The Experimental Study Of Lentiviral Vector Mediated GAP-43Gene-modified Bone Mesenchymal Stem Cells In The Treatment Of Rat Optic Nerve Injury

Optic nerve injury is a common clinical ocular trauma and often leads topermanent loss of visual acuity. It is one of the blind eye diseases. At present， mainlywe often use the drug to treat this disease in clinical such as high-dose steroidtherapy，but the treatment effect is not obvious.For its extent of poor visual acuityand more side effects， restoring eyesight after optic nerve injury becomes acomplex question which need us to overcome， and to find an effective treatmentis a concern of ophthalmic scholars.After optic nerve injury pathological studies confirmed the main optic nervepathology is the degeneration of retinal ganglion cells (RGCs) axonal，it lead toedema and apoptosis of retinal ganglion cells. Therefore， the most important thingis to promote the regeneration of RGCs after optic nerve injury， and select theappropriate method to suppress apoptosis is a prerequisite for its survival. of ganglioncellsThe study on bone marrow mesenchymal stem cells (BMSCs) has become acentral issue， such and inducing their differentiation in a variety of conditions intodifferent cells and transplantation in the treatment of different diseases， but theapplication of research reports on ophthalmology is not much. GAP-43is a nervegrowth-associated protein， is closely related to neural development andregeneration. This study will combine GAP-43with bone marrow mesenchymal stemcells in both hot research together， through the establishment of the optic nerveinjury model in rats， explore lentivirus-mediated GAP-43gene transfection of mesenchymal stem cell transplantation in the treatment of large feasibility rat opticnerve injury.Part one Construction of Lentiviral-mediated GAP-43Overexpression andInhibition of the Expression VectorsObjective:To construct lentiviral-mediated GAP-43overexpression and inhibition ofexpression vectors.Methods:1、The PLenti6/v5-GP-43Plasmid was set up by the digestion of NotI and NsiIdouble restriction enzyme and ligation of T4ligase， and then the Plasmid wastransformed inio Top10cells. Several clones were picked for enzymatic digestion andonly the clones with correct result were chosen for sequencing. Co-transfecting the293T cells with the PLenti6/V5-GP-43and the Packaging mixture byLipofectamine2000，the purified plasmid from the positive clone was used to producethe lentivirus..Viral supernatant was harvested and titer was determined.2、Construction lentivirus-mediated inhibition plasmid. Construct for the fourtarget genes (Gap43-rat-619， Gap43-rat-844， Gap43-rat-1215， Gap43-rat-678)，everyone of the four interfering plasmids and the vector with the high expression ofGAP-43gene were chosen to infected the293cells. The most effective plasmid wasused to construct the lentiviral vector by means of recombination technique. Thenthe lentiviral vector and packaging plasmid (mix) were also used to infected293Tcell. NEXT， the lentiviral with viral titer determined was harvested after the viralfluid was concentrated by ultra filtration.Results:1、 V5-GAP-43sequence is correct.by restriction enzyme digestion andsequencing.293T cells were harvested after a viral packaging storage solution，viraltiter is2*108TU/ml.2、V3-shRNA-GAP-43sequence is correct.by restriction enzyme digestion andsequencing.293T cells were harvested after a viral packaging storage solution，viral titer is2*108TU/ml.Conclusion:We constructed GAP-43gene overexpression and inhibition of expression oflentiviral vectors successfully and confirmed the efficiency of virus titers in the293Tcells.Part two Experimental Study of Lentivirus-mediated GAP-43GeneTransfection Bone Marrow Mesenchymal Stem CellsObjective:To culture and identify rat bone marrow-derived mesenchymal stem cells，andto detect the over-expression and morphological changes after GAP-43genetransfected to BMSCS，Methods:The simple adherent method was adopted in isolating and culturing experimentsof bone marrow mesenchymal stem cells(BMSCs).The inverted microscope wasused to observe cells. The flow cytometry was used in checking cell mark antigens.Lentivirus carrying GAP-43infected BMSCs (MOI=20)， using qPCR and Westernblot to detect GAP-43expression levels. Then observe morphological changes ofBMCSs after transfection，detect the mRNA ’s expression by RT-PCR.The results:The morphological characteristics of the major of the BMSCs were unified intopolygonal or fusiform，germinated into a spiral shape. Both high expression of CD90、CD44and low expression of CD l1b and CD34were detected by the flow cytometry.We confirmed exogenous GAP-43expression in GAP-43-BMSCs by qPCR andWestern blot..3days after GAP-43gene transfection， BMSCs cell body began toshrink， refraction enhanced cell protrusions， and differentiate into the typicalneuron-like cell，RT-PCR detection of NSE，nestin，NF，βⅢ-tubulin are positiveexpression.Conclusion:The simple adherent method was a feasible way to isolate，culture and purify BMSCs successfully. It layed foundation for later experimental procedures of thisresearch.Lentiviral-mediated GAP-43can efficiently infect BMSCs， anddifferentiate into neuron-like cells.Part Three Experimental Study of Bone Marrow Mesenchymal Stem CellsDifferentiate into Neuron-like Cells after Inhibition of GAP-43GeneObjective:To observe bone marrow mesenchymal stem cells differentiate into neuron-likecells after inhibition of GAP-43geneMethods:Lentiviral-mediated GAP-43-shRNA transfected bone marrow mesenchymalstem cells， to determine the concentration of puromycin， using qPCR and Westernblot to detect GAP-43expression levels. Observe morphological changes of BMSCsafter differentiation， using RT-PCR to detect expression changes of NSE， nestin，NF， βⅢ-tubulin mRNA.Results:Lentiviral-mediated GAP-43-shRNA transfected bone marrow mesenchymalstem cells in the retina condition differentiation was induced， qPCR and Westernblot results show， the expression of GAP-43， NSE， nestin， NF， β Ⅲ-tubulinmRNA were significant low levels in GAP-43-shRNA group compared with thenegative control group (BMSC group)，the difference was statistically significant (p<0.05).Conclusion:The ability of BMSCs differentiation into neuron-like cells was significantlyweakened when GAP-43gene was inhibited.Part Four The Experimental Study of Lentiviral Vector Mediated GAP-43Gene-modified Bone Mesenchymal Stem Cells in the Treatment of Optic NerveInjury in RatsObjective:To investigate the mechanism of lentiviral vector mediated GAP-43 gene-modified bone mesenchymal stem cells in the treatment of optic nerve injury inrats， expected to provide new ideas for the treatment of optic nerve damage， andexplore a new way.Methods:The rat model of optic nerve injury was set up by optic nerve clamping. The120rats were randomly divided into sham operation group (Sham group)， solvent controlgroup (PBS group)，no-load lentiviral group (GFP/BMSCs group)，GAP-43fusiongene lentivirus group (GAP-43/BMSCs) and shRNA-GAP-43fusion gene lentivirusgroup (shRNA-GAP-43/BMSCs group). The24rats of each group were divided intofour sub-groups on the3rdd，7thd，14thd， and28thd after the operation.5ul1×PBSsolution or1×105/number of cells was injected only into the subretinal space.UsingHE staining to detect retinal pathology after transplantation using RT-PCR andWestern-blot to detect expression changes of GAP-43mRNA.Results:IN the3rdd，7thd，14thd，28thd days group after optic nerve injury model，retinal pathology changes in GAP-43/BMSCs group were significantly lighter thanthe other groups. In addition， compared with the sham group， PBS group， GFP/BMSCs group， shRNA-GAP-43/BMSCs group， mRNA and protein expressionof retinal tissue in GAP-43/BMSCs group，GAP-43gene expression increased，thedifferences were statistically significance (P <0.05).Conclusion:We constructed the optic nerve injury in rats successfully. Lentiviral-mediatedGAP-43-transfected BMSCs transplanted into the subretinal space can expresseGAP-43gene very efficiently， it plays a role in nerve repair process.