Experimental Study Of Lif Gene Modified Human Umbilical Cord Mesenchymal Stem Cells Transplantation In The Treatment Of Early Diabetic Retinopathy In Rats

Objective:To observe the effects of LIF-hUCMSCs on diabetic rats animal model of early retinopathy in immune and neurotrophic factor expression,and the curative effect on retinopathy.To investigate the feasibility of LIF-hUCMSCs for DR treatment and to accumulate experience for MSCs transplantation for DR treatment.Methods:hUCMSCs were obtained by trypsin digestion and cultured in vitro,the detection of the surface marker of immune flow cytometry,parallel detection of the cell cycle,adipocytes and osteoblasts differentiation of hUCMSCs was induced.hUCMSCs stably transformed cell line expressing LIF was constructed.Fluorescence quantitative PCR verified expression effect.MTT,PNPP,Annexin V-FITC/PE method of LIF-hUCMSCs compared with hUCMSCs proliferation,differentiation and apoptosis,ELISA detection of IFN-?,IL-4 and NT-4 content,the migration ability of Transwell cells after coculture with RMEC cells was examined by experiments.STZ induced diabetic rat model by intraperitoneal injection,the experimental animal were divided into four groups:normal control group?A group?,diabetic control group?B group?,the control of hUCMSCs injection group?C group?and LIF-hUCMSCs injection group?D group?;After 3 months of modeling,HE of retinopathy was detected and treated successfully after intervention.4 weeks after intravitreal injection,the retinal function of F-ERG detection,FITC-dextran examination of retinal blood vessels,HE observation of the retinal structure,the expression of PCR and WB method to detect the expression of ADPN and hs-CRP,ELISA were detected in NT4.Results:1.Flow cytometry was used to detect the expression of hUCMSCs,CD90positive,CD34 and CD45 negative;Cell cycle analysis showed that it has strong ability of division and proliferation.Adipogenic differentiation and osteogenic differentiation showed its potential of multi-directional differentiation in vitro.2.A lentiviral vector carrying LIF gene was successfully constructed,with a titer of 1.92×10¹⁰?TU/L?.3.Building LIF-hUCMSCs of stabletransfection cell lines;Over expression of LIF group compared with the negative control group,no significant difference detected by MTT?P>0.05?,PNPP had significant difference?P<0.01?test method,Annexin V-FITC/PE double staining method to detect LIF expression significantly inhibit cell apoptosis,ELISA methods to detect LIF overexpression group compared with the negative control group,IFN-?content decreased,IL-4 and NT-4 were significantly increased,the difference was statistically significant?P<0.01?;After coculture with RMEC,the number of migrating cells per field was 70.66 in LIF-hUCMSCs group and36.33 in hUCMSCs group,and the difference was statistically significant?P<0.01?.4.After intraperitoneal injection of STZ blood glucose greater than 16.7 mmol/L reached the standard model,HE showed that the diabetic group internal limiting membrane structure is not complete,reduce the number of ganglion cells;Intravitreal injection after 4 weeks,F-ERG record C and D group compared with B group,D group compared with C group,Rod-R a and b peaks decrease in latent time and amplitude increase,Ops amplitude increased significantly?P<0.01?;FITC-dextran showed C,D group compared with B group,retinal vascular dilatation and reduced tortuosity improvement.The retinal HE showed that the C and D groups compared with the B group,the cells arranged neatly,and the nerve fiber layer edema reduced.Fluorescence quantitative PCR detection and WB method,B,C,D group compared with A group the expression of APN and hs-CRP increase,C,D compared with B group and D group compared with C group increased the expression of APN,decreased expression of hs-CRP;ELISA detection of A group compared with B,C,D group,the difference of NT-4 were statistically significant?P<0.01?,C,D group compared with B group,D group compared with C group,the differences were statistically significant?P<0.05?.Conclusion:1.MSCs isolated from human umbilical cord has high purity,strong proliferation and differentiation potential.2.Compared with hUCMSCs,IFN-?decreased,IL-4 and NT-4 increased by LIF-hUCMSCs;LIF can significantly reduce the differentiation and apoptosis of hUCMSCs,enhance proliferation and migration ability.3.LIF-hUCMSCs can upregulate the expression of APN and NT-4,reduce the expression of hs-CRP in the retina of diabetic rats,improve the retinal function of diabetic rats,and have broad application prospects in the field of DR therapy.