论文部分内容阅读
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 study the mechanism of the effect of respiratory syncytial virus (RSV) nonstructural protein (NS) 1 on the expression of apoptosis / anti-apoptotic genes in human lung adenocarcinoma of the human lung (A549). Methods RSV-NS1 expression plasmid (p NS1) was constructed, and si RNA (si RNA-bcl-2, si RNA-NS1, mismatch sequence) was synthesized to make its complex. A549 cells were selected for plasmid transfection to obtain pNS1 A549 cells, sibcl-2-p NS1 A549 cells, si RNA-p NS1 A549 cells and mismatch sequence A549 cells. Plasmid regulation of apoptosis [p NS1 transfection group (sub-subgroup 2), mismatch control group] and suppressor gene experiments [si NS1 intervention group, RSV infection group, mismatch control group] were performed. Western blot was used to detect the protein expression of Bax and Bcl-2 at 0,24,48,72 h after transfection of pNS1 into A549 cells. Flow cytometry was used to detect the apoptosis rate of cells after NS1 silencing Death rate. Results Bax and Bcl-2 were expressed in A549 cells. The expression of Bax protein was down-regulated and the expression of Bcl-2 was significantly up-regulated after transfected with p NS1 in A549 cells. The protein expression in the group was significantly different (P < 0.05). The apoptosis rate of NS1 transfected sub-group 1 was significantly lower than that of the control group (P <0.05). After transfection with sibcl-2, the expression of Bax protein was significantly up-regulated by p NS1 transfection and peaked at 24 h and continued for 72 h. The apoptosis rate was significantly increased (P <0.05). RSV infection significantly inhibited cell apoptosis within 72 h (P <0.05). However, the apoptosis rate of RSV infection group was significantly lower than that of the control group (P> 0.05). Conclusion Respiratory syncytial virus NS1 can delay the apoptosis of A549 cells by up-regulating the anti-apoptotic gene bcl-2 and down-regulating the apoptosis gene bax.