稳定表达的人GDNF基因对PC12细胞的分化作用研究

THE HUMAN GDNF STABLE EXPRESSION IN ENGINEERED CELLS INDUCED DIFFERENTIATION OF PC12 CELLS

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摘要目的　利用自行构建的 BHK- h GDNF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L ipofecam ine将构建的真核表达载体 p TARGET/ h GDNF(± )转染 BHK- 2 1细胞 ,在含有 G418的选择培养基中筛选出稳定表达人 GDNF的 BHK- h GDNF基因工程细胞。用免疫组织化学的方法检测 BHK- h GDNF基因工程细胞中人 GDNF的表达。并且用 BHK- h GDNF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 p TARGET/ h GDNF(± )的酶解消化和测序结果正确 ;用正向真核表达载体 p TARGET/ h GDNF(+)转染 BHK- 2 1细胞后免疫组织化学的结果证明 BHK- h GDNF细胞能够表达人 GDNF;BHK- h GDNF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- h GDNF基因工程细胞中表达的人 GDNF可以促进 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 induced to differentiate to neuronal cell with culture supernatant of BHK GDNF cells. Conclusions The human GDNF can be expressed by constructed BHK GDNF engineered cells, and human GDNF is able to induce the differentiation of PC12 cells to neuronal cells.