【作者】 杨丽；

【导师】 杨作成；

【作者基本信息】 中南大学 ， 临床医学， 2013， 硕士

【摘要】 目的观察CVB3感染Hela细胞后促凋亡基因Bax、Bim Caspase-3及caspase-9mRNA和蛋白表达的变化。方法将Hela细胞分为未予CVB3感染的对照组和CVB3感染的病毒组,取3h、6h、9h、12h、24h共5个时间点,采用RT-PCR、 Western Blot分别检测Bax、Bim、Caspase-3及caspase-9mRNA和蛋白的表达。结果1.Bax、Bim、Caspase-3及Caspase-9mRNA在对照组和病毒组Hela细胞中均有表达,且在各自组内差异均无显著性(P>0.05)；病毒组Bax、Bim mRNA表达在24h时间点均低于对照组(P<0.05)。对照组与病毒组中Caspase-3、Caspase-9mRNA在组内各时间点间的表达差异均无显著性(P>0.05)；病毒组Caspase-9mRNA表达较在24h时间点较对照组降低(P<0.05),而Caspase-3mRNA表达组间各时间点比较差异无显著性(P>0.05)。2. Bax、Bim、Caspase-3及Caspase-9蛋白表达在对照组中各时间点均有表达,但差异均无显著性(P>0.05)。在病毒组中,Bax蛋白在24h时间点表达下降,与组内各时间点比较差异均有显著性(P<0.05),且病毒组表达明显低于对照组(P<0.05)；Bim蛋白表达随感染时间延长而下降(P<0.05),且组间比较9h、12h、24h时间点蛋白表达病毒组低于对照组(P<0.05)。病毒组Caspase-3及Caspase-9蛋白表达均在24h点明显降低(P<0.05),且低于对照组24h时间点(P<0.05)；此外在对照组中Cleaved Caspase-9蛋白表达在12h点明显高于其他各时间点(P<0.05)；病毒组Cleaved Caspase-9蛋白表达在3h及6h点与9h、12h、24h时间点间比较差异有显著性(P<0.05),组间比较9h、12h时间点明显高于对照组(P<0.05)、而24h点明显低于对照组(P<0.05)；将Cleaved Caspase-9蛋白表达值与Caspase-9蛋白表达值比较,对照组中Cleaved Caspase-9/Caspase-9表达在12h时间点增高,差异有显著性(P<0.05),病毒组中Cleaved Caspase-9/Caspase-9表达在3h、6h、9h各时间点比较,差异无显著性(P>0.05),但是随感染时间的延长表达增强,至12h时间点出现增高,24h时间点明显增强。结论1.CVB3感染Hela细胞后Bax、Bim mRNA在24h时间点表达降低；Bax蛋白表达在病毒感染后24h时间点明显降低,而Bim蛋白表达随感染时间延长而下降,至24h时间点明显下降。2.CVB3感染Hela细胞后Caspase-3mRNA表达无明显变化,而Caspase9mRNA在24h时间点表达降低；Caspase-3、Caspase-9蛋白表达均在病毒感染后24h时间点降低,而Cleaved Caspase-9蛋白表达在病毒感染后12h时间点最强。3. Bax、Bim、Caspase-3、Caspase-9均在CVB3诱导的Hela细胞凋亡中发挥作用。更多还原

【Abstract】 Objective To observe the mRNA and protein expressions of Bax, Bim, Caspase-3and Capase-9in Coxsackie virus B3(CVB3) infected Hela cells.Methods HeLa cells were divided into the control group (without CVB3infection) and CVB3infection group. The mRNA and protein expressions of Bax, Bim, Caspase-9and Capase-3were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western Blot methods at3,6,9,12and24hour postinfection (pi).Results1.The mRNA expressions of Bax and Bim were checked in both control group and CVB3infection group, of which there was no statistical significance in either of control group or CVB3infection group respectively at different time points (all P>0.05). However, the mRNA expressions of Bax and Bim in CVB3infection group were lower than that in control group at24h pi (all P<0.05). The mRNA expressions of Caspase-3and Capase-9were measured in both control groups and CVB3infection groups, of which there were no statistical significances in either of control groups or CVB3infection groups of Caspase-3and Caspase-9respectively at different time points (all P>0.05); The mRNA expression of Caspase-9in CVB3infection group was lower than that in control goup at24h pi (P<0.05), but the mRNA expression of Caspase-3in CVB3infection group was no statistical significances(P>0.05) at every time points.2. The protein expressions of Bax, Bim, Caspase-3and Caspase-9were measure both in control group and CVB3infection group, while there was no statistical significance in control group at different time points (P>0.05). In the CVB3infection group, the protein expression of Bax decreased at24h pi (P<0.05), of which in CVB3infection group was lower than that in control group at24h pi (P<0.05). The protein expression of Bim decreased with the differents infection time points, which shown that protein expressions in CVB3infection groups were lower than that in control goups at9h,12h and24h respectively (P<0.05). The protein expressions of Caspase-3and Capase-9decreased at24h pi (P<0.05), of which in CVB3infection group were lower than that in control group at24h (P<0.05). In control group, the protein expression of Cleaved Caspase-9at12h was higher than that of other time points. In CVB3infection group, the protein expression of Cleaved Caspase-9were higher at3h pi and6h pi than that at9h pi,12h pi and24h pi. And the protein expressions of Cleaved Caspase-9at9h pi,12h pi in CVB3groups were higher than that in control groups, while24h pi was lower than that in control(P<0.05).In the control group, The protein expression value of the Cleaved Caspase-9compared with the Caspase-9were incread at12h time point(P<0.05), in the CVB3infection groups, the Cleaved Caspase-9/Caspase-9expression in3h、6h、9h at each time point was no significant differentce (P>0.05),but the Cleaved Caspase-9/Caspase-9expression became increasing at12h、24h pi (P<0.05)。Conclusions1. The mRNA expressions of Bax and Bim in CVB3infection groups were downregulated in24h pi times point respectively. The protein expression of Bax in24h pi was lower than that in other time points. The protein expression of Bim decreased with the time extension after CVB3infection, and the protein expression dcreased significantly in24h pi time point.2. The mRNA expression of Caspase-3was no change at every time point aftern CVB3infection, while the mRNA expression of Caspase-9was downregulated in24h pi times point. The protein expressions of Caspase-3and Caspase-9in24h pi were lower than that in another time points. The protein expression of Cleaved Caspase-9in12h pi was highest.3. The proteins of Bax, Bim, Caspase-3, Caspase-9played roles in CVB3-induced apoptosis in Hela cells.更多还原