摘要
目的:构建并鉴定凋亡相关基因——PNAS-2的短发夹RNA(shRNA)质粒表达载体,将其转染U937细胞,筛选后检测细胞凋亡的变化。方法:shRNA质粒载体pRNAT U6.1/NEO经限制性内切酶BamHⅠ及HindⅢ双酶切后,连接带有BamHⅠ及HindⅢ黏性末端的shRNA插入片段,转化感受态细菌,并行测序鉴定。将阳性shRNA质粒载体进行细胞转染,合并使用G418及流式细胞仪(FCM)筛选出GFP阳性细胞,应用半定量PCR鉴定shRNA封闭效果;用Annexin V-APC/7-AAD标记后,使用FCM及激光共聚焦显微镜检测细胞凋亡。结果:测序鉴定证实,PNAS-2特异的shRNA质粒表达载体构建正确;合并使用G418及FCM分选出GFP阳性细胞后,半定量PCR鉴定示shRNAⅠ、Ⅱ、Ⅲ组对PNAS-2的抑制率分别为78.1%、75.4%及51.4%。FCM显示shRNA对照组的细胞凋亡率为(3.52±1.25)%;而shRNAⅠ组为(9.23±1.88)%(P<0.01);shRNAⅡ(8.85±1.80)%(P<0.05);shRNAⅢ组为(7.19±2.09)%(P>0.05)。激光共聚焦显微镜观察显示,shRNA对照组细胞凋亡率为7.3%;shRNAⅠ组为14.7%;shRNAⅡ为13.8%;shRNAⅢ为10.3%。结论:测序结果表明,本研究成功构建了PNAS-2的shRNA质粒表达载体。半定量PCR显示shRNA抑制PNAS-2表达效果佳;抑制PNAS-2后可促使细胞凋亡,提示该基因是抗细胞凋亡基因。
Objective To constructed recombinant plasmids containing short hairpin RNA of PNA S-2 in order to suppress the expression of PNA S-2 gene in U937 cell line, and after identified them, to transfect the recombinant plasmid to U937 cell line then purified cells, and detect cell apoptosis ratio changes. Methods PNAS-2 specific oligonucleotides were designed and synthesized and the oligonucleotides and pRNAT U6.1/NEO vectors were then digested by both re- stricted endoenzymes BamH I and Hindlll. The constructed recombinant plasmids were transformed into TOP10 chemically competent E.Coli cells, sequencing to validate them. After transformation, the vectors were collected and then the recombinant plasmids were transfected to U937 cell line and purified the transfected cells through use G418 together with flow cytometer（FCM） sorting according to GFP, which was a marker of pRNAT U6.1/NEO vectors and could express in the transfected cells. After harvesting the purified cells, PNA S-2 gene expression was detected by semi-quantitative reverse transcriptase polymerse chain reaction technique. The cells were stained by Annexin V-APC/7-AAD to detect the cell apoptosis by confocal microscopy and FCM. Results DNA sequencing showed the sequences of the recombinant plasmids were constructed correctly. Confocal microscopy showed that the recombinant plasmids to U937 cell line were successfully transfected. The gene suppression rate were 78.1%,75.4% and 51.4% in shRNA Ⅰ, Ⅱ and Ⅲ group, respectively. Apoptosis ratios of confocal microscopy were 7.3% in shRNA control group, 14.7% in shRNA Ⅰ group,13.8% in shRNA Ⅱ group and 10.3% in shRNA Ⅲ group. Apoptosis ratio increase after PNAS-2 has been completely depleted （P〈0.05）. Conclusions The successfully constructed recombinant plasmids contain short hairpin RNA of PNA S-2 and could be transfected them to U937 cell line. The short hairpin RNA could efficiently suppress PNAS-2 expression in the U937 cell line. The PNAS-2 had a function of anti-apoptosis.
出处
《诊断学理论与实践》
2007年第5期422-426,共5页
Journal of Diagnostics Concepts & Practice
基金
国家自然科学基金资助项目(30271660)
