CN106755104A - A kind of adeno-associated virus 2 of carrying genes of Pim 1 and its application in crushed optic nerve is repaired - Google Patents

A kind of adeno-associated virus 2 of carrying genes of Pim 1 and its application in crushed optic nerve is repaired Download PDF

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CN106755104A
CN106755104A CN201611114485.2A CN201611114485A CN106755104A CN 106755104 A CN106755104 A CN 106755104A CN 201611114485 A CN201611114485 A CN 201611114485A CN 106755104 A CN106755104 A CN 106755104A
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许家军
张守梅
王栋
刘芳
黄婷婷
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Second Military Medical University SMMU
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Abstract

The present invention relates to gene engineering technology field, the inherent power of regeneration of retinal ganglion cell after optic nerve injury is low, is the key factor that visual function is difficult to recover, and how effectively to treat the great difficult problem that always clinical medicine faces.The invention provides a kind of carrier of recombinant adeno-associated virus 2 and its construction method of the carrying genes of Pim 1, the described carrier of adeno-associated virus 2 is carried such as SEQ ID NO:The DNA sequence dna of the genes of Pim 1 shown in 1.The present invention also provides application of the carrier of recombinant adeno-associated virus 2 for carrying the genes of Pim 1 in crushed optic nerve medicine is repaired in preparation treatment.The present invention provides new powerful measure and theoretical foundation for clinical treatment optic nerve injury, to CNS diseases and the treatment for damaging also important in inhibiting.

Description

A kind of adeno-associated virus 2 of carrying Pim-1 genes and its repair crushed optic nerve in Application
Technical field
It is a kind of adeno-associated virus 2 of carrying Pim-1 genes specifically the present invention relates to gene engineering technology field Preparing the application during crushed optic nerve medicine is repaired in treatment.
Background technology
After central nervous system (CNS) disease and damage, nerve regneration is extremely difficult, how to carry out effectively treatment one It is directly great difficult problem that clinical treatment is perplexed.Optic nerve is a part of CNS.After optic nerve injury, axon regeneration difficulty Main cause includes retinal ganglial cells (RGCs) mortality, in RGCs power of regeneration is low, guiding axon regeneration Cell lack, suppress microenvironment, the deficiency of neurotrophic factor etc. of regeneration.The inherent Regenerated energy of RGCs after optic nerve injury Power is low, is to regenerate the key factor for unsuccessfully causing optic atrophy and blindness after optic nerve injury.At present, although clinically pin Extensive work, but its actual unsatisfactory curative effect have been carried out in treatment to crushed optic nerve, it is difficult to allow patient to recover eyesight.
Emerged in an endless stream on improving the achievement in research in RGCs in power of regeneration in the recent period：Benowitz seminar passes through acupuncture Hinder crystalline lens, intravitreal crystalline protein activates the inherent regeneration potential of RGCs, and make aixs cylinder is regenerated by optic nerve Squeezing traumatic part position reach distal section nerve, but quantity and growth potential it is limited (referring to document [1] de Lima S, Koriyama Y, Kurimoto T,et al.Fulllength axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.Proc Nat Acad Sci U S A,2012； 109(23):9149-54).By activating the B-Raf of RAF-MEK paths, particularly joint knocks out PTEN to O ' Donovan etc., together When effectively promote the regeneration after optic nerve in rat (referring to document [2] O'Donovan KJ, Ma K, Guo H, et al.B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.J Exp Med,2014；211(5):801-14).Yungher Deng the adeno-associated virus 2 and cAMP analogs of mouse intravitreal injection PTEN-shRNA, CNTF, Regenerating Axons far up to optic chiasma and SCN simultaneously forms cynapse (referring to document [3] Yungher BJ, Luo X, Salgueiro Y, Blackmore MG, et al.Viral vector-based improvement of optic nerve regeneration: characterization of individual axons’growth patterns and synaptogenesis in a visual target.Gene Therapy,2015；22:811-821).Nawabi etc. is to PTEN knock-out mice intravitreal injections The adeno-associated virus 2 of DCLKs, axon regeneration far up to optic chiasma (referring to document [4] Nawabi H, Belin S, Cartoni R, et al.Doublecortin-like kinases promote neuronal survival and induce growth cone reformation via distinct mechanisms.Neuron,2015；88:704-719.).Therefore, seek The target molecule and gene for looking for regulation to regenerate, and will be combined with the factor of influence regeneration in power of regeneration in enhancing RGCs, promote Optic nerve regeneration, with bright clinical landscapes.
Moloney murine leukemia virus proviral integration site (provirus integration site for Moloney murine leukemia virus, Pim-1) it is proto-oncogene, serine/threonine protein kitase is expressed, belong to Calcium-calmodulin regulation kinases.It express kinases be widely present in each histocyte, such as thymus gland, marrow, colon, testis, Prostate, angiocarpy, cerebral cortex, hippocampus and retina etc., Pim-1 be many cell factors or growth factor (including IL-2, GCSF, EGFR, HGF etc.) signal path (including JAK-STAT, AKT, MAPK, NF-kB etc.) common downstream effect molecule, and Phosphospecific substrate, participate in cell propagation, differentiation, survive etc. (bibliography [5] Mondello P, Cuzzocrea S, Mian M.Pim kinases in hematological malignancies:where are we now and where are we goingJ Hemat Oncol,2014；7:95).
Pim-1 can participate in vascularization (referring to document [6] Zippo A, De Robertis A, Bardelli M, et al.Identification of Flk-1target genes in vasculogenesis:Pim-1is required for endothelial and mural cell differentiation in vitro.Blood,2004；103:4536-4544； Referring to document [7] Walpen T, Peier M, Haas E, et al.Loss of pim1imposes a hyperadhesive phenotype on endothelial cells.Cell Physiol Biochem,2012,30(4):1083-96), promote VSMC (VSMCs) is bred and is survived (referring to document [8] Willert M, Augstein A, Poitz D M, et al.Transcriptional regulation of Pim-1kinase in vascular smooth muscle cells and its role for proliferation.Basic Res Cardiol,2010,105(2):267-77；Referring to document [9]Meloche J,Paulin R,Courboulin A,et al.RAGE-dependent activation of the oncoprotein Pim1plays a critical role in systemic vascular remodeling processes.Arterioscler Thromb Vasc Biol,2011,31(9):2114-24；Referring to document [10] Chen J,Yin H,Jiang Y,et al.Induction of microRNA-1by myocardin in smooth muscle cells inhibits cell proliferation.Arterioscler Thromb Vasc Biol,2011,31:368- 375；Referring to document [11] Paulin R, Courboulin A, Meloche J, et al.Signal transducers and activators of transcription-3/pim1axis plays a critical role in the pathogenesis of human pulmonary arterial hypertension.Circulation,2011,123 (11):1205-15；Referring to document [12] Paulin R, Meloche J, Jacob MH, et al.Dehydroepiandrosterone inhibits the Src/STAT3constitutive activation in pulmonary arterial hypertension.Am J Physiol Heart Circ Physiol,2011,301: H1798-H1809) and cardiac progenitor cell (CPCs) propagation survive and delay its aging (referring to document [13] Hu S, Yan G, Xu H,et al.Hypoxic preconditioning increases survival of cardiac progenitor cells via the Pim-1kinase-mediated anti-apoptotic effect.Cir J,2014,78(3): 724-731；Referring to document [14] Samse K, Emathinger J, Hariharan N, et al.Functional effect of Pim1depends upon intracellular localization in human cardiac progenitor cells.J Biol Chem,2015,290(22):13935-47；Referring to document [15] Cottage CT, Bailey B, Fischer KM,et al.Cardiac progenitor cell cycling stimulated by pim- 1kinase.Circ Res,2010,106(5):891-901), promote CPCs differentiation (referring to document [16] Iwakura T, MohritT,Hamatani T,et al.STAT3/Pim-1signaling pathway plays a crucial role in endothelial differentiation of cardiac resident Sca-1+ cells both in vitro and in vivo.J Mol Cell Cardiol,2011,51(2):207-14；Referring to document [17] Tufan H, Zhang XH,Haghshenas N,et al.Cardiac progenitor cells engineered with Pim-1(CPCeP) develop cardiac phenotypic electrophysiological properties as they are co- cultured with neonatal myocytes.J Mol Cell Cardiol,2012,53(5):695-706), protection is received Cardiac muscle is damaged (referring to document [18] Muraski JA, Rota M, Misao Y, et al.Pim-1regulates cardiomyocyte survival downstream of Akt.Nat Med,2007,13(12):1467-75；[19] Stumpner J,Smul TM,Redel A,et al.Desflurane-induced and ischaemic postconditioning against myocardial infarction are mediated by Pim- 1kinase.Acta Anaesthesiol Scand,2012,56(7):904-13；[20]Din S,Mason M,Volkers M,et al.Pim-1preserves mitochondrial morphology by inhibiting dynamin-related protein 1translocation.Proc Natl Acad Sci,2013,110(15):5969-74)。
In nervous system, Pim-1 is present in fetal mice occipital lobe visual cortex, olfactory cortex, corpus straitum and retina and embryo In tire zebra fish retina, and can promote above-mentioned neuromechanism development (referring to document [21] Eichmann A, Yuan L, Breant C,et al.Developmental expression of pim kinases suggests functions also outside of the hematopoietic system.Oncogene,2000,19(9):1215-24；[22]Yin J,Shine L,Raycroft F,et al.Inhibition of the Pim1oncogene results in diminished visual function.PLoS One,2012,7(12):e52177).Rat pyriform lobe cortex, hippocampus knot Structure, corpus straitum, dorsal thalamus expression Pim-1 mRNA and albumen, epileptic attack, long term potentia￣tion can induce above-mentioned zone respectively Pim-1 mRNA expression high (bibliography [23] Feldman JD, Vician L, Crispino M, et al.KID-1, a protein kinase induced by depolarization in brain.J Biol Chem,1998,273(26): 16535-43；[24]Konietzko U,Kauselmann G,Scafidi J,et al.Pim kinase expression is induced by LTP stimulation and required for the consolidation of enduring LTP.Embo J,1999,18(12):3359-69；[25]Feldman JD,Vician L,Crispino M,et al.Seizure activity induces PIM-1expression in brain.J Neurosci Res,1998,53 (4):502-9), in rat cell line PC12 cells, PC12 cells receive KCl and adenyl cyclase activator forskolin (Fk) after induction treatment, Pim-1 albumen rises after 1h, and 2-4h peaks, and normal level (bibliography [26] is recovered after 6h Liu W,Feldmann JD,Machado HB,et al.Expression of depolarization-induced immediate early gene proteins in PC12cells.J Neurosci Res,2003,72(6):670-8), In vitro study shows that Pim-1 promotes the release of PC12 cells catecholamines such as dopamine, norepinephrine and IL-6 (bibliography [27] Glazova M, Aho TL, Palmetshofer A, et al.Pim-1kinase enhances NFATc activity and neuroendocrine functions in PC12cells.Brain Res Mol Brain Res, 2005,138(2):116-23).To sum up prompting Pim-1 take part in the depolarising and plasticity regulation of neuron and releasing for mediator Put.In the mouse brain cortex neuron cultivated in vitro, Pim-1 is passed through after having mediated mouse brain cortex neuron DNA damage The death caused by NF-kB and Cdc25A is (referring to document [28] Zhang Y, Parsanejad M, Huang E, et al.Pim- 1kinase as activator of the cell cycle pathway in neuronal death induced by DNA damage.J Neurochem,2010,112(2):497-510), it is (12-72h) and new in early days after acute cerebral ischemia in rats In raw rat brain Hypoxic ischemic, intracerebral Pim-1 mRNA and protein expression are raised, and take part in granulocyte collection after rat cerebral ischemia The anti-Neuron Apoptosis of G-CSF and promote neuronal survival process (referring to document [29] Solaroglu I, Tsubokawa T,Cahill J,et al.Anti-apoptotic effect of granulocyte-colony stimulating factor after focal cerebral ischemia in the rat.Neuroscience, 2006,143(4):965-74；[30]Yata K,Matchett GA,Tsubokawa T,et al.Granulocyte- colony stimulating factor inhibits apoptotic neuron loss after neonatal hypoxia-ischemia in rats.Brain Res,2007,1145:227-38).These promptings Pim-1 take part in impaired The survival of neuron and apoptosis.
With reference to the dependent interaction of above-mentioned Pim-1, particularly its substantial connection and optic nerve injury backsight with visual development Nethike embrane changes in gene expression (see embodiment 2), it is probably to influence the key molecule in RGC in power of regeneration to point out Pim-1, therefore false If compared to above-mentioned path such as JAK-STAT, AKT is adjusted, the single developed by molecule in MAPK adjusts the gene with Pim-1 as target Expression, may play the effect of " supporting ten with ", can more effectively strengthen in impaired RGC in power of regeneration.
The research of current Pim-1 focuses mostly in terms of tumour, myocardial preservation and regeneration, and the research in nervous system is still It is few, there is not yet Pim-1 genes and the correlative study for repairing crushed optic nerve, also there are no document report structure one kind and carry Pim- The carrier of recombinant adeno-associated virus 2 of 1 gene treats crushed optic nerve and other neurotrosises.
The content of the invention
It is an object of the invention to provide a kind of adeno-associated virus 2 of carrying Pim-1 genes, its construction method, and its Prepare the application during crushed optic nerve medicine is repaired in treatment.
The present invention crushes 5s at 2mm after SD rat balls using interim aneurysm clips, to make optic nerve squeezing Wound model.
The present invention is had found in normal newborn rat in the detection of the genetic chip and Real-time PCR of rat retina In retina, Pim-1 mRNA expressions are significantly higher than Normal Adult Rat, about 1.93 times；3 days after optic nerve injury, depending on Pim-1 mRNA up-regulateds in nethike embrane, are 1.53 times of normal adult mouse, are down to below normal level by 14 days. Western Blot results show, in normal newborn rat retina, Pim-1 protein expression levels are big apparently higher than normal adult 3.24 times of mouse, about Normal Adult Rat；And 3 days after optic nerve in rat of growing up, intraretinal Pim-1 protein expressions Raise, about the 1.98 of Normal Adult Rat times, be just down to below normal level by 7 days, 21 days still low compared with normal level.It is immune Groupization result shows that 1d and 3d after optic nerve injury, the expression of ganglionic layer of retina Pim-1 substantially increase, afterwards gradually Lower.With reference to the dependent interaction of above-mentioned Pim-1, particularly its substantial connection with visual development and and its gene damaged in optic nerve The change of retina expression after wound, and the inherent growth ability of newborn rat pattern of retinal ganglion cells is strong, points out in optic nerve injury The overexpression of early stage row Pim-1 recombinant adeno-associated virus 2 treatment afterwards may promote the survival of compromised retinal ganglion cell and regarding god The regeneration of warp.
Technical scheme, mainly builds the carrier of recombinant adeno-associated virus 2 of Pim-1, and in adult SD rats In optic nerve squeezing wound model, the carrier transfected into rat retina of recombinant adeno-associated virus 2 of intravitreal Pim-1, in vivo Observe and inquire into the effect that crushed optic nerve is repaired in targeting Pim-1 gene therapies.
To achieve these goals, a kind of the first aspect of the present invention, there is provided the restructuring gland related diseases of carrying Pim-1 genes Malicious 2 carriers, the described carrier of adeno-associated virus 2 is carried such as SEQ ID NO:The DNA sequence dna of the Pim-1 genes shown in 1.
The described carrier of adeno-associated virus 2 selects the pUC pUCs of AAV tri-, by AAV carriers pAOV-CAGMINI-eGFP-2A- MCS-3FLAG, packaging plasmid pAAV-RC and helper plasmid pHelper are constituted；Genes of interest Pim-1 is opened by CAGMINI promoters It is dynamic；Pim-1 gene insertion vector MCS polyclone enzyme enzyme sites, C-terminal band 3 × Flag label proteins of Pim-1.
The second aspect of the present invention, there is provided the structure of the carrier of recombinant adeno-associated virus 2 of above-mentioned carrying Pim-1 genes Method, specifically includes following steps：
The clone of the carrier of recombinant adeno-associated virus 2 of 1.Pim-1 genes builds
According to Pim-1 genes (GenBank ID:NM017034 following PCR primer) is designed and synthesized：
Pim-1 sense primers：5’-CATGGTCCTG CTGGAGTTCG TG-3’
(SEQ ID NO:2)；
Pim-1 anti-sense primers：5’-CATAGCGTAA AAGGAGCAAC A-3’
(SEQ ID NO:3)；
Using PCR method from containing such as SEQ ID NO:Pim-1 gene cDNA clones template shown in 1 carries out Pim-1 mesh Gene magnification；PAOV-CAGMINI-eGFP-2A-MCS-3FLAG carriers and Pim-1 gene PCR products are used into Nhel enzymes Carrier is cut, the purifying of digestion products is carried out after digestion is complete, the gland relevant viral vector after digestion and Pim-1 gene PCRs are produced Thing is attached reaction, and above-mentioned endonuclease bamhi is accurately connected on the NheI sites of pAOV expression vectors by coupled reaction, PAOV-CAGMINI-eGFP-2A-Pim-1-3FLAG expression vectors are obtained, after obtaining connection product, product impression is transformed into In state Escherichia coli, the transformant to growing carries out the clone identification of the plasmid of Pim-1 genes adeno-associated virus 2, electrophoresis result Positive is positive colony, it was demonstrated that genes of interest is oriented to be connected into purpose carrier；The clone positive to PCR identifications gives birth to again Thing is sequenced and analyses and compares, the GenBank ID in sequence and database:NM_017034 sequences are consistent, compare correct, explanation Plasmid construction success；To build and go endotoxin kit to extract by ultrapure with fluorescin eGFP expression plasmids, lead to Transfection reagent lipo2000 transfection 293T cells are crossed, is transfected as green is glimmering by fluorescence microscope to cell after 24 hours The carrier transfection expression of recombinant adeno-associated virus 2 success of light, as Pim-1 genes.
The recombinant adeno-associated virus 2 of 2.Pim-1 genes are packed
Adeno-associated virus packaging system uses three pUC pUCs, by packaging plasmid pAAV-RC and helper plasmid pHelper, The expression plasmid pAOV-CAGMINI-eGFP-2A-Pim-1-3FLAG of recombinant adeno-associated virus 2 with Pim-1 genes passes through transfection Reagent lipo2000 cotransfection 293T cells, before transfection, AAV-293 cell fusions degree need to reach more than 80%, after transfection 72h, Cell is collected by centrifugation, then cell is cracked, after 4 DEG C of 53,000rmp high speed centrifugations 30h, collected rich in restructuring gland related diseases The cell supernatant of malicious 2 particles, obtains the concentrate of the recombinant adeno-associated virus 2 of high titre, after packing, -80 after being concentrated to it DEG C preserve, finally using quantitative PCR determine virus titer.
The requirement that inside and outside is tested to adeno-associated virus titre is different, can obtain different titers in production process according to demand Adeno-associated virus particle, for corresponding experiment.
The third aspect of the present invention, there is provided the carrier of adeno-associated virus 2 of above-mentioned carrying Pim-1 genes is repaiied in preparation treatment Application in multiple crushed optic nerve medicine.
The vehicle treatment crushed optic nerve of adeno-associated virus 2 of the carrying Pim-1 genes built with the present invention.Use first The carrier transfected into rat retina of adeno-associated virus 2 of Pim-1 genes, reapplies interim aneurysm clip and prepares optic nerve and not exclusively damage Wound model, the effect repaired to crushed optic nerve after observation Pim-1 gene overexpressions and mechanism.
Application of the carrier of adeno-associated virus 2 of Pim-1 genes in treatment reparation crushed optic nerve medicine is prepared is carried, The adeno-associated virus 2 of Pim-1 genes can transfect RGCs, clinically, specifically can be using intravitreal medicine transfection RGCs To carry out the gene therapy after optic nerve injury.
Gland relevant viral vector AAV is a kind of acapsular single-stranded wire deficient DNA virus, and the DNA of AAV can be with The form low frequency of dsDNA is integrated into the chromosome of host, and there is AAV carriers latence to stablize, host range is wide, transfection effect The features such as rate is high, expression time is long, immune response is weak, therefore be considered as that the virus for experiments in vivo safest at present is carried Body, is particularly suited for ocular tissue's experiment.There is more than 100 kinds of AAV parting now, be designed to recombinant viral vector there are 12 kinds, Wherein AAV2 types serotype adeno-associated virus because possessing stronger transfecting and specificity in ocular tissue, as treatment optic nerve Damage the most frequently used serumal carrier.This seminar early stage is received with carrying the vehicle treatment of adeno-associated virus 2 of Neuritin genes Optic nerve is damaged, 70% is may be up in the transfection efficiency of RGCs, and obtain good therapeutic effect.
The present invention is based on Pim-1 is in thymus gland, marrow, colon, testis, prostate, angiocarpy, cerebral cortex, hippocampus and regards Unique effect in the tissue such as nethike embrane, with adeno-associated virus 2 (AAV2) carrier for carrying Pim-1 genes, targets Pim-1 gene mistakes In power of regeneration in expression enhancing RGCs, crushed optic nerve is repaired, observe its survival and apoptosis, optic nerve axons to RGCs again The effect of raw and visual performance recovery etc..
Optic nerve is a part of central nervous system (CNS), thus frequently as the preferable portion that research CNS is damaged and regenerated Position.Therefore the reparation after research optic nerve injury and regeneration, new powerful measure and reason will be provided for clinical treatment optic nerve injury By basis, to CNS diseases and the treatment for damaging also important in inhibiting.
Brief description of the drawings
Fig. 1 is the preparation of optic nerve Incomplete injury model
A is 2mm places after adult male SD rats optic nerve eyeball, and 5 seconds are crushed with making with interim mini aneurysm clips Squeezing wound model.B is the optic nerve of the damage 2 weeks of RITC marks, is damage zone at left arrow meaning, and its aixs cylinder is polymerized to group Block, it is distal end portion that right side square frame is signified, and C figures are visible after amplification has optic nerve axons to pass through, and prompts for Incomplete injury model, Scale=100 μm.
Fig. 2 is adeno-associated virus AAV2-Pim-1-eGFP transfected into rat RGCs
4 weeks after rat intravitreal AAV2-Pim-1-eGFP, row inner nuclear layer retina.The big portion of wherein A display retinas Point cell and the upper recombinant adeno-associated virus 2 of nerve fibre transfection, express green fluorescence, and transfection efficiency is up to 50%.B shows AAV2- Pim-1-eGFP Successful transfections RGCs and transfection efficiency are up to 71%.It is thin that C displays AAV2-Pim-1-eGFP hardly transfects star-like colloid Born of the same parents.D displays AAV2-Pim-1-eGFP transfects a part of amakrine.A, B, C scale=50 μm；D scale=15 μm.
Fig. 3 is overexpression of the Pim-1 in retina after transfection
A represents expression of the Pim-1 mRNA in each group retina after normal and optic nerve injury.Vitreum is injected respectively 4 weeks after physiological saline, AAV2-GFP (empty carrier) and AAV2-Pim-1 (materials are normal), optic nerve squeezing wound model is carried out Make, after 2 weeks (materials are damage), detect Pim-1 mRNA in sham-operation group, empty carrier group, treatment with Real-time PCR Expression (n=6, * P in group and sham-operation group, merely damage group, empty carrier group, treatment group<0.05).B represents Pim-1 albumen Expression in each group retina after normal and optic nerve injury.Vitreum difference injecting normal saline, AAV2-GFP and AAV2- 4 weeks after Pim-1, the making that optic nerve squeezes wound model is carried out, detect Pim-1 albumen normal with Western blot after 2 weeks With expression (n=6, * * the * P in each group retina after optic nerve injury<0.001, * * P<0.01).
Fig. 4 is survival effect of the Pim-1 recombinant adeno-associated virus 2 to ganglionic layer of retina cell
Wherein A is dyeed using HE, observes sham-operation group, merely damage group, empty carrier group and treatment group's pattern of retinal ganglion cells The survival condition of confluent monolayer cells；B is quantitative result (n=6, * * P<0.01)；Scale=100 μm.
Fig. 5 is survival effect of the Pim-1 recombinant adeno-associated virus 2 to RGCs
Wherein A uses SABC (gamma synuclein specific marker RGCs), observation sham-operation group, simple damage Hinder the survival condition of group, empty carrier group and treatment group RGCs；B is the quantitative result (n that each group ganglionic layer of retina RGCs is counted =6, * * P<0.01)；Scale=100 μm.Wherein C marks RGCs, each group inner nuclear layer retina to observe from view using FITC-CTB Film center marks the survival condition of RGCs to periphery diverse location FITC-CTB；D：Counting (the n=of RGCs on each group retina 6, * * P<0.01)；Scale=20 μm.
Fig. 6 is influence of the Pim-1 recombinant adeno-associated virus 2 to ganglionic layer of retina Apoptosis
Wherein A is dyeed using the TUNEL of each group eyeball paraffin section, observes the apoptosis feelings of ganglionic layer of retina cell Condition, nucleus is the cell (shown in arrow) of apoptosis in brown color；B is the counting of each group ganglionic layer of retina Apoptosis (n=6, * P<0.05)；Scale=100 μm.
Fig. 7 is the structure figure of the expression plasmid carrier of Pim-1 recombinant adeno-associated virus 2.
Fig. 8 is the line chart of the expression plasmid carrier of II type adeno-associated virus eGFP and Pim-1 recombinant adeno-associated virus 2.
Specific embodiment
The specific embodiment that the present invention is provided is elaborated with reference to embodiment.
Embodiment 1：
1. the acquisition of genes of interest fragment
The sequence of Pim-1 genes is inquired about from GenBank, VectorNTI software Design primers is used, with Pim-1 gene Cs DS The cDNA clone in area is template, and Pim-1 genetic fragments are expanded using PCR.
PCR amplifying target genes：Using the PrimeSTAR enzymatic amplification genes of interest of high-fidelity, reaction system and condition are such as Under：
Table 1：Amplification system with Pim-1 gene cDNA clones as template
The expression vector establishment of 2.Pim-1 recombinant adeno-associated virus 2
(1) digestion of the expression vector of adeno-associated virus 2 and genes of interest PCR primer
By pAOV-CAGMINI-EGFP-2A-MCS-3FLAG carriers (being purchased from Addgene companies), enzyme is carried out using NheI Cut, while PCR primer is carried out into digestion using NheI restriction endonucleases, carrier and genes of interest are reclaimed after agarose gel electrophoresis identification DNA。
Table 2：PAOV-CAGMINI-eGFP-2A-MCS-3FLAG carriers and Pim-1 gene PCR product endonuclease reaction bodies System
(2) coupled reaction
The carrier of adeno-associated virus 2 and the PCR primer of Pim-1 genes after digestion is carried out according to the reaction system in table 1 Coupled reaction.
Table 3：Homologous recombination reaction system
The digestion products of carrier and genes of interest are mixed, 4 DEG C connect 12 hours, connection product is used to convert.
(3) the connection liquid conversion of digestion products
1) take a competent cell to be placed on ice, rear addition to be dissolved 10 μ l connection liquid is being placed after mixing in ice 30min。
2) it is placed in hot compress 90s in 42 DEG C of thermostat water baths.
3) quickly it is transferred into ice bath, cell is cooled down 2-3min.
4) add 900 μ lLB nutrient solutions, place on 37 DEG C of shaking tables, 1h.
5) transformed bacteria solution for taking appropriate amount is coated on LB agar plates.
6) plate, 37 DEG C, 16h in constant incubator are inverted.
(4) the plasmid positive clone identification of Pim-1 genes adeno-associated virus 2
1) transformant grown on picking flat board is resuspended in 10 μ l LB nutrient solutions, and therefrom taking 1 μ l and doing template carries out bacterium colony PCR is identified.
2) PCR reaction systems：
Table 4：PCR reaction systems
By above-mentioned system configurations it is good after mix into performing PCR, 94 DEG C, 5min；94 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 1.5min, 30 circulations；72 DEG C, 10min；4 DEG C, ∞.
3) 1.5% agarose gel electrophoresis identification PCR primer, positive colony is selected according to the result that electrophoresis is positive, is entrusted Nanjing Jin Sirui biological order-checkings company is sequenced, gene size 939bp, such as SEQ ID NO:Shown in 1, sequencing result shows Pim-1 The clone of gene number 1 is the correct positive colony of identification, can enter the experiment flow of the packaging of adeno-associated virus 2.
(5) endotoxin plasmid extraction is gone
Extracted using Axygen plasmid extractions kit and remove endotoxin plasmid, specific extraction steps are as follows：
1) the 5ml bacterium solutions of incubated overnight 2ml is glycerol stock guarantor, and remaining 3ml is at twice in a 1.5ml centrifuge tube 12,000 × g is centrifuged 1 minute, thoroughly removes supernatant；
2) 250 μ l bacterial suspensions are added, oscillator fully resuspended bacterium is subsequently added into 250 μ l bacterial lysates, overturns Mix 4-6 times；Note：Can not be vigorously mixed, contaminating genomic DNA otherwise occurs；
3) 250 μ l bacterial lysates are added, 350 μ l neutralizers is added in five minutes, gently overturned and mix 6-8 times；12, 000 × g, is centrifuged 10 minutes；
4) supernatant is transferred in small scale purification post, small scale purification post is inserted in waste collection pipe, 12,000 × g centrifugations 1 Minute；Precipitation should not be drawn onto, genomic DNA and protein contamination otherwise occurs；The waste liquid abandoned in collecting pipe, by small scale purification Post is inserted in collecting pipe, plus 700 μ l bacterial lysates, in small scale purification post, 12,000 × g is centrifuged 1 minute；Repeat the step One time；The waste liquid abandoned in collecting pipe, small scale purification post is put into same branch collecting pipe, and 12,000 × g is centrifuged 1 minute thoroughly Removal bacterial lysate；
5) small scale purification post is put into new clean 1.5ml centrifuge tubes, plus 50 μ l 2.5mM Tris-HCl wash-outs Liquid, after room temperature is placed 1 minute, 12,000 × g is centrifuged 1 minute；Solution in centrifuge tube is extracted plasmid；
6) extracting plasmid is identified with EcorI and XhoI digestions, determine that digestion post-fragment size is correct, measurement plasmid is dense Degree, for adeno-associated virus packaging.
The poison packaging of 3.Pim-1 gene glands related diseases 2
(1) the carrier system plasmid essential information of adeno-associated virus 2
The Viral Packaging System be three pUC pUCs, by AAV carriers pAOV-CAGMINI-EGFP-2A-Pim-1-3FLAG, Packaging plasmid pAAV-RC and helper plasmid pHelper compositions (all plasmids are purchased from Addgene companies).Genes of interest Pim-1 Started by CAGMINI promoters, the expression property with nerve-specific.Pim-1 genes are inserted into the polyclonal positions of MCS of carrier Point, the C-terminal of gene connects 3 × Flag.Packaging contains the adeno-associated virus 2 of genes of interest Pim-1 respectively, and without genes of interest The control adeno-associated virus 2 (as shown in Figure 8) of Pim-1.
(2) plasmid transfection and viral purification
1) by 3 × 106Individual AAV-293 cells are inoculated in 10cm2In Tissue Culture Dish, 37 DEG C, 5%CO2Trained in incubator Support, be used to transfect after 48h.
2) the AAV-293 cells of passage are checked before transfecting, degrees of fusion reaches 70%-80%.
3) concentration of plasmid is adjusted to 1mg/ml with the TE buffer solutions of PH=7.5, according to needed for packaging tray number is calculated Rotaring redyeing system and plasmid consumption, draw three kinds of each 10 μ l of plasmid and are placed in centrifuge tube, add the 0.3M CaCl of 1ml2, gently mix Close.
4) by mixed DNA/CaCl2/ HBS solution is added drop-wise on cell culture, gently rocks thin during addition simultaneously Born of the same parents' culture plate so that solution tries one's best homogeneous distribution in the medium, 37 DEG C, 5%CO26h is cultivated in incubator.
5) after transfection terminates, fresh culture medium 10ml is changed, continues to cultivate 66-72h.
6) by produce poison cell be collected into the centrifuge tube of 15ml together with culture medium, 200g, 3min, centrifugal separating cell and Supernatant, supernatant is deposited in addition, and cell is resuspended with 1ml PBS.
7) cell suspending liquid is shifted repeatedly in dry ice ethanol bath and 37 DEG C of water-baths, freeze thawing four times.After each freeze thawing slightly Plus concussion.
8) 10000g centrifugations removal cell fragment, centrifuged supernatant is transferred in a new centrifuge tube.
9) 40%PEG8000 is added in supernatant until final concentration 8%, 2h is placed on ice and (is mixed one back and forth per 15min It is secondary), 2500g centrifugation 30min remove supernatant, and precipitation cracks supernatant and merges after using PBS resuspended with cell.
10) 3000g centrifugations 30min, supernatant is transferred in another centrifuge tube (should not have cell fragment).
11) DNA of Benzonase nuclease digestions removal residual is added, in 37 DEG C of incubations after being sufficiently mixed 30min, is filtered with 0.45 μm of filtering head, takes out filtrate.
12) to solid CsCl (density 1.41g/ml) is added in viral concentration liquid, vibration is dissolved.
13) sample is added in ultracentrifugation pipe, is filled up centrifuge tube remaining space with the CsCl solution for preparing in advance.
14) 175000g centrifugations 24h, forms density gradient, and the sample of different densities is collected in distribution in order, collects AAV The component of grain, repeats said process once.
15) 4ml ionized waters are added in Amicon-15 ultrafiltration apparatus, the virus liquid that density gradient centrifugation is obtained is added To in ultrafiltration apparatus, plus PBS to cumulative volume be 4ml.
16) 1500g centrifugations 5-10min, a residual body product is checked per 5min, until final volume is 200-250 μ l.
17) liquid will be filtered it will be collected into and disinfect together, filter membrane will be put back into device.
18) Jia 1 × PBS dilution concentrations after virus make volume for 4ml, repeat said process 3 times, centrifugal ultrafiltration pipe makes Final volume is about 0.5ml, is sampled for virus titer detection.
19) after virus liquid packing, -80 DEG C of preservations.
(3) titer determination of adeno-associated virus 2
Virus titer is determined using quantitative PCR：
1) gradient dilution standard items plasmid and testing sample are to original content 10-5, 10-6, 10-7, 10-8。
2) reaction system of quantitative PCR
Table 5：Quantitative PCR reaction system
3) the μ l of reaction solution 15 are added in each reacting hole, the template of 5 μ l is subsequently adding.
4) machine on, annealing temperature is set to 60 DEG C, obtains Ct values according to standard operation and calculates the copy number in AAV samples.
Embodiment 2：
1. optic nerve squeezes the foundation of wound model
After every group of experimental animal routine disinfection, abdomen is carried out according to the every chloraldurate of rat 10% (0.4ml/100mg) Chamber is anaesthetized, the left eye of preserved skin rat after anesthesia, is placed under microscope and is performed the operation.Chou is cut off at corneal limbus with microscissors Film, then carries out blunt separation until after ball, blood vessel and its hetero-organization should not be injured in operating process in order to avoid causing with microforceps Bleeding, fully exposes optic nerve, and 5s (concrete operations reference the present inventor is crushed using interim aneurysm clip at 2mm after ball Document [31] Wang R, Xu J, Xie J, et al.Hyperbaric oxygen preconditioning are delivered promotes survival of retinal ganglion cells in a rat model of optic nerve crush.J Neurotrauma.2010(27):763-770.) simulation clinic causes optic nerve Incomplete injury (such as Fig. 1 institutes Show).
2. experimental animal packet
(1) sham-operation group：Exposure optic nerve is not damaged, intravitreal injection physiological saline；
(2) damage group merely：Exposure optic nerve is simultaneously damaged, intravitreal injection physiological saline；
(3) empty carrier group：Damage optic nerve, intravitreal injection AAV2-GFP；
(4) treatment group：Damage optic nerve, intravitreal injection AAV2-Pim-1；
The overexpression of Pim-1 after the transfected into rat retina of 3.Pim-1 recombinant adeno-associated virus 2 and transfection
After intravitreal AAV2-Pim-1 4 weeks, total body perfusion materials are fixed on 4% paraformaldehyde after taking out eyeball In 1 day, saccharose gradient dehydration after, row frozen section, then respectively with RGCs specific marker things gamma-synuclein, star The immunofluorescence dyeing of type spongiocyte specific marker thing GFAP, it is found that AAV2-Pim-1 transfects most RGCs, efficiency About more than 70%；And hardly transfect star spongiocyte.After intravitreal AAV2-Pim-1 4 weeks, total body perfusion takes Material, is fixed in 4% paraformaldehyde, row inner nuclear layer retina after 4-6 hours after taking out eyeball, after DPAI dye cores, fluorescence microscope Observation, it is seen that most cells and nerve fibre on AAV2-Pim-1 transfection retinas, in green fluorescence；With amakrine After specific marker thing HPC-1 fluorescent co-locations, it was observed that the amakrine of an AAV2-Pim-1 transfection parts is (such as Fig. 2 institutes Show).
Using immunohistochemistry, the method for Real-time PCR and Western blot observation Pim-1 gland related diseases After malicious 2 transfected into rat retinas, Pim-1 molecules, Pim-1 mRNA and Pim-1 the albumen each group after normal and optic nerve injury Overexpression situation (as shown in Figure 3) in retina.
Protective effect of the 4.Pim-1 overexpression to crushed optic nerve and its RGCs
(1) influence of the Pim-1 overexpression to RGCs confluent monolayer cells numbers
HE is dyeed：The eyeball of each group is taken out through flowing water overnight, dehydration, dimethylbenzene is transparent, FFPE, row HE after section Dyeing, observes RGCs confluent monolayer cells quantity from form.2 weeks after quantitative result display optic nerve injury, Pim-1 restructuring gland related diseases Poison 2 increased the quantity (as shown in Figure 4) of RGCs confluent monolayer cells.
(2) survival of the Pim-1 overexpression to RGCs is acted on
1) immunohistochemistry：After the perfusion of each group SD rat bodies, the embedding of eyeball routine paraffin wax is taken, primary antibody uses gamma Synuclein rabbit polyclonal antibodies, are not added with primary antibody group as negative control in addition.DAB colour developing observation RGCs survival conditions.Depending on god 2 weeks after damage, survival of the Pim-1 recombinant adeno-associated virus 2 to RGCs has facilitation (as shown in Fig. 5 A, B).
2) CTB marks RGCs：Each group rat first 2 days, the fluorescein-labeled choleratoxin B subunit of intravitreal injection of materials (CTB) after, animal survives 2 days, row inner nuclear layer retina after total body perfusion materials retina is in cross petal centered on regarding nipple Optic cup is cut off, retina is separated, is placed under fluorescence microscope after anti-fluorescence decay agent mounting by ganglionic layer of retina upward Observe and take pictures.Each quadrant takes 3 visuals field (20 × 10) and takes pictures, and respectively in central, the middle and periphery of retina, and uses Image-Pro Plus6.0 image processing softwares carry out cell count to the RGCs that CTB is marked, and average.Optic nerve injury 2 weeks afterwards, the survival (as shown in Fig. 5 C, D) of the RGCs of retina is promoted after Pim-1 overexpression.
(3) detection of the Pim-1 overexpression to RGCs confluent monolayer cells apoptosis situations
Row TUNEL dyeing after each group retina paraffin section, nucleus is the cell of apoptosis in brown color, observes Pim-1 To the apoptosis situation of RGCs confluent monolayer cells after overexpression, 2 weeks after quantitative result display optic nerve injury, Pim-1 restructuring gland related diseases Malicious 2 overexpression reduce RGCs confluent monolayer cells apoptosis numbers, there is inhibitory action (as shown in Figure 6) to Apoptosis.
(4) effect of the Pim-1 recombinant adeno-associated virus 2 to optic nerve regeneration
In rat intravitreal rhodamine (RITC), optic nerve of being drawn materials after 2 days observes the feelings of RGCs axon regenerations Condition.Optic nerve is placed in 4%PFA, and 4 DEG C of fixations overnight, sink to the bottom through the dehydration of 30% sucrose.Optic nerve is placed in has used freezing-microtome On the OCT glue for cutting flat with, embedded with OCT glue afterwards, -20 DEG C freeze.Section is longitudinally continuous in -20 DEG C of freezing-microtomes, thickness is 9 μm.37 DEG C of baking boxs are dried, the situation of fluorescence microscopy Microscopic observation RITC spike aixs cylinders after fluid mounting.
The effect that 5.Pim-1 overexpression recovers to visual function
1) pupillary light reflex
Each group animal (sham-operation group, merely damage group, empty carrier group, treatment group, n=6), 2 weeks after damage, after anesthesia, After darkroom adapts to 30min, will not perform an operation branch hole eyeball masking foil lucifuge, with certain light boosting operation branch hole eyeball, adopt Taken pictures (experimental technique bibliography [32] de Lima S, Koriyama Y, Kurimoto T, et with stereomicroscope al.Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.Proc Natl Acad Sci U S A,2012(109):9149- 54).Kinescope: Kinescope Expert software start recording pupil of left eye is contracted to the time of minimum from maximum, and detects every group of minimum pupil Diameter and maximum pupil diameter ratio, be repeated once every 5min, be at least repeated 5 times.
2) flash visual evoked potential
After each group Animal Anesthesia, the electric drill for being about 5mm drill bits with diameter first causes cranium in the cerebral cortex occipital lobe area on right side Cranial defect, but endocranium can not be damaged, rat is then fixed on stereotaxic apparatus, stimulation parameter is set：The a width of 5- of ripple 10ms, amplitude is 10v, and time delay is 32s, is superimposed 30 times respectively, selects the waveform of stabilization, records the latent of VEP P ripples Volt phase and wave amplitude, detect its flash visual evoked potential.Specific method (referring to document [33] Pangratz-Fuehrer S, Kaur K,Ousman SS,et al.Functional rescue of experimental ischemic optic neuropathy with alphaB-crystallin.Eye(Lond),2011(25):809-17)。
Below the preferred embodiment to the invention is illustrated, but the invention be not limited to it is described Embodiment, those of ordinary skill in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
SEQUENCE LISTING
<110>Second Military Medical University, PLA
<120>A kind of adeno-associated virus 2 of carrying Pim-1 genes and its application in crushed optic nerve is repaired
<130> /
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 939
<212> DNA
<213>Artificial sequence
<400> 1
atgctcttgt ccaagatcaa ctccctggcc cacctgcgcg cagccccttg caacgacctg 60
cacgccaaca agctggcgcc gggcaaagag aaggagcccc tggagtcgca gtaccaggtg 120
ggcccgctgt tgggcagcgg tggcttcggc tcggtctact cgggcatccg cgtcgccgac 180
aacttgccgg tggccatcaa gcacgtggag aaggaccgga tttccgactg gggggaactg 240
cccaacggca cccgagtgcc catggaagtg gtcctgctga agaaggtgag ctcgggcttc 300
tcgggcgtca ttagacttct ggactggttc gagaggcccg atagtttcgt gctgatcctg 360
gagaggcccg aacccgtgca agacctcttc gacttcatca ccgagcgagg agccctccag 420
gaggagctgg cccggagctt cttctggcag gtgctggagg ccgtgcggca ttgccacaac 480
tgcggggttc tccaccgcga catcaaggac gagaacatct taatcgacct gaaccgcggc 540
gaactcaaac tcatcgactt cgggtcgggg gcgctgctca aggacacagt ctacacggac 600
tttgacggaa cccgagtgta cagtcctcca gagtggattc gctaccatcg ctaccacggc 660
aggtcggctg ctgtttggtc cctggggatc ctgctctatg acatggtctg cggagatatt 720
ccatttgagc acgacgaaga gatcgtcaag ggccaagtgt actttaggca aagggtctct 780
tcagaatgtc aacatcttat tagatggtgc ctgtccctga gaccatcgga ccggccctcc 840
tttgaagaaa tccagaacca tccgtggatg caggatgttc tcctgcccca ggccaccgcc 900
gagattcatc tgcacagcct gtcaccatca cccagcaaa 939
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
catggtcctg ctggagttcg tg 22
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
catagcgtaa aaggagcaac a 21

Claims (4)

1. a kind of carrier of recombinant adeno-associated virus 2 of carrying Pim-1 genes, it is characterised in that the described carrier of adeno-associated virus 2 It is to carry such as SEQ ID NO:The DNA sequence dna of the Pim-1 genes shown in 1.
2. the carrier of recombinant adeno-associated virus 2 of carrying Pim-1 genes according to claim 1, it is characterised in that described The carrier of adeno-associated virus 2 selects the pUC pUCs of AAV tri-, by AAV carriers pAOV-CAGMINI-eGFP-2A-MCS-3FLAG, packaging Plasmid pAAV-RC and helper plasmid pHelper is constituted；Genes of interest Pim-1 is started by CAGMINI promoters；Pim-1 genes are inserted Enter carrier MCS polyclone enzyme enzyme sites, C-terminal band 3 × Flag label proteins of Pim-1.
3. a kind of construction method of the carrier of recombinant adeno-associated virus 2 of carrying Pim-1 genes as claimed in claim 2, it is special Levy and be, comprise the following steps：
A) clone of the carrier of recombinant adeno-associated virus 2 of Pim-1 genes builds that to design and synthesize PCR primer as follows：Pim-1- is just To primer such as SEQ ID NO:Shown in 2；SEQ ID NO in Pim-1- reverse primers:Shown in 3；
Using PCR method from containing such as SEQ ID NO:Pim-1 gene cDNA clones template shown in 1 carries out the base of Pim-1 mesh Gene-amplification；PAOV-CAGMINI-eGFP-2A-MCS-3FLAG carriers and Pim-1 gene PCR products are carried using Nhel digestions Body, carries out the purifying of digestion products after digestion is complete, the gland relevant viral vector after digestion and Pim-1 gene PCR products are entered , be accurately connected to above-mentioned endonuclease bamhi on the NheI sites of pAOV expression vectors by coupled reaction by row coupled reaction, is obtained PAOV-CAGMINI-eGFP-2A-Pim-1-3FLAG expression vectors, after obtaining connection product, are transformed into competence big by product In enterobacteria, the transformant to growing carries out the clone identification of the plasmid of Pim-1 genes adeno-associated virus 2, and electrophoresis result is positive Be positive colony, it was demonstrated that genes of interest is oriented to be connected into purpose carrier；The clone positive to PCR identifications carries out biometric again Sequence and analyses and comparison, if the GenBank ID in sequence and database:NM_017034 sequences are consistent, compare correctly, illustrate matter Grain is successfully constructed；To build and go endotoxin kit to extract by ultrapure with fluorescin eGFP expression plasmids, pass through Transfection reagent lipo2000 transfects 293T cells, is transfected as green is glimmering by fluorescence microscope to cell after 24 hours The carrier transfection expression of recombinant adeno-associated virus 2 success of light, as Pim-1 genes；
B) recombinant adeno-associated virus 2 of Pim-1 genes are packed
Adeno-associated virus packaging system uses three pUC pUCs, by packaging plasmid pAAV-RC and helper plasmid pHelper, with The expression plasmid pAOV-CAGMINI-eGFP-2A-Pim-1-3FLAG of recombinant adeno-associated virus 2 of Pim-1 genes is tried by transfecting Agent lipo2000 cotransfection 293T cells, before transfection, AAV-293 cell fusions degree need to reach more than 80%, after transfection 72h, will Cell is collected by centrifugation, and then cracks cell, after 4 DEG C of 53,000rmp high speed centrifugations 30h, collects and is rich in recombinant adeno-associated virus 2 The cell supernatant of particle, obtains the concentrate of the recombinant adeno-associated virus 2 of high titre after being concentrated to it, added in concentrate Glycerine, final concentration of 5%, after packing, -80 DEG C of preservations finally determine virus titer using quantitative PCR.
4. a kind of carrier of recombinant adeno-associated virus 2 of carrying Pim-1 genes as claimed in claim 1 or 2 is repaiied in preparation treatment Application in multiple crushed optic nerve medicine.
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