| Objective:To evaluate the effects of pigment epithelium derived factorgene-modified human umbilical cord mesenchymal stem cells(PEDF-MSCs) on the expression of PEDF and vascular endothelial growth factor(VEGF) and protective effects in early diabetic retinopathy of rats.Methods: h UCMSCs were isolated from human umbilical cord tissue using trypsin digestion method. h UCMSCs surface marker expression was then evaluated using flow cytometry. The PEDF lentiviral vectors(LV-PEDF) were conducted using gene recombination and then sequenced. h UCMSCs were transfected by LV-PEDF with MOI 10,30,50, respectively. Real-time PCR and ELISA methods were used for detecting the expression of PEDF and VEGF. Diabetes was induced by intraperitoneal injection(ip) of streptozotocin(STZ). The rats were divided into five groups: normal control(N), diabetic control(D), PBS-treated(B), GFP-MSC-treated(G) and PEDF-MSC-treated diabetic(P) group. For the P and G group, the rats were treated with PEDF-MSCs and GFP-MSCs, respectively; for the B group, the rats were treated with normal PBS; for the N and D group, the rats were observed only. 2 weeks after intravitreal injection, PEDF-MSCs and GFP-MSCs were observed wether they would survive in the eyes, respectively. The changes of retina in different groups were detected by HE staining, and the thickness of inner plexiform layer(IPL), inner nuclear layers(INL), and outer nuclear layers(ONL) were measured by computer-based image analytical system.The expression of PEDF and VEGF were compared by real-time PCR and immunohistochemistry among all groups, respectively.The changes of retinal vascular morphology and tortuosity dilation were observed after the intravitreal treatment.Results: Results showed that the expression of CD105, CD73, CD90 were positive, while the expression of CD34, CD45, CD11 b, CD19 and HLA-DR were negative. By the analysis of gene sequencing and comparison(1257/1257=100%), the sequencing result was consistent with the target sequence. The best MOI was 50 and the transfer efficiency was up to 72.1%. Real time-PCR results showed that PEDF m RNA expression levels was higher in PEDF-MSCs than the control group(P<0.01). ELISAresults showed that, after transfection, PEDF protein expression in the supernatant of PEDF-MSCs were higher than the control group. The total success rate of intraperitoneal injection of STZ was 95%. 2 weeks after intravitreal injection, green fluorescence was observed in the vitreous of P and G group rats under fluorescence microscope; no obvious green fluorescence was found in the retina. HE staining showed that, 1 months of diabetes, edema of the nerve fiber layer(NFL) and thickening of IPL appeared, 4 months of diabetes, NFL edema was aggravated, retinal blood vessels dilated, structure of IPL loosed, INL cells arranged in disorder. 6 months of diabetes, NFL edema was aggravated, the local INL and the outer nuclear layer( ONL) thinning appeared. 2 months after intravitreal injection of PEDF-MSCs, IPL INL and edema was significantly reduced, ONL layers of cells arranged regularly. 2 months after intravitreal injection, the thickness of IPL in P group was significantly decreased; the thickness of INL and ONL was higher. Retinal vascular morphology analysis showed that, 2 months after treatment, vascular area in the peripheral retina of P group was significantly decreased. Immunohistochemical staining results showed that 2 months after intravitreal treatment, in P group, the average optical density values of PEDF were improved; the average optical density values of VEGF were decreased. Real-time PCR test results showed that, 2 months after treatment, the expression level of PEDF m RNA in P group were improved. The expression levels of VEGF m RNA were decreased.Conclusion: The authors have demonstrated that PEDF-MSCs can survive in eyes with diabetes, which can upregulate the expression of PEDF and downregulate the expression of VEGF. Intravitreal transplantation of PEDF-MSCs can reverse the changes of injured neuroretina, prevent the retinal cell death and maintain normal retinal thickness in diabetic rats. |
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