【摘要】 目的建立慢病毒介导的胶质细胞系源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)表达系统,体外感染骨髓基质细胞,检测过表达GDNF对蛋白酶抑制剂引起的PC12细胞损伤的神经保护作用。方法经双酶切和T4连接酶构建pLenti6/V5-GDNF表达质粒,经293FT细胞包装产生高滴度病毒。用RT-PCR、ELISA和免疫细胞化学方法检测感染骨髓基质细胞(bone marrow stromal cells, BMSCs)后GDNF的表达,并检测过表达GDNF对蛋白酶抑制剂乳胞素(lactacystin)引起的PC12细胞损伤的保护作用。结果成功构建pLenti6/V5-GDNF表达质粒,获得高滴度具有感染能力的病毒储存液(5.6×105TU/mL)。BMSCs体外被感染后能大量分泌GDNF(接近800pg/mL),过表达GDNF能减轻乳胞素(10μmol/L)引起的PC12细胞损伤。结论慢病毒介导的GDNF转染骨髓基质细胞后能分泌具有生物学活性的GDNF,对蛋白酶体抑制剂引起的PC12细胞损伤有保护作用。更多还原

【Abstract】 Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neuro- trophic factor (GDNF), and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfect- ing it into bone marrow stromal cells (BMSCs). Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×105 TU/mL. After transduction, GDNF-BMSCs successfully secreted GDNF to supernatant with higher concentration (800 pg/mL) than BMSCs did (less than 100 pg/mL). The supernatant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin (10 μmol/L). Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.更多还原