摘要
目的研究呼吸道合胞病毒(RSV)非结构蛋白(NS)1调节人肺Ⅱ型腺上皮癌细胞(A549)凋亡基因/抗凋亡基因表达的作用机制。方法构建RSV-NS1表达质粒(p NS1),合成si RNA(si RNA-bcl-2、si RNA-NS1、错配序列),制作其复合物。选用A549细胞,进行质粒转染,获得p NS1 A549细胞、sibcl-2-p NS1 A549细胞、si RNA-p NS1 A549细胞、错配序列A549细胞。进行质粒对凋亡调节实验[p NS1转染组(分2亚组),错配对照组]和抑制基因实验[si NS1干预处理组、RSV感染组、错配对照组]。应用Western blot检测p NS1转染A549细胞后0、24、48、72 h Bax及Bcl-2的蛋白表达水平、流式细胞仪测细胞凋亡率;流式细胞仪测NS1沉默后的细胞凋亡率。结果 Bax和Bcl-2在A549细胞均有表达,当p NS1转染A549细胞后,Bax蛋白表达随时间逐渐降低,Bcl-2表达明显上调,组内蛋白表达量差异有统计学意义(P<0.05)。p NS1转染亚1组细胞凋亡率较错配对照组显著下降(P<0.05)。经sibcl-2转染后的细胞,p NS1转染显著上调Bax的蛋白表达,24 h达到高峰,并持续表达72 h;细胞凋亡率显著增加(P<0.05)。RSV感染组在72 h内显著抑制细胞凋亡(P<0.05),而在si NS1干预处理组,细胞凋亡率虽然均较错配对照组降低,但无统计学意义(P>0.05)。结论呼吸道合胞病毒NS1通过上调抗凋亡基因bcl-2、下调凋亡基因bax延迟A549细胞凋亡。
Objective To investigate the mechanism of respiratory syncytial virns（RSV） nonstructural protein（NS） 1 in regulating apoptosis related genes in human lung cancer type 11 epithelial（A549） cells. Methods The expression plasmid for RSV-NS1 （pNSI） and synthesized siRNAs（siRNA-bcl-2, siRNA-NS1, mismatch sequence）, were transfected into A549 cells and obtained in pNS1 A549 cells, sibcl-2-pNS1 A549, siRNA-pNS1 A549 cells and mismatched sequence A549 cells. The regulation of apoptosis in pNS1 transfected group （divided into 2 subgroups） and mismatch control group, and gene expression in siNS1 intervention treatment group, RSV infection group and mismatch control group were investigated. The Bax and Bcl-2 protein levels were detected by Western blot at 0-hour, 24-hour, 48-hour and 72-hour after pNS1 transfection, and the apoptotic rate was measured by flow cytometry. Results Bax and Bcl-2 were both expressed in A549 cells. In pNS1 transfected A549 cells, Bax protein expression decreased with time while Bc1-2 expression significantly increased, and the difference in protein expression between the cells was statistically significant（P 〈 0.05）. Compared to mismatch plasmid control group, the apoptotic rate was significantly decreased in pNS1 transfected cells（P 〈 0.05）. After sibcl-2 transfection, the Bax protein expression was significantly up-regulated with pNS1 transfection, which reached the peak at 24-hour and sustained expression for 72-hour, and apoptotic rate increased significantly（P 〈 0.05）. RSV infection significantly inhibited the apoptosis of A549 cells within 72-hour, while siNS1 intervention led to lower apoptotic rate as compared with that of mismatch control group, but the difference was not statistically significance （P 〉 0.05）. Conclusion It is demonstrated that respiratory syncytial virus NS1 up-regulates anti-apoptotic gene bcl-2 and down-regulates the apoptotic gene bax to delay A549 cell apoptosis.
出处
《生物医学工程与临床》
CAS
2015年第1期60-65,共6页
Biomedical Engineering and Clinical Medicine
基金
国家自然科学基金资助项目(81170005)
