is transfected by Lipofectamine2000 transfection reagent, reduces the expression of Spy1 in mSOD1 cell, The expression for detecting GAP-associated protein GAP in its cell activity and DNA damage approach changes.By mSOD1 cell in contain 10% tire ox It is cultivated in the DMEM complete medium of serum, cell is divided into three groups, respectively mSOD1 groups of cells (Control group), feminine gender RNA interference group (si-NC group) and specificity Spy1 interference group (si-Spy1 group), when cell fusion is to 60%~70%, Cell is transiently transfected using Lipofectamine2000, after transfection 48 hours, cell is collected and carries out, use Annexin-PE/7-AAD apoptosis kit detects apoptosis, flow cytometry analysis.As a result as shown in figure 4, the results show that The generation that Spy1 significantly facilitates apoptosis is knocked out, based on early apoptosis.By statistical analysis, wither in si-Spy1 group cell Dying rate is 77.70%, and in si-NC group, apoptosis rate 4.27% has apparent statistical difference (Fig. 4 D, E).In order into The viability that one step detects Spy1 knockout influence cell is related with DNA damage.After being transfected 48 hours in mSOD1 cell, collect Cell extraction RNA and cell protein carry out subsequent experimental, pass through qRT-PCR and Western bloting method, detection Correlative protein expression is horizontal in Spy1 and DNA damage approach.The result shows that after transfection Spy1 siRNA interference sequence, In mSOD1 cell, Spy1 mRNA expression is significantly reduced, and p53 mRNA expression obviously increases (Fig. 4 C).DNA damage In approach, phosphorylated protein p-ATM, p-ATR, p-Chk1, p-Chk2 and p53 expression quantity is obviously increased, and non-phosphorylating egg White ATM, ATR, Chk2, Chk1 are without significant change (Fig. 4 A, B).Therefore, the laggard one-step activation DNA damage of interference Spy1 is anti- It answers.

3.4, which are overexpressed Spy1 in mSOD1 cell, enhances cell viability, inhibits DNA damage reaction

By reducing Spy1 in the expression of mSOD1 cell, cell activity decline, DNA damage reaction is further to be activated, Promote apoptosis.So, it by being overexpressed the expression of exogenous Spy1, is overexpressed using MTT method detection Spy1 Influence to mSOD1 cell activity, and the detection of application Western blotting method is to the GAP-associated protein GAP of DNA damage approach Expression, pass through qRT-PCR detect Spy1 and p53 expression variation.MCherry-Spy1-pEZ-M95 plasmid wink When transfection transfected using Lipofectamine2000 reagent.After culture 48 hours, carries out fluorescence microscope detection and clearly turn Contaminate efficiency.As a result as shown in figure 5, compared with control group (NSC34 cell, pLV cell, wtSOD1 cell), the work of mSOD1 cell Property be substantially reduced, by transiently transfect Spy1 plasmid after, in the mSOD1 cell of transfection Spy1 be in high expression status, using MTT Method detection reduces as it can be seen that being overexpressed Spy1 and can partially reverse since hSOD1G93A transfects caused cell activity.It is overexpressed Cell is collected after 48 hours, extracts its albumen, using the detection of Western blotting method in DNA damage approach, phosphoric acid The expression for changing albumen p-ATM, p-ATR, p-Chk1, p-Chk2 is decreased obviously compared with the control group, and has statistics meaning Adopted (Fig. 5 A), rather than phosphorylated protein ATM, ATR, Chk1, Chk2 expression is without significant change, and p53 albumen, mRNA Level significantly reduces (Fig. 5 B, C, E).Mtt assay detects cell activity, and the SOD1 of G93A mutation leads to the activity drop of NSC34 cell It is low, and such case can partially be over-expressed Spy1 and reverse (Fig. 5 D).In the past the study found that Spy1 promotes cell Proliferation to make With, and when participating in DNA damage effect, be and activate the activity of CDK2 and play a role by conjunction with CDK2, and In the inhibition apoptotic process that Spy1 is mediated, Spy1 and CDK2 form Spy1/CDK2 compound.So in ALS cell model In, whether Spy1 interacts with CDK2 and plays and inhibit apoptotic effect.It is detected by co-immunoprecipitation method, is existed as the result is shown In the mSOD1 cell of mSOD1 cell and transient transfection Spy1 plasmid, Spy1 with CDK2 co-precipitation, then illustrates In ALS mSOD1 cell model, Spy1 is to interact with CDK2 and play and inhibit apoptotic effect.

3.5 are overexpressed hSOD1G93A gene in NSC34 cell, inhibit the expression of Spy1

In previous experiments as it can be seen that compared with NSC34 cell, pLV cell, wtSOD1 cell, the table of Spy1 in mSOD1 cell It is reduced up to level, is the expression for inhibiting Spy1 gene after hSOD1G93A gene due to being overexpressed to further clarify, NSC34 cell gradient transiently transfects the plasmid containing hSOD1G93A, by EGFP-SOD1G93A-pcDNA3.1 plasmid with every hole 0 μ g, 1 μ g, 2 μ g, 3 μ g, the concentration of 4 μ g are transfected using Lipofectamine2000, and it is aobvious to carry out fluorescence after transfection 48 hours Micro mirror detects its transfection efficiency.Cell is collected, cell protein and mRNA are lifted.The result shows that with mutation is continuously increased After hSOD1G93A gene, the mRNA and protein expression level of Spy1 constantly reduces (Fig. 6 A, C, D, E), and is examined by MTT method Cell activity is surveyed as it can be seen that cell activity reduces (Fig. 6 B) as transfection hSOD1G93A mrna concentration increases.Therefore, clearly turn Dye hSOD1G93A gene can reduce the expression (Fig. 6) of Spy1.

On the basis of the above embodiments, by the Spy1 polymer nano granules prepared by way of tail vein injection In (80 μ g/ are only) injection SOD1G93A ALS Mice Body, injection in every five days is primary, and continuous four times, in last time injection two days Afterwards to SOD1G93A ALS mouse and control group mice carry out disease time, life cycle observation and Behaviors survey (tail-suspention test, Weight and turn-club test) (Wang P, Zheng X, Guo Q, Yang P, Pang X, Qian K, Lu W, Zhang Q, Jiang X.Systemic delivery of BACE1 siRNA through neuron-targeted nanocomplexes for Treatment of Alzheimer's disease.J Control Release. 279 (2018): 220-233.), upper On the basis of the experimental data for stating embodiment, it is believed that these three polymer nano granules can play the hair for postponing ALS mouse The sick time extends life cycle and improves the effect of behavioral function.

The above is only the preferred embodiment of the present invention, it is noted that for those skilled in the art, Without departing from the invention herein, several improvement and supplement can also be made, these improve and supplement also should be regarded as it is of the invention Protection scope.

Sequence table

<110>Harbin Medical University

<120>purposes of Spy1 gene and its expression albumen in treatment amyotrophic lateral sclerosis

<130> KLPI181067

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 933

<212> DNA

<213> human

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Claims (10)

1. the purposes of cell cycle-regulatory gene Spy1 and its expression albumen in preparation treatment amyotrophic lateral sclerosis drug.

2. purposes as described in claim 1, which is characterized in that the drug is by inhibiting DNA damage response pathway and increasing Add the survival of neuron, caused amyotrophic lateral sclerosis is mutated by SOD1G93A for improving.

3. purposes as described in claim 1, which is characterized in that the drug is to pass through nano-carrier combination Spy1 gene system Standby obtained nano particle, gel or emulsion, the nano particle is including but not limited to polymer nano granules, lipid nanometer Grain, carbon nano-particle, metal nanoparticle.

4. cell cycle-regulatory gene Spy1 and its synergist for expressing albumen are treated in amyotrophic lateral sclerosis drug in preparation Purposes.

5. purposes as claimed in claim 4, which is characterized in that the drug is by inhibiting DNA damage response pathway and increasing Add the survival of neuron, caused amyotrophic lateral sclerosis is mutated by SOD1G93A for improving.

6. purposes as claimed in claim 4, which is characterized in that the synergist be cell cycle-regulatory gene Spy1 and its Express agonist, upper adjustment or the stabilizer of albumen.

7. purposes as claimed in claim 4, which is characterized in that the synergist, which refers to, any can be improved Spy1 gene or egg The white stability of activity, raising Spy1 gene or albumen, the expression of up-regulation Spy1 gene or albumen, increase Spy1 gene or egg The substance of white effective acting time, the synergist are compound, chemical small molecule or biomolecule.

8. purposes as claimed in claim 7, which is characterized in that the biomolecule can be DNA or RNA, or regulation The viral product of Spy1 gene or protein expression.

9. a kind of for treating the polymer nano granules of amyotrophic lateral sclerosis, which is characterized in that be prepared by the following method It obtains:

(1) diblock copolymer Mal-PEG-PDMAEMA is synthesized；

(2) by the diblock copolymer Mal-PEG-PDMAEMA of synthesis and sulfydryl CGN peptide or Tet1 peptide in phosphate buffer The ratio of 1:1 and effect 4 hours at room temperature in molar ratio, reaction solution is dialysed with the bag filter of MWCO 5ka, and dialyzate is Distilled water obtains CGN-PEG-PDMAEMA and Tet1-PEG-PDMAEMA product after freeze-drying；

(3) CGN-PEG-PDMAEMA and Tet1-PEG-PDMAEMA is dissolved respectively in DEPC water, then with Spy1 gene It mixes, is vortexed 30 seconds in the ratio that N/P is 1-10:1, then cultivate obtain the polymer nanocomposite after twenty minutes at room temperature Grain.

10. purposes of the polymer nano granules as claimed in claim 9 in preparation treatment amyotrophic lateral sclerosis drug, In, it is preferred that the polymer nano granules are used for by inhibiting DNA damage response pathway and increasing the survival of neuron Improve amyotrophic lateral sclerosis caused by being mutated by SOD1G93A.