大鼠GDNF基因的克隆表达及对PC12工程细胞的影响  

Cloning,expression of rat GDNF gene and it's effects on PC12-engineered cells

作　　者：王丽梅[1] 魏传垠 陈雪红[3] 张磬[4] 陈哲宇[1] 丁达夫[4] 路长林[1] 

机构地区：[1]第二军医大学神经生物学教研室,上海200433 [2]山东淄博卫生学校外科教研室,淄博255200 [3]青岛大学医学院病理生理学教研室,青岛266021 [4]中国科学院生物化学与细胞研究所蛋白质组学重点实验室,上海200031

出　　处：《基础医学与临床》2003年第3期263-268,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金 (30 0 0 0 0 4 8) ;上海市青年科技启明星计划 (0 1QB14 0 0 1) ;国家教育部优秀青年教师资助计划项目 (30 0 70 16 7) ;国家重点基础研究"973"项目 (19990 5 4 0 0 5 )

摘　　要：克隆与表达大鼠胶质细胞源性神经营养因子 (glialcellline derivedneurotrophicfactor,GDNF)并观察其对PC12工程细胞的影响。从新生 4天的SD大鼠海马组织中提取总RNA ,通过RT PCR方法 ,扩增出GDNFcDNA。构建表达质粒pET GDNF ,转化大肠杆菌BL2 1(DE3) ,IPTG诱导GDNF表达并在Ni2 + NTA柱上用一步复性法纯化和复性。把PCDNA3 0 GFRα1和pcDNA3 0 RET质粒双转染入PC12细胞 ,G4 18筛选稳定克隆 ,构建PC12工程细胞。用GDNF刺激工程细胞 ,3天后观察其存活和分化。大鼠GDNFcDNA的克隆与表达获得成功 ,纯化和复性的重组GDNF蛋白可显著增强PC12 GFRα1 RET工程细胞的存活和分化。初步明确GDNF诱导PC12工程细胞存活和分化是通过RET 依赖途径。To obtain glial cell line derived neurotrophic factor (GDNF) cDNA and recombinant GDNF protein and to observe its effects on PC12 engineered cells of GDNF. Using RT PCR method, the cDNA encoding the mature rat GDNF was isolated with total RNA extracted from newborn SD rat hippocampus tissue. The expression plasmid pET GDNF was constructed by inserting GDNF cDNA into plasmid pET 28a(+) containing T7 promoter and then transformed into E.coli BL21(DE3). The rat GDNF protein was highly expressed by IPTG induction. GDNF protein was purified by Ni 2+ chelation affinity chromatography and pcDNA3.0 GFRα1/pcDNA3.0 RET plasmid were transfected into PC12 cells respectively to constructe PC12 engineered cells. Stimulated PC12 engineered cells with recombinant GDNF protein and examined survival as well as differentiation after 3 days. Rat GDNF cDNA and recombinant GDNF protein were successfully obtained. Purified and refolded GDNF protein significantly promoted the survival and induced the differentiation of PC12 engineered cells. GDNF promoted the survival and induce the differentiation of PC12 engineered cells through RET dependent signaling.

关 键 词：大鼠 GDNF基因 胶质细胞源 神经营养因子 PC12工程细胞 影响 基因表达 反转录—聚合酶链反应 

分 类 号：R346[医药卫生—基础医学]