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目的应用基于聚合酶链式反应(PCR)技术的改良消减杂交方法克隆肿瘤凋亡相关基因,验证维甲酸调控生物细胞增殖、分化,诱导多种肿瘤细胞凋亡的作用。方法以全反式维甲酸(all-transretinoicacid)诱导前列腺癌DU-145细胞、早幼粒白血病HL-60细胞和乳腺腺癌MCF-7细胞产生凋亡,在此基础上,采用基于PCR技术的改良消减杂交方法克隆肿瘤细胞凋亡相关基因。结果在维甲酸诱导前列腺癌DU-145细胞、早幼粒白血病HL-60细胞和乳腺腺癌MCF-7细胞产生凋亡过程中,有TNF、泛素、热休克蛋白和C-erbB-2基因参与,并成功克隆出多条可能与凋亡密切相关的未知基因,均被GenBank收录,登录号为AF174394,AF144056,AF141882。结论这种基于PCR技术的改良消减杂交方法对于差异表达基因的克隆是十分有效的。
Objective To clone the genes related to apoptosis by improved subtractive hybridization based on polymerase chain reaction (PCR) and verify the effect of retinoic acid on the proliferation and differentiation of biological cells and induce the apoptosis of various tumor cells. Methods Apoptosis was induced by all-transretinoic acid in prostate cancer DU-145 cells, promyelocytic leukemia HL-60 cells and breast adenocarcinoma MCF-7 cells. On the basis of this, PCR-based Cloning of tumor cell apoptosis related genes by improved subtractive hybridization. Results Apoptosis of DU-145 cells, HL-60 cells and MCF-7 cells of breast adenocarcinoma cells were induced by retinoic acid. TNF, ubiquitin, heat shock protein and C-erbB-2 And successfully cloned a number of unknown genes that may be closely related to apoptosis. All of them were reported by GenBank with accession numbers AF174394, AF144056 and AF141882. Conclusion This improved subtractive hybridization based on PCR technique is very effective for the cloning of differentially expressed genes.