锌指蛋白146下调对荧光素酶标记的胶质瘤细胞GL261-luc活性的影响

Effect of Zinc Finger Protein 146 Down Regulation on Activity of the Luciferase Stable GL261-luc Cells

目的观察DNA损伤修复蛋白——锌指蛋白146(RNF146)下调对荧光素酶稳转胶质瘤细胞株GL261-luc活性的影响。方法通过干涉慢病毒感染的方法,下调荧光素酶稳转胶质瘤细胞株GL261-luc中RNF146的表达,经实时荧光定量聚合酶链式反应(qRT-PCR)和Western blot检测GL261-luc细胞中RNF146的敲减效率。Western blot检测RNF146下调后GL261-luc细胞中荧光素酶蛋白的表达水平。比较第1代、第15代和第30代GL261-luc细胞中荧光素酶活性。结果RNF146干涉慢病毒感染能够下调GL261-luc细胞中RNF146基因表达约为85.7%(P<0.01);Western blot检测结果也显示GL261-luc细胞RNF146的蛋白表达下调超过85.0%(P<0.001)。RNF146下调不影响荧光素酶基因的表达、酶的活性和酶的稳定性(P均>0.05)。结论用慢病毒感染下调GL261-luc细胞RNF146表达,不影响荧光素酶的表达、活性及稳定性,能够用于后续小动物体内成瘤后的活体成像研究。

Objective To confirm the effect of zinc finger protein 146(RNF146)down-regulation on the activity of a luciferase stable gliomas cell line GL261-luc.Methods The expression of RNF 146 in GL261-luc cells was down regulated with shRNA-RNF146 lentivirus.The knockdown efficiency of RNF146 in GL261-luc cells was confirmed with qRT-PCR and Western blot.The expression of luciferase protein in GL261-luc cells was detected using Western blot.The luciferase activities of the 1st,15th and 30th generation GL261-luc cells were compared.Results The expression of RNF146 was down-regulated 85.7%in the GL261-luc cells with shRNA-RNF146 lentivirus treatment(P<0.01);Western blot also showed that the expression of RNF146 in GL261-luc cells was significantly down-regulated by more than 85.0%(P<0.001).RNF146 down-regulation did not affect the gene expression,the activity and the stability of luciferase gene(P all>0.05).Conclusion The down regulation of RNF146 in the lentivirus-interfering GL261-luc cells didn’t affect luciferase expression,activity and stability in these cells.Therefore it can be used in the imaging study of tumor-bearing mice.