【作者】 熊怀林；

【导师】 余崇林；

【作者基本信息】 泸州医学院 ， 人体解剖学与组织胚胎学， 2009， 硕士

【摘要】 目的:探讨胶质源性神经营养因子( glial cell line-derived neural factor,GDNF)基因修饰的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)表达GDNF蛋白的特点及其对缺氧复氧损伤神经细胞的保护作用。方法:体外培养SD大鼠BMSCs,将pAdEasy-1-pAdTrack-CMV-GDNF重组腺病毒感染BMSCs,用酶联免疫吸附剂测定(Enzyme-linked immunosorbnent assay,ELISA)法检测GDNF基因修饰的BMSCs(BMSCs/GDNF)和BMSCs分泌GDNF蛋白的特点。培养SD乳大鼠大脑皮层神经细胞,制备神经细胞缺氧复氧损伤模型:将原代培养5d的神经细胞放入三气培养箱内缺氧4h,然后移入CO2细胞培养箱内复氧继续培养,根据复氧时间的不同分为12h、1d、2d、3d、5d共5个亚组。将BMSCs/GDNF或BMSCs诱导成神经元样并与缺氧复氧损伤的神经细胞共培养,用细胞凋亡-Hoechst33258染色法检测BMSCs/GDNF对缺氧复氧损伤神经细胞凋亡的影响,用免疫荧光化学(immunofluorescent-chemistry,IFC)法检测共培养细胞表达生长相关蛋白-43(growth-associated protein-43,GAP-43)的特点。结果:(1)GDNF基因修饰的BMSCs(BMSCs/GDNF)的细胞培养上清液中GDNF蛋白含量稳定表达在(129.28±12.39)pg/ml左右,各时间点之间的差异无统计学意义;而BMSCs基本不表达GDNF蛋白。两者相比, GDNF蛋白表达的差异具有统计学意义(P<0.01)。(2)与BMSCs相比,BMSCs/GDNF分泌的GDNF蛋白能显著降低共培养细胞中缺氧复氧损伤神经细胞的凋亡率(12h～5d, P<0.05)。(3)诱导后的BMSCs/GDNF或BMSCs与缺氧复氧损伤的神经细胞均表达GAP-43蛋白,BMSCs/GDNF共培养组的GAP-43蛋白表达明显高于BMSCs共培养组,其差异具有统计学意义(1d～5d,P<0.05)。结论:GDNF基因修饰的BMSCs能稳定表达GDNF蛋白,且能明显减轻因缺氧复氧对神经细胞造成的损伤,减少神经细胞的凋亡,诱导后的BMSCs/GDNF能表达和促进缺氧复氧神经细胞表达GAP-43蛋白。更多还原

【Abstract】 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.更多还原