CN103224956A - Type-II adeno-associated virus carrying neuritin genes and application thereof in restoring optic nerve injuries - Google Patents

Abstract

The invention belongs to the technical field of gene engineering and nerve injuries. Optic nerve injuries are common eye diseases and external injuries, and cause the optic nerve atrophia and blindness of patients when the optic nerve injuries are severe, and at present, the clinic actual curative effect is unsatisfactory. The invention provides a type-II adeno-associated virus carrying neuritin genes and a building method thereof, the invention also further provides the application of the type-II adeno-associated virus carrying neuritin genes in preparing medicaments for restoring optic nerve injuries.

Description

A kind of II type adeno-associated virus and application in repairing optic nerve injury thereof of carrying the Neuritin gene

Technical field

The invention belongs to genetically engineered and nerve injury technical field, be specifically related to a kind of II type adeno-associated virus and application in repairing optic nerve injury thereof of the Neuritin of carrying gene.

Background technology

Optic nerve injury is many eye diseases and wound, as glaucoma, optic nerve ischemia, contusion injuries, fracture of optic canal etc., causes visual function infringement common effect link, and the atrophy that can cause patient's optic nerve when serious is with blind.Although a large amount of research work have been carried out in the treatment of optic nerve injury, its clinical practice curative effect is unsatisfactory, is difficult to allow the patient recover eyesight according to present medical technique level.Retinal ganglial cells (retinal ganglion cells, RGCs) inherent regenerative power is low, be that regeneration failure causes optic atrophy and blind key factor behind the optic nerve injury, how carry out effective reversal therapies and be always one of difficult problem that ophthalmic industry faces.

Recent studies show that, find target spot accurately and strengthen the inherent regenerative power of impaired RGC, regeneration that can promote that optic nerve is long-term, enlivens and arrival superior colliculus and lateral genicular body are (referring to document [1] Sun F, Park KK, Belin S, et al.Sustained axon regeneration induced by co-deletion of PTEN and SOCS3.Nature.2011; 480 (7377): 372-5.; [2] Kurimoto T, Yin Y, Omura K, et al.Long-distance axon regeneration in the mature optic nerve:contributions of oncomodulin, cAMP, and pten gene deletion.J Neurosci.2010; 30 (46): 15654-63.), and to a certain degree functional rehabilitation is arranged (referring to document [3] de Lima S, Koriyama Y, Kurimoto T, et al.Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.Proc Natl Acad Sci USA.2012; 109 (23): 9149-54.), but be applied to clinical still far, still need seek and can be applicable to the clinical key factor.

Neuritin has another name called Candidate Plasticity-related 15(candidate plasticity-related gene 15, CPG15), is the newcomer in the neurotrophic factor family.Neuritin mainly is expressed in the neural system, Neuritin has unique neurotrophic effect, can keep neuronal cell survival (referring to document [4] Putz U, Harwell C and Nedivi E.Soluble CPG15 expressed during early development rescues cortical progenitors from apoptosis.Nat Neurosci.2005; 8 (3): 322-31.), promote the growth of nervous process (referring to document [5] Cappelletti G, Galbiati M, Ronchi C, et al.Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells.J Neurosci Res.2007; 85 (12): 2702-13.; [6] Nedivi E, Wu GY and Cline HT.Promotion of dendritic growth by CPG15, an activity-induced signaling molecule.Science.1998; 281 (5384): 1863-6.) and the cynapse maturation (referring to document [7] Cantallops I, Haas K and Cline HT.Postsynaptic CPG15 promotes synaptic maturation and presynaptic axon arbor elaboration in vivo.Nat Neurosci.2000; 3 (10): 1004-11.), regulate synaptic plasticity (referring to document [8] Wibrand K, Messaoudi E, Havik B, et al.Identification of genes co-upregulated with Arc during BDNF-induced long-term potentiation in adult rat dentate gyrus in vivo.Eur J Neurosci.2006; 23 (6): 1501-11.), and be that neurotrophic factor is (referring to document [9] Naeve GS, Ramakrishnan M, Kramer R, et al.Neuritin:a gene induced by neural activity and neurotrophins that promotes neuritogenesis.Proc Natl Acad Sci USA.1997; 94 (6): 2648-53.; [10] Lee KH, Ryu CJ, Hong HJ, et al.CDNA microarray analysis of nerve growth factor-regulated gene expression profile in rat PC12 cells.Neurochem Res.2005; 30 (4): 533-40.) and nervous activity (referring to document [11] Nedivi E, Fieldust S, Theill LE, et al.A set of genes expressed in response to light in the adult cerebral cortex and regulated during development.Proc Natl Acad Sci USA.1996; 93 (5): 2048-53.; [12] Lee WC and Nedivi E.Extended plasticity of visual cortex in dark-reared animals may result from prolonged expression of cpg15-like genes.J Neurosci.2002; 22 (5): 1807-15.; [13] Harwell C, Burbach B, Svoboda K, et al.Regulation of cpg15 expression during single whisker experience in the barrel cortex of adult mice.J Neurobiol.2005; 65 (1): the common downstream factor that 85-96.) plays a role, mediation male sex hormone and electricity irritation promote the necessary and key molecule of neurotization (referring to document [14] Sharma N, Marzo SJ, Jones KJ, et al.Electrical stimulation and testosterone differentially enhance expression of regeneration-associated genes.Exp Neurol.2010; 223 (1): 183-91.; [15] Marron TU, Guerini V, Rusmini P, et al.Androgen-induced neurite outgrowth is mediated by neuritin in motor neurones.J Neurochem.2005; 92 (1): 10-20.).

The rat ischemia brain injury is (referring to document [16] Han Y, Chen X, Shi F, et al.CPG15, a new factor upregulated after ischemic brain injury, contributes to neuronal network re-establishment after glutamate-induced injury.J Neurotrauma.2007; 24 (4): 722-31.; [17] Rickhag M, Teilum M and Wieloch T.Rapid and long-term induction of effector immediate early genes (BDNF, Neuritin and Arc) in peri-infarct cortex and dentate gyrus after ischemic injury in rat brain.Brain Res.2007; 1151:203-10.), the rat brain diffuse axonal injury (referring to document [18] He Yalong, He Xiaosheng, Zhang Xiang, etc. the expression of candidate's plasticity-genes involved 15 in the rat brain diffuse axonal injury. Chinese Neurological Surgery magazine .2010; 26 (2): the 186-188.) up-regulated of Neuritin in the brain such as grade, prompting Neuritin plays an important role in neural network reconstruction process.Xi Shaosong etc. are in the model of acute spinal cord injury, inject recombinant human Neuritin through subarachnoid space, can promote rat acute spinal cord injury (improvement Allen punch method) back to hinder the expression of district's axon regeneration associated protein neurofilament protein 200 and growth associated protein 43 (GAP-43), and the recovery that can promote the rat hindlimb motor function is (referring to document [19] Xi Shaosong, Liu Weigang, Zhang Hong, et al.Neuritin albumen to rat acute spinal cord injury after the effect of neuron axon regenerated. Chinese reconstruction surgical magazine .2009; 23 (10): 1219-1223.).These all point out Neuritin neurally reach maturity, trauma repair, pathology takes place and treatment in play an important role, application prospect is extensive.

Calendar year 2001, Krishnamoorthy etc. adopt the rat retina cell system (RGC5) of 1 day rat retina ganglion cell transfection ψ 2 E1A of birth virus foundation just (referring to document [20] Krishnamoorthy RR, Agarwal P, Prasanna G, et al.Characterization of a transformed rat retinal ganglion cell line.Brain Res Mol Brain Res.2001; 86 (1-2): 1-12.), since setting up clone, because its growth vigor is vigorous, being easy to cultivate, being widely used in experiment in vitro research by people, also is simultaneously the best instrument of research optic nerve injury and reparation.

Still it is relevant with the reparation optic nerve injury not have bibliographical information Neuritin gene at present, and also not seeing has the II type gland relevant viral vector of a kind of Neuritin of carrying gene of bibliographical information structure to treat optic nerve injury.

Summary of the invention

The object of the present invention is to provide a kind of II type gland relevant viral vector of the Neuritin of carrying gene.Another object of the present invention is to provide the application of this II type gland relevant viral vector that carries the Neuritin gene in preparation treatment reparation optic nerve injury medicine.

The present invention is on (RGC5) basis at the rat retina cell of employings such as Krishnamoorthy 1 day rat retina ganglion cell transfection ψ 2E1A of birth virus foundation just, adopts acrylamide damage RGC5 first, to simulate intravital optic nerve injury model.

The inventor finds behind the optic nerve injury 7 days in the gene chip of rat optic nerve, 5 times of the Neuritin down-regulated expressions in the optic nerve; Optic nerve crushed get different time points after 9 seconds respectively and observe the expression of retina neuritin and change, preliminary observation is obvious to interior 3 days of damage back short period of time and 7 days down-regulated expressions, 13 days up-regulateds moved closer to normal level by 28 days but still flat low than normal water.The down-regulated expression of the early stage Neuritin of optic nerve injury, prompting gives the treatment of Neuritin recombinant protein or Neuritin recombinant virus may produce provide protection and help optic nerve to the survival of retinal ganglion cell regeneration in early days at optic nerve injury.

Technical scheme of the present invention, it mainly is the II type gland relevant viral vector that makes up Neuritin, and be in the acrylamide damage model of RGC5 at retinal ganglial cells, utilize the II type gland relevant viral vector infection ganglion cell of Neuritin, observation in vitro is also inquired into effect and the mechanism that RGC5 is repaired in the Neuritin gene therapy.Set up rat optic nerve injury model, intravitreal injection is carried the II type adeno-associated virus of Neuritin gene in the row socket of the eye, observes and inquire into the effect and the mechanism of Neuritin gene repair optic nerve injury at body.

The Neuritin gene below also claims NRN1 gene or Nrn1 gene.

A first aspect of the present invention provides a kind of II type gland relevant viral vector of the Neuritin of carrying gene, and this gland relevant viral vector is the dna sequence dna that carries the Neuritin gene shown in SEQ ID NO:1.Described II type gland relevant viral vector is selected AAV three pUC pUCs for use, is made up of AAV carrier pAOV-CAG-eGFP, packaging plasmid pAAV-RC and helper plasmid pHelper.Goal gene NRN1 is started by the CAG promotor, has the expression character of nerve-specific.The NRN1 gene can be chosen in eGFP N end or C end or 3 * Flag C end and insert when inserting, also can select eGFP is replaced, or eGFP-3 * Flag replaces simultaneously.When albumen is inserted in eGFP N end, there is the flexible small peptide of GTGGGGSG to interconnect between eGFP and the NRN1 gene.

A second aspect of the present invention provides a kind of construction process of II type gland relevant viral vector of the Neuritin of carrying gene, and concrete steps are as follows:

1.Neuritin the structure of gene II type glandular associated virus expression vector

According to Neuritin(NRN1) gene (GenBank:BC002683.2) designs and synthesizes following PCR primer:

NRN1-forward primer: 5 '-CGACGCGTATGGGACTTAAGTTGAAC-3 '

（SEQ?ID?NO:2）；

NRN1-reverse primer: 5 '-TTTGCGGCCGCTCAGAAGGAAAGCCA-3 '

（SEQ?ID?NO:3）；

Use PCR method from containing amplification NRN1 gene C DS district in NRN1 gene cDNA (1551 bp are shown in SEQ ID NO:1) the clone template; With II type gland relevant viral vector and goal gene difference double digestion, purifying enzyme is cut behind the product and to be carried out orientation with NRN1 gene PCR product and be connected or recombinate, above-mentioned endonuclease bamhi accurately is connected on the Kpn I/Sal I site of pAOV expression vector by ligation, obtain the pAOV-Neuritin expression vector, its product transformed competence colibacillus bacterium, the clone who grows is carried out the Nrn1 gene PCR identify, PCR is accredited as the male clone, prove goal gene orientation be connected into the purpose carrier.Again PCR is identified that the male clone checks order and analyses and compares, and compares the correct fusion protein expression plasmid carrier that successfully constructs that is.The fusion protein expression vector that builds is carried out the ultrapure intracellular toxin extracting of going, and calcium changes method and changes plasmid over to the 293T cell, passes through fluorescence microscope fusion rotein eGFP after 36-48 hour.

2.Neuritin gene adeno-associated virus packing

Preparation packaging plasmid pAAV-RC and helper plasmid pHelper, with Neuritin gene II type glandular associated virus expression vector pAOV-Neuritin-eGFP cotransfection 293T cell, be replaced by perfect medium after the transfection in 8 hours, cultivate after 48 hours, II type adeno-associated virus particulate cell conditioned medium liquid is rich in collection, obtain the II type adeno-associated virus concentrated solution of high titre after it is concentrated, adopt coubling dilution to detect virus titer after infecting the Hela cell.

Different to the requirement of II type adeno-associated virus titre in the body with experiment in vitro, can concentrate the II type adeno-associated virus particle that obtains different titers in the production process excessively.

A third aspect of the present invention provides the application of the above-mentioned II type gland relevant viral vector that carries the Neuritin gene in preparation treatment reparation optic nerve injury medicine.

The RGC5 that the Neuritin gene II type that the carries adeno-associated virus treatment that makes up with the present invention damages: at first adopt Neuritin gene II type adeno-associated virus to infect RGC5 clone, use acrylamide again and cause RGC5 damage, observe behind the Neuritin gene overexpression damaging effect and the mechanism of back RGC5.Employing Neuritin albumen has provide protection to the survival that damages back RGC5, reduces the early stage apoptosis in late period that reaches of cell by the activity that suppresses Caspase 3.

Carry the application of II type gland relevant viral vector in preparation treatment reparation optic nerve injury medicine of Neuritin gene, but Neuritin II type adeno-associated virus transfection retinal ganglion cell, clinically, specifically can adopt this medicine transfection retinal ganglial cells of intravitreal injection to carry out the gene therapy of optic nerve injury.

Gland relevant viral vector AAV is a kind of nonencapsulated strand wire deficient dna virus, AAV is acknowledged as safest virus vector, the DNA of AAV can be integrated in host's the karyomit(e) with the form of dsDNA, keeps a kind of stable latent state, host range wide, good stability.The inventor is a carrier with the slow virus early stage; made up the Neuritin slow virus; finding that its infection rate to the ganglion cell is lower, mainly is to infect retinal pigment epithelium, may mainly be to realize its provide protection to the ganglion cell by the paracrine of Neuritin.And the infection rate of II type adeno-associated virus in the retinal ganglion cell can be up to 90%, carries the II type gland relevant viral vector of Neuritin gene by structure, can bring into play the provide protection of Neuritin to the retinal ganglion cell better.

The present invention is based on neurotrophic effect and the variation of the expression in neural wound, disease and the effect of Neuritin uniqueness, with Neuritin is that target is to RGC5 damage carrying out gene therapy, to strengthen the inherent regenerative power of RGC, improve its survival and regeneration situation.And infer that it has tangible effect to the survival of RGC5 and the key link of regenerate this optic nerve injury and disease; give multiple NTF or multiple therapy methods protection retina R GC and promote optic nerve regenerated curative effect thereby meet or exceed, for the clinical treatment optic nerve injury provides strong means and theoretical foundation.

Optic nerve is actually the part of central nervous system (CNS) owing to its special structure and physiological property.So the reparative regeneration behind the research optic nerve injury, also the treatment to CNS disease and damage has significance.

Description of drawings

Fig. 1 is that staurosporin (Staurosporine) is induced differentiation retinal ganglial cells system,

Wherein A is inductive RGC5 not; B is that staurosporin (Staurosporine) induces the RGC5(after the differentiation to adopt staurosporin (Staurosporine) that retinal ganglial cells is induced differentiation, is sophisticated neuron cell.) scale=50 μ m.

Fig. 2 is the acrylamide damage retinal ganglial cells system of different concns

Wherein A is the photo under the phase microscope, shows the effect of different concns acrylamide to retinal ganglion clone (RGC5) damage 24h; B be the different concns acrylamide to after the RGC5 damage the longest projection variation, 0.2mM is to the existing obvious damaging action of the longest projection of RGC5; C is the influence of different concns acrylamide to the survival of RGC5, and the RGC5 survival influence of 0.2mM damage back is not remarkable.Therefore, the acrylamide that the present invention intends selecting 0.2mM for use is to simulate intravital optic nerve injury model, and this concentration has the obvious impairment effect to projection, and less to ganglion cell's influence of surviving.Scale=20 μ m.

Fig. 3 is that the CCK8 method is surveyed the provide protection of recombinant human Neuritin to the survival of RGC5 after damaging.

Fig. 4 is that flow cytometry is surveyed apoptosis

Wherein A is a normal group; B is simple damage group; C is a Neuritin treatment group; D is the CNTF positive controls.Early apoptosis and late period apoptosis show that all Neuritin treatment group can reduce the apoptosis of damage back RGC5, provide protection is the most obvious, is better than the CNTF positive controls.

Fig. 5 is an II type adeno-associated virus AAV2-eGFP transfection rat retinal ganglion cell

Wherein A shows AAV2-eGFP success transfection rat retinal ganglion cell; B shows the retinal ganglial cells aixs cylinder of AAV2-eGFP mark; Scale=50 μ m.

Fig. 6 is Neuritin II type adeno-associated virus expression plasmid vector construction figure.

Fig. 7 is the line chart of II type adeno-associated virus eGFP plasmid and Neuritin II type adeno-associated virus expression plasmid carrier.

Embodiment

Describe the present invention below in conjunction with embodiment and accompanying drawing.But the following example should not regarded limitation of the scope of the invention as.

Embodiment 1:

1.Neuritin gene II type glandular associated virus expression vector makes up

(1) enzyme of II type glandular associated virus expression vector and goal gene is cut

Adopt pAOV-CAG-eGFP carrier (available from Addgene company), use Not I, Mlu I to carry out enzyme and cut, the carrier after enzyme is cut uses when preparing vector construction.CDNA clone with NRN1 gene C DS district is that template adopts pcr amplification NRN1 gene fragment, and product uses Not I, Mlu I to carry out enzyme and cut, and reclaims carrier and goal gene DNA after agarose gel electrophoresis is identified.

(2) ligation

The II type gland relevant viral vector after enzyme cut and the PCR product of NRN1 gene carry out ligation according to the reaction system in the table 1.

Table 1: ligation system

In 4 ℃, ligation in 12 hours preparation clone connects liquid, prepares to transform after the above reagent mix.

(3) transform, choose the clone and identify

Adopt Axygen plasmid extraction test kit to extract plasmid, concrete extraction steps is as follows: the 5ml bacterium liquid of incubated overnight is done glycerol stock with 2ml and is protected, and residue 3ml is centrifugal 1 minute of 12,000 * g in a 1.5ml centrifuge tube at twice, thoroughly removes supernatant; Add 250 μ l bacterial suspensions, the abundant resuspended bacterium of vibrator; Add 250 μ l bacterial lysates, put upside down mixing 4-6 time; Attention: can not acutely mix, pollute otherwise genomic dna can occur; Add and add 350 μ l neutralizers in the 250 μ l bacterial lysates five minutes, put upside down mixing 6-8 time gently; 12,000 * g, centrifugal 10 minutes; Supernatant is transferred in a small amount of purification column, and purification column inserts in the waste collection pipe centrifugal 1 minute of 12,000 * g in a small amount; Be not drawn onto precipitation, otherwise genomic dna and protein contamination can occur; Abandon the waste liquid in the collection tube, purification column inserts in the collection tube in a small amount, adds 700 μ l bacterial lysates in a small amount of purification column, centrifugal 1 minute of 12,000 * g; Repeat this step 1 time; Abandon the waste liquid in the collection tube, purification column is put into same collection tube in a small amount, and 12,000 * g thoroughly removed bacterial lysate in centrifugal 1 minute; The a small amount of purification column is put into new clean 1.5ml centrifuge tube, add 50 μ l 2.5mMTris-HCl elutriants, room temperature was placed after 1 minute, centrifugal 1 minute of 12,000 * g; Solution in the centrifuge tube is extractive plasmid; The gained plasmid adopts PCR to identify and Not I and Mlu I double digestion are identified, enzyme is cut product and selected positive colony and send order-checking.

Entrust the order-checking of the biological order-checking of Nanjing Jin Sirui company, 1551 bp, shown in SEQ ID NO:1, sequencing result shows that NRN1 gene number 1 clone for identifying correct positive colony, can enter the experiment flow of II type adeno-associated virus packing.

2.Neuritin gene II type adeno-associated virus packing

(1) II type gland relevant viral vector system plasmid essential information

This virus packets assembling system is three pUC pUCs, forms (all plasmids are all available from Addgene company) by AAV carrier pAOV-CAG-eGFP, packaging plasmid pAAV-RC and helper plasmid pHelper.Goal gene Nrn1 is started by the CAG promotor, has the expression character of nerve-specific.The Nrn1 gene can be chosen in eGFP N end or C end or 3 * Flag C end and insert when inserting, also can select eGFP is replaced, or eGFP-3 * Flag replaces simultaneously.When albumen is inserted in eGFP N end, there is the flexible small peptide of GTGGGGSG to interconnect between eGFP and the Nrn1 gene.Packing contains the II type adeno-associated virus of goal gene Nrn1 respectively, and the II type adeno-associated virus that does not contain goal gene Nrn1 is with in contrast.(as Fig. 6, shown in Figure 7)

(2) plasmid transfection

The II type adeno-associated virus packing cell transfection 293T cell (available from Shanghai cell institute of the Chinese Academy of Sciences) of tryptic digestion logarithmic phase, (each plating cell is about 2-2.5 * 10 to be re-seeded into the 10cm Tissue Culture Dish ⁶), 37 ℃, 50ml/L CO ₂Cultivate in the incubator; Three kinds of plasmid dna solutions in the preparation II type adeno-associated virus packaging system; Add sterilized water and be settled to 1800 μ l, add CaCl again ₂(2.5mol/L) solution 200 μ l, mixing adds 2 * BBS buffer salt solution, 2000 μ l, and room temperature is placed 20-25min.

Transfection when cell density reaches 60%～70% is transferred in the nutrient solution that contains monolayer cell mixing with each plasmid and calcium phosphate mixed solution, discard the nutrient solution that contains the transfection miscellany after cultivating after 6-8 hour, add PBS 15ml, discard behind the jog, repeat this step 3 time; Add the cell culture fluid 6ml that contains 10% foetal calf serum in every bottle of cell, continue to cultivate 48 hours; Collect 72 hours 293T cell conditioned medium liquid of transfection; In 4 ℃, the centrifugal 10min of 4000g collects the supernatant liquor after centrifugal with the supernatant liquor collected; Supernatant liquor is filtered with 0.45 μ m filter; In 40ml ultracentrifugation pipe, 4 ℃, the centrifugal 2hrs of 25000rpm/min; Then being deposited in 4 ℃ of dissolvings with ice PBS liquid or the resuspended virus of 500 μ lDMEM substratum spends the night.

(3) II type adeno-associated virus titer determination

Adopt by hole dilution titer assay method: measure the day before yesterday,, add 1 * 10 in each 96 hole for measuring the required 293T cell bed board of titre ⁵Individual cell, volume are 100 μ l.According to the expection titre of virus, prepare 7-10 aseptic Ep pipe.The fresh culture that in each pipe, adds 90 μ l.Get virus stock solution used to be determined 10 μ l and join in first pipe, behind the mixing, get 10 μ l and join in second pipe.Continue an identical operations pipe to the last.Choose required cell hole, inhale and remove 90 μ l substratum.Add the good viral solution of dilution.Putting into incubator cultivates.After 24 hours, add fresh culture 100 μ l.After 4 days, the observation of cell upgrowth situation, and collect cell and carry out flow cytometry mensuration titre.

Calculate the titre formula according to the flow cytometer detected result at last:

The last like cell number of titre (TU/ml)=100000() * volume (ml) of the percentage ratio/viral supernatant liquor of GFP positive cell.

Embodiment 2:

1.RGC5 cultivation and induce differentiation

RGC5 is that the retinal ganglial cells of one day rat of birth just is (referring to document [20] Krishnamoorthy RR, Agarwal P, Prasanna G, et al.Characterization of a transformed rat retinal ganglion cell line.Brain Res Mol Brain Res.2001; 86 (1-2): 1-12.), fully matured does not at first adopt staurosporin that it is induced, to be divided into sophisticated neuron cell (Fig. 1).Do not induce preceding RGC5 to adopt the serum cultivation RGC5 cell of high sugared DMEM substratum and 5% to place and contain 5%CO ₂, 37 ℃ incubator, cell passed once generation in one day.Adopt the staurosporin of 326nM to induce during use 24 hours.

2.Neuritin the foundation of gene overexpression and RGC5 damage model

When RGC5 is cultured to 80% fusion, add the II type adeno-associated virus amount and a certain amount of polybrene that are fit to that have touched concentration in advance, change normal substratum after 24 hours into, adopt the expression of fluorescence microscope RFP after 96 hours, transfection efficiency can reach about 90% (Fig. 5), other adopt Western blot and real-time fluorescence quantitative PCR can be observed 10-12 that the cell expressing that changes the Neuritin gene over to is about control group doubly about, become merits and demerits to express.Adopt acrylamide to damage crossing the RGC5 that expresses the Neuritin gene, concentration is 200nM, damages 24 hours (Fig. 2).This RGC5 group is Neuritin gene overexpression group.

It is as follows that other establishes group: (1) normal control group only adds staurosporin and induces, damage; (2) simple damage group is promptly only induced and is damaged and do not add any treatment group; (3) contrast virus group is the contrast virus of the GFP that do not contain the Neuritin gene, and processing mode is with Neuritin gene overexpression group; (4) Neuritin protein for treatment group, the Neuritin albumen pre-treatment of adding 300ng/ml added once every one day before the acrylamide damage, and other handle the same; (5) ciliary neurotrophic factor (CNTF) positive controls, the CNTF of adding 30ng/ml before damage.Put mutually during observation and be 1 day, 2 days, 3 days, 4 days, 5 days after the damage.

3.Neuritin gene II type adeno-associated virus is to damaging provide protection and the mechanism of back RGC5

(1) detection of RGC5 cell survival rate

Behind the RGC5 transfection Neuritin gene II type adeno-associated virus, with 5 * 10 ³The density in/hole is planted in 96 orifice plates, plant that density is about 70% after one day, the staurosporin that adds 316nM is induced differentiation 24 hours, the back adopts the acrylamide of 200nM to damage 24 hours, change normal substratum into, each group of experiment respectively after damage different time phases adopt the CCK8 method to measure its survival.Add 10 μ l CCK8 in per 100 μ l nutrient solutions, behind the 90min in 450nm place survey its absorbance (Fig. 3).

(2) detection of RGC5 apoptosis situation

Behind the RGC5 transfection Neuritin gene II type adeno-associated virus, with 1 * 10 ⁵The density in/hole is planted in the culture dish of 90 * 10mm, after the cytogamy to 80%, the staurosporin of 316nM is induced differentiation 24h, then the damage of the acrylamide of 200nM is 24 hours, change normal substratum into, each group of experiment respectively after damage different time phases adopt flow cytometry to measure its apoptosis situation (Fig. 4).

(3) observation of RGC5 cell process quantity and length variations

Behind the RGC5 transfection Neuritin gene II type adeno-associated virus, with 2 * 10 ⁴Plant in 24 orifice plates in/hole, and after the cytogamy to 70%, the staurosporin of 316nM is induced differentiation 24 hours, and then the damage of the acrylamide of 200nM is 24 hours, changes normal substratum into, and each group of experiment is being taken pictures under the different time phases phase microscope after the damage respectively.Adopt Image J to the projection quantity of RGC5 and length is measured and statistical analysis.

(4) survival and growth associated protein change of Expression in the RGC5 cell

Behind the RGC5 transfection Neuritin gene II type adeno-associated virus, with 1 * 10 ⁵The density in/hole is planted in the culture dish of 90 * 10mm, after the cytogamy to 80%, the staurosporin of 316nM is induced differentiation 24 hours, then the damage of the acrylamide of 200nM is 24 hours, change normal substratum into, each group of experiment is different time phases extraction albumen after damage respectively, adopts Western Blot and immunocytochemistry to detect the proteic expression changing conditions of STAT3, PI3/Akt, MAPK signal path and Caspase3 respectively, to illustrate the Neuritin mechanism of action.

Embodiment 3:

1. crossing of transfection retinal ganglion cell and Neuritin expressed in the II type adeno-associated virus body

Adopt intravitreal injection Neuritin II type adeno-associated virus, one week back employing, 4% Paraformaldehyde 96 total body perfusion mouse, get retina shop sheet, adopt immunohistochemical methods β-III-tubulin dyeing or choleratoxin B subunit (CTB) injection to locate the retinal ganglion cell altogether to observe the transfection effect, transfection efficiency is about (as shown in Figure 5) more than 90%.

The proteic situation of crossing expression and expression and localization of Neuritin after the method observation Neuritin II type adeno-associated virus transfection of employing Western blot and immunohistochemical methods.

2. the foundation of optic nerve injury model

The present invention adopts optic nerve to crush model, about 1mm place crushes 9s after using the Yasargil cerebral aneurysm to be sandwiched in bull rat optic nerve eyeball, concrete operations have been delivered document Wang R with reference to the inventor, Xu J, Xie J, et al.Hyperbaric oxygen preconditioning promotes survival of retinal ganglion cells in a rat model of optic nerve crush[J] .J Neurotrauma.2010 (27): 763-770., simulate the clinical incomplete damage that causes.

Laboratory animal is grouped into: Neuritin II type adeno-associated virus group, contrast virus group, physiological saline group.The time point of damage is: 2 weeks, 4 weeks, 8 weeks.

3. optic nerve injury is repaired the index of correlation detection

(1) fluorescent mark of retinal ganglion cell, Nissl's staining and counting

Each treated animal (Neuritin II type adeno-associated virus group, contrast virus group, the physiological saline group, n=5), get damage back different time points (2 weeks respectively, 4 weeks, 8 weeks), adopt the choleratoxin B subunit (CTB) of Hamliton microsyringe intravitreal injection band fluorescein that the retinal ganglion cell is carried out mark, total body perfusion is fixed after 48 hours, draw materials, retina shop sheet is behind the optic disk of location, thereon, down, nose, look nipple 1/6 by distance in four quadrants of temporo, 3/6, respectively get 2 high power fields at random and take pictures at 5/6 place, carry out ganglion cell counting, the cell density (cell count/high power field) of each group is compared parallel statistical analysis.

Can carry out Nissl's staining subsequently: toluidine blue dye liquor 5min, 75% ethanol 2min, 95% ethanol 2min, 100% ethanol 5min time, mounting is observed after dimethylbenzene is transparent again.Simple microscope is observed down, carry out ganglion cell counting (get in the visual field that volume is big, the particulate state nissl bodies is arranged in the kytoplasm, nuclear staining is shallow and the tangible cell of kernel is the retinal ganglion cell) as stated above, the cell density (cell count/high power field) of each group is compared parallel statistical analysis.

(2) optic nerve aixs cylinder fluorescent mark and nerve growth related protein (GAP-43) immunofluorescence dyeing

Each treated animal (Neuritin II type adeno-associated virus group, contrast virus group, physiological saline group, n=5), get respectively damage back different time points (2 weeks, 4 weeks, 8 weeks), adopt intravitreal injection CTB that the optic nerve aixs cylinder is carried out mark, total body perfusion is fixedly drawn materials after 48 hours, after fix 4 hours, the dehydration of 30% sucrose, to the capable frozen section of optic nerve, observe and add up the regeneration aixs cylinder quantity and the distribution thereof of mark, with the quantity of the aixs cylinder of relatively surviving.

The optic nerve frozen section carries out the dyeing of GAP43, to show the axonal regeneration activity.At first PBS washes 5 minutes * 3 times, 0.4%Triton-X100 rupture of membranes 10 minutes, sealing is 1 hour under the 10% serum room temperature, dries section, one anti-(GAP-43) 4 ℃ of overnight incubation, next day, PBS washed 5 minutes * 3 times, fluorescent mark two anti-room temperature reactions 1 hour, and PBS washes 5 minutes * 3 times, DAPI dyed nuclear 5 minutes, PBS washes 5 minutes * 3 times, and glycerine carbonate mountant mounting is observed down in fluorescent microscope.

(3) pupillary light reflex and visual evoked potential

Optic nerve crushes and is incomplete damage model, adopts pupillary light reflex and visual evoked potential to show the sight function situation of each treated animal.Each treated animal (Neuritin II type adeno-associated virus group, contrast virus group, the physiological saline group, n=5), get damage back different time points (2 weeks respectively, 4 weeks, 8 weeks), after the anesthesia, in the darkroom, the branch hole eyeball masking foil lucifuge of will not performing an operation, with certain light boosting operation branch hole eyeball, adopt stereoscopic microscope (experimental technique reference [22] the de Lima S that takes pictures, Koriyama Y, Kurimoto T, et al.Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors[J] .Proc Natl Acad Sci US A.2012 (109): 9149-54.).The ratio of measuring its latent period and minimum pupil diameter respectively is to disclose the function of its pathways for vision.

Visual evoked potential (experimental technique reference [23] Pangratz-Fuehrer S, Kaur K, Ousman S S, et al.Functional rescue of experimental ischemic optic neuropathy with alphaB-crystallin[J] .Eye (Lond) .2011 (25): 809-17.), after each treated animal anesthesia, rat is fixed on the stereotaxic apparatus, opens cranium and is positioned the brain occipital lobe cortical area, detects its flash visual evoked potential.

More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.

Claims (4)

1. an II type gland relevant viral vector that carries the Neuritin gene is characterized in that, this II type gland relevant viral vector is the dna sequence dna that carries the Neuritin gene shown in SEQ ID NO:1.

2. a kind of II type gland relevant viral vector that carries the Neuritin gene according to claim 1, it is characterized in that, described II type gland relevant viral vector is selected AAV three pUC pUCs for use, is made up of AAV carrier pAOV-CAG-eGFP, packaging plasmid pAAV-RC and helper plasmid pHelper; Goal gene NRN1 is started by the CAG promotor; The NRN1 gene is chosen in eGFP N end or C end or 3 * Flag C end and inserts when inserting, perhaps select eGFP is replaced, and perhaps eGFP-3 * Flag replaces simultaneously; When albumen is inserted in eGFP N end, there is the flexible small peptide of GTGGGGSG to interconnect between eGFP and the NRN1 gene.

3. construction process that carries the II type gland relevant viral vector of Neuritin gene as claimed in claim 2 is characterized in that concrete steps are as follows:

A) structure of the II type glandular associated virus expression vector of Neuritin gene

It is as follows to design and synthesize the PCR primer:

The NRN1-forward primer is shown in SEQ ID NO:2;

In the NRN1-reverse primer shown in the SEQ ID NO:3;

Use PCR method from containing just like amplification NRN1 gene C DS district the NRN1 gene cDNA clone template shown in the SEQ ID NO:1; With II type gland relevant viral vector and goal gene difference double digestion, purifying enzyme is cut behind the product and to be carried out orientation with NRN1 gene PCR product and be connected or recombinate, above-mentioned endonuclease bamhi accurately is connected on the Kpn I/Sal I site of pAOV expression vector by ligation, obtain the pAOV-Neuritin expression vector, its product transformed competence colibacillus bacterium, the clone who grows is carried out the Nrn1 gene PCR identify, PCR is accredited as the male clone, prove goal gene orientation be connected into the purpose carrier; Again PCR is identified that the male clone checks order and analyses and compares, and compares the correct fusion protein expression plasmid carrier that successfully constructs that is; The fusion protein expression vector that builds is carried out the ultrapure intracellular toxin extracting of going, and calcium changes method and changes plasmid over to the 293T cell, passes through fluorescence microscope fusion rotein eGFP after 36-48 hour;

B) the II type adeno-associated virus of Neuritin gene packing

Preparation packaging plasmid pAAV-RC and helper plasmid pHelper, with Neuritin gene II type glandular associated virus expression vector pAOV-Neuritin-eGFP cotransfection 293T cell, be replaced by perfect medium after the transfection in 8 hours, cultivate after 48 hours, II type adeno-associated virus particulate cell conditioned medium liquid is rich in collection, obtain the II type adeno-associated virus concentrated solution of high titre after it is concentrated, adopt coubling dilution to detect virus titer after infecting the Hela cell.

4. the application of II type gland relevant viral vector in preparation treatment reparation optic nerve injury medicine of carrying the Neuritin gene as claimed in claim 1 or 2.

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