BDNF基因修饰淋巴细胞的蛋白表达及对PC12细胞的增殖和H_2O_2损伤的影响

Effect of lymphocytes modified by brain-derived neurotrophic factor gene on the proliferation and the H_2O_2-induced apoptosis of PC12 cell

目的 研究脑源性神经营养因子 (brain derivedneu rotrophicfactor,BDNF)基因修饰淋巴细胞的蛋白表达及对PC12细胞的增殖和H2 O2 损伤的影响。方法 采用Lipofec tAMINE将重组大鼠BDNFcDNA导入PA317细胞 ,获取高滴度的病毒上清转染大鼠淋巴细胞 ,流式细胞仪 (FCM)和免疫组化检测BDNF的表达 ,MTT法检测PC12细胞增殖 ,PI染色流式细胞仪 (FCM)检测PC12细胞凋亡。结果 BDNF基因修饰的大鼠淋巴细胞高表达BDNF蛋白 ,其培养上清可促进PC12细胞增殖并能抑制H2 O2 诱导PC12细胞凋亡。结论 BDNF基因修饰淋巴细胞能高表达和分泌具有生物活性的BDNF。

AIM To study the expression of brain-derived neurotrophic factor (BDNF) in lymphocytes modified by BDNF gene and its effect on the proliferation and the H 2O 2-induced apoptosis of PC12 cells for the transfer of ex vivo therapeutic gene into central nervous system (CNS) tissue with atraumatic means. METHODS Recombined BDNF cDNA plasmid was transfected by LipofectAMINE into the packing cell line PA317 and G418-resistant clones with highest titer was selected. Rat lymphocytes were infected repeatedly with virus supernatant. The expression of BDNF in rat lymphocytes was assayed by FCM and immunohistochemistry. The proliferation of PC12 cells was evaluated by MTT assay. H 2O 2-induced apoptosis of PC12 cells was determined by PI stain flow cytometry. RESULTS BDNF expressed in rat lymphocytes genetically modified and the concentration of BDNF in the conditioned medium of engineered lymphocytes had a linear correlation with the proliferation of the PC12 cells. The supernatant of lymphocytes modified by BDNF gene decreased the apoptosis of PC12 cells induced by H 2O 2. CONCLUSION Rat lymphocytes genetically modified can express and secret active BDNF in vitro.