Experimental Research On Co-cultivation Of Bone Marrow Mesenchymal Stem Cells Modified By GDNF Gene And Anoxia/Reoxygen Neural Cells

Objective:To study the characteristics of protein expression of bone marrow mesenchymal stem cells (BMSCs) modified by glial cell line-derived neural factor(GDNF)gene and the protection on Anoxia/Reoxygen neural cells. Methods: BMSCs in SD rats were cultured in vitro. PAdEasy-1-pAdTrack-CMV-GDNF was transfected to the BMSCs (BMSCs/ GDNF). The method of Enzyme-linked immunosorbnent assay (ELISA) was used to detect GDNF protein expressing of BMSCs/GDNF and BMSCs. The difference between them was analysed. The cortical neural cells which derived from newborn SD rats were cultured. The model of anoxia/reoxygenation of cultured neural cells were established. Neural cells which were primary cultured for 5d were set in the anoxic incubator for 4h. After that, they were put into CO2 incubator for reoxygen and continued culturing. According to the difference of reoxygen time, the neural cells were divided into 12h,1d,2d,3d and 5d five subgroups. BMSCs/GDNF or BMSCs which had been induced to neural cells direction were co-cultured with the anoxia/reoxygen neural cells. Hoechst33258 dying method on neural cells apoptosis was used to observe the difference between BMSCs/GDNF and BMSCs protecting injured neural cells and resisting neural cells apoptosis. Immunofluorescent-chemistry (IFC) method was used to observe the characteristics of co-culturing cells expressing growth-associated protein-43(GAP-43). Results:(1) The content of GDNF protein in culturing cell supernatant of BMSCs/GDNF express stably about (129.28±12.39) pg/ml from 3d to 12d. But BMSCs almost didn't express GDNF protein. Comparing with BMSCs, BMSCs/GDNF expressed GDNF protein. The dramatically difference between BMSCs/GDNF and BMSCs possessed statistical significanc(eP<0.01). (2) Comparing with BMSCs, the GDNF protein were secreted by BMSCs/GDNF could reduce remarkably the rate of anoxia/reoxygen neural cell apoptosis(12h～5d, P<0.05).(3) BMSCs/GDNF or BMSCs which had been induced to neural cell derection and anoxia/reoxygen neural cells also expressed GAP-43 protein. GAP-43 protein expressing in BMSCs/GDNF co-culturing group was higher than that in BMSCs co-culturing group. The difference had statistical significance(1d～5d, P<0.05). Conclusion: BMSCs/GDNF could express stably GDNF protein. BMSCs/GDNF could alleviate neural cell injury obvously because of anoxia/Reoxygenation and decrease neural cell apoptosis. BMSCs/GDNF could express and promote anoxia/reoxygen neural cells expressing the GAP-43 protein.