Effect Of Human Umbilical Mesenchymal Stem Cells Transplanted Into Vitreous Cavity Of Diabetic Rat

Objective To investigate the effects on retina after human umbilical cord mesenchymal stem cells (hUMSCs) transplanted into the vitreous cavity of diabetic rats.Methods The umbilical cord from full-term neonates delivered abdominally were collected in sterile condition. hUMSCs were obtained by enzyme digestion and cultured in DMEM/F12 medium. Surface antigen of hUMSCs was detected by flow cytometry. Seventy-eight adult SD rats were randomly divided into two groups: diabetes and normal control. After high blood glucose continued 6 weeks,2μl DMEM/F12 and hUMSCs suspensions labeled by red fluorescence PKH-26 were immediately transplanted into the left eye, and the right eye as control eyes, which were injected with equivalency DMEM/F12. The diabetic group was divided into 1 W, 2 W,4 W group according to the different time of transplantation. The animals were killed with depth of anaesthesia at different time points. The survival, migration, and integration of hUMSCs transplanted were observed by immunofluorescence microscopy. The changes of retina in different groups were detected by HE staining, and the thickness of inner nuclearl layers/outer nulcear layers were measured by computer-based image analytical system. The expression of GFAP was compared between hUMSCs and DMEM/F12 groups by immunofluorescent labeling. The expressions of mRNA and proteins of GFAP, NSE and rhodopsin were assayed by semi-quantitative real time PCR and Western-Blot respectively.Results1.Mesenchymal stem cells derived from human umbilical cord were spindle shaped adherent cells. They have strongly proliferation ability. Flow cytometry analysis showed that hUMSCs from the standard inbred strains were positive for the surface, antigens CD29, CD105(SH2), CD73(SH3); negative for CD14, CD34, CD31, CD45, HLA-DR. The rate of positive cells exceeded 95%, which tested the purity of cells was high very much. 2. After transplantation, hUMSCs labeled by PKH-26 were mostly present along with ILM and only a few cells were integrated into GCL. The thickness of INL was significant differences between diabetes and normal groups (P<0.05). There were significant differences between U4&D4 groups (P<0.05); no significant after transplanted 1W or 2 W (P>0.05). There were no significant differences about the thickness of ONL between every two of groups (P>0.05).3. HE staining showed that inner retina changed in DM campared with normal. There were no significant differences between U1 and D1; RGCs reduced in U2&D2, and INL & ONL were thinner in D2; INL was thinner, NFL was flatter, and ONL was more confused in D4 than in U4.4. Immunofluorescence of GFAP was detected significant differences between hUMSCs and DMEM/F12 groups after transplanting 2 or 4 weeks.5. The expressions of GFAP, NSE and rhodopsin mRNA and proteins were detected significant differences between diabetes and normal groups.1 week after transplantation, the expressions of GFAP, NSE and rhodopsin mRNA were detected no significant differences between hUMSCs and DMEM/F12 by semi-quantitative real time PCR (P>0.05); while 2 and 4 weeks after transplantation, the expressions of GFAP and rhodopsin mRNA were significant differences(P<0.05), after 4 weeks, there was significant differences in the expressions of NSE mRNA (P<0.05).Conclusions1. STZ induced diabetic rat is a reliable animal model which can be used to imitate human nonproliferation diabetic retinopathy.2. hUMSCs transplanted into vitreous cavity can survival, migrate and integrate into injured retina.3. hUMSCs transplantation may be a valuable neuroprotection tool for treating retina influenced by inducing relative genes and proteins in retina.