| Objective: The application of lentiviral transfection after overexpression of CXCR4 rat bone marrow mesenchymal stem cells(BMSCs)by intravitreal injection of transplanted into diabetic rats,observe the expression of CXCR4-BMSCs increased distribution in the retina,which has a better therapeutic effect,and to explore its therapeutic mechanism of possibly retinopathy of diabetic rats the.Through transplanting overexpressing CXCR4 bone marrow mesenchymal stem cells(BMSCs)into diabetic sprague-dawley rats by intravitreal injection,to observe the therapeutic effects,and investigate the probably mechanisms of BMSCs therapeutic for providing a new theory basis for clinical application of MSCs in future.Methods: 1.BMSCs were isolated and cultured,and were transfered to the third passage and verified by flow cytometry.2.BMSCs were infected by lentivirus constructed with CXCR4.The expression of CXCR4 gene was examined through immunofluorescence,western blot and q-PCR.To detect BMSCs proliferation by MTS cell proliferation assay.The chemotaxis of BMSCs was detected by Transwell chamber,and to observed whether the migration ability of BMSCs was enhanced after transfection of CXCR4 gene under SDF-1 induction.The apoptosis degree of BMSCs was examined by flow cytometry,and to observe whether the degree of apoptosis of BMSCs was changed after transfection of CXCR4 gene.3.The third generation of CXCR4 overexpressing BMSCs was transplanted into 4W diabetic rats induced by STZ,and the general situation of blood glucose,body weight and so on before transplantation and after transplantation in 1W,2W,3W rats were detected.All rats were devided into normal control group(normal group),diabetic rats control group(Blank group),phosphate buffered saline treatment group(PBS group),untreated BMSCs injection treatment group(native group),injection of GFP virus infection in BMSCs treatment group(GFP group),injection of CXCR4-GFP virus infection in BMSCs group(group CXCR4).At post injection 1st week,2nd week and 3rd week,retina tissues of rats were carried out HE pathologic histology examination.The expression of Rhodopsin,NSE and inflammatory cytokines IL-6,TNF-? protein of retina of rats were examined by western blot and immunohistochemisty.Results: 1.BMSCs were isolated and cultured from rat bone marrow cells.They were high purity,uniform shape and have the typical characteristic of MSC.2.After infected by lentivirus constructed with CXCR4,BMSCs showed overexpression of CXCR4 protein.The BMSCs remain have in vitro amplification ability and immature status after lentivirus CXCR4 transfection.The proliferation ability of CXCR4-BMSCs were not significantly different from control group(P > 0.05).The migratory cells amount of CXCR4-BMSCs were more the control group(P<0.05).The apoptosis level CXCR4-BMSCs were not significantly different from control group(P > 0.05).3.Compared with the control group,the degree of retinal inflammation was significantly reduced after intravitreal transplantation of CXCR4-BMSCs.The expression of Rhodopsin,NSE protein was significantly increased than control group,the expression of inflammatory cytokines IL-6,TNF-protein was significantly decreased than control group examined by western blot and immunohistochemisty(P<0.05).Conclusion: 1.It was the first time to report that transplanting BMSCs infected by lentivirus constructed with CXCR4 treated the diabetic rat model.After trasfection,the migration and homing capacity of overexpression CXCR4 BMSCs were increased significantly.It can maximize the therapeutic capacity of MSCs.2.Overexpression CXCR4 BMSCs could better reduce the inflammatory reaction in retinopathy of diabetic rats.It can participate in the induction of the expression of related proteins in the retina,so as to better play the role of repair of retinal injury.These provided a new theory basis for clinical application of gene-modified MSCs for treatment of diabetic retinopathy in future. |
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