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稳定表达的人GDNF基因对PC12细胞的分化作用研究 |
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焦建伟 田竟生 祁亚慧 赫翠珍 商永磊 林嘉友
【摘要】 目的 利用自行构建的BHK-hGDNF基因工程细胞，研究GDNF是否具有促进大鼠嗜铬细胞瘤细胞系PC12细胞分化的作用。 方法 从人胎儿脑组织中提取总RNA，用RT-PCR的方法克隆人GDNF基因；利用Lipofecamine将构建的真核表达载体pTARGET／hGDNF(±)转染BHK-21细胞，在含有G418的选择培养基中筛选出稳定表达人GDNF的BHK-hGDNF基因工程细胞。用免疫组织化学的方法检测BHK-hGDNF基因工程细胞中人GDNF的表达。并且用BHK-hGDNF基因工程细胞培养的上清液培养PC12细胞，观察GDNF是否具有促进大鼠嗜铬细胞瘤细胞系PC12细胞分化作用。 结果 构建的正向和反向真核表达载体pTARGET／hGDNF(±)的酶解消化和测序结果正确；用正向真核表达载体pTARGET／hGDNF(+)转染BHK-21细胞后免疫组织化学的结果证明BHK-hGDNF细胞能够表达人GDNF；BHK-hGDNF基因工程细胞培养上清液可以促进PC12细胞分化。 结论 构建的BHK-hGDNF基因工程细胞中表达的人GDNF可以促进PC12细胞分化。
【关键词】 胶质细胞源性神经营养因子；基因工程细胞；细胞分化
【中图分类号】 Q593.2；R329.2+8；R329.2+1 【文献标识码】 A 【文章编号】 0529-1356(2000)03-246
THE HUMAN GDNF STABLE EXPRESSION IN ENGINEERED
CELLS INDUCED DIFFERENTIATION OF PC12 CELLS
JIAO Jian-wei TIAN Jing-sheng QI Ya-hui HE Cui-zhen SHANG Yong-Lei LIN Jia-you
（Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences,Beijing 100005，China）
（Beijing Institute of Neuroscience, Capital University of Medical Sciences, Beijing 100054,China）
【Abstract】 Objective To explore differentiation of PC12 cells by using stable expression engineered BHK-hGDNF cells. Methods We obtained GDNF DNA from human fetal brain tissues by RT-PCR and two mammalian expression vectors were constructed. The two mammalian expression vectors pTARGET／hGDNF (±) were transfected into BHK-21 cells mediated by lipofecamine. The GDNF stable expression in BHK-GDNF cells was detected by immunohistochemical staining method. To detect if GDNF can induce differentiation of PC12 cells, the PC12 cells were cultured in the supernatant of BHK-GDNF cells culture medium. Results Identifications of pTARGET／hGDNF(±) were confirmed by digestion of endonuclease and sequence analysis; The immunohistochemical staining positive cells were observed in BHK-21 cells that were transfected with pTARGET／hGDNF(+). The PC12 cell can be in[1] [2] [3] 下一页
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