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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 PC12细胞分化
OBJECTIVE: To investigate whether GDNF can promote the differentiation of rat pheochromocytoma cell line PC12 by using self-constructed BHK-h GDNF genetically engineered cells. Methods Human GDNF gene was cloned by RT-PCR from human fetal brain. BHK-2 cells were transfected with pTARGET / h GDNF (±) by L ipofecam ine BHK-h GDNF genetically engineered cells stably expressing human GDNF were screened in selection medium containing G418. Human GDNF expression in BHK-h GDNF genetically engineered cells was examined by immunohistochemistry. And PC12 cells were cultured in supernatant of BHK-h GDNF gene engineering cells to observe whether GDNF promotes the differentiation of rat pheochromocytoma cell line PC12 cells. Results The positive and negative eukaryotic expression vector pTARGET / h GDNF (±) was constructed by digestion and sequencing. The positive eukaryotic expression vector pTARGET / h GDNF (+) was transfected into BHK-2 1 Post-cellular immunohistochemistry results demonstrated that BHK-h GDNF cells can express human GDNF; BHK-h GDNF engineered cell culture supernatant can promote PC12 cell differentiation. Conclusions Human GDNF expressed in BHK-h GDNF gene-engineered cells can promote the differentiation of PC12 cells