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目的探讨PRDM5基因在前列腺癌细胞中的抑癌作用。方法采用聚合酶链反应(PCR)、限制性内切酶酶切、T4DNA连接酶连接等方法,克隆PRDM5基因并插入慢病毒载体中。将携带PRDM5基因的慢病毒质粒(或阴性对照质粒)与慢病毒包装质粒通过脂质体法共转染293T细胞,收集病毒上清,感染人前列腺癌细胞株22Rv1。用蛋白质印迹法检测细胞PRDM5的表达,细胞倍增实验和平板克隆实验检测细胞增殖和克隆形成能力,软琼脂克隆形成实验检测细胞非锚着依赖性生长能力。结果成功构建PRDM5重组慢病毒载体,并包装获得慢病毒上清。将PRDM5重组慢病毒载体感染22Rv1细胞后,筛选得到稳定表达细胞株,蛋白质印迹法结果显示该细胞株能稳定表达外源性PRDM5蛋白。过表达PRDM5的前列腺癌22Rv1细胞的增殖能力[倍增时间:(52.5±1.4)h vs(44.0±1.3)h]、克隆形成能力[克隆形成数:(1 114±98)/皿vs(1 361±123)/皿]和非锚着依赖性生长能力[克隆形成数:(94.6±8.7)/孔vs(154.0±3.5)/孔]均低于阴性对照组(P<0.05)。结论前列腺癌22Rv1细胞中PRDM5的过表达具有抑制肿瘤细胞增殖、克隆形成和非锚着依赖性生长的能力。
Objective To investigate the tumor suppressor effect of PRDM5 gene in prostate cancer cells. Methods PRDM5 gene was cloned by polymerase chain reaction (PCR), restriction endonuclease digestion and T4 DNA ligase, and inserted into lentiviral vector. 293T cells were co-transfected with lentiviral plasmid (or negative control plasmid) carrying PRDM5 gene and lentiviral packaging plasmid, and the virus supernatant was collected and infected into human prostate cancer cell line 22Rv1. The expression of PRDM5 was detected by Western blotting. Cell proliferation and colony formation ability were detected by cell doubling assay and plate cloning assay. Soft agar colony formation assay was used to detect the non-anchorage-dependent growth of cells. Results The PRDM5 recombinant lentiviral vector was successfully constructed and packaged to obtain the lentivirus supernatant. After the 22DM1 cells were infected with PRDM5 recombinant lentiviral vector, the stable expression cell lines were screened. The results of Western blotting showed that the cell lines stably expressed exogenous PRDM5 protein. Proliferation of prostate cancer 22Rv1 cells overexpressing PRDM5 [doubling time: (52.5 ± 1.4) h vs. (44.0 ± 1.3) h], clonogenicity [Clone formation number: (1114 ± 98) / dish vs ± 123) / plate] and non-anchorage-dependent growth ability [clonogenicity: (94.6 ± 8.7) / well vs (154.0 ± 3.5) / well] were lower than those in the negative control group (P <0.05). Conclusion The overexpression of PRDM5 in prostate cancer 22Rv1 cells has the ability to inhibit tumor cell proliferation, colony formation and non-anchorage-dependent growth.