CVB3对Hela细胞高尔基体及其细胞周期的影响

发布时间：2018-07-03 08:02

本文选题：柯萨奇病毒 + CVB3　；参考：《南昌大学》2009年硕士论文

【摘要】： 目的: 探讨CVB3(Coxsackievirus B3)对Hela(Henrietta Lacks strain of cancer cells)细胞高尔基体及其细胞周期的影响,为研究CVB3的致病机制奠定基础。方法: 将Hela细胞株作为敏感宿主细胞进行以下实验,用MOI(multiplicity of infection)为5的CVB3感染细胞: 1、免疫荧光双标检测病毒感染后0 h、1.5 h、3 h、4.5 h和6 h等时间点VP1、GM130(Golgi matrix protein 130)和GRASP65(Golgi reassembly stacking protein 65)在细胞中的定位; 2、选择CVB3感染0 h、1.5 h、3 h、4.5 h、6 h、7.5 h、9 h、10.5 h、12 h、13.5 h、15 h、16.5 h、18 h、21 h和24 h等时间点收集细胞,进行总蛋白定量后,Western Blot检测VP1、GM130和GRASP65蛋白的表达变化; 3、分别选择G1/S期或G2/M期同步化细胞释放0 h、3 h、6 h和9 h等时间点,流式细胞仪检测CVB3病毒感染组与正常细胞组之间的细胞周期差异。结果: 1、免疫荧光双标结果显示: (1)GM130和GRASP65的免疫荧光双标结果显示:GM130与GRASP65之间存在明显共定位,正常Hela细胞高尔基体位于近核区,高尔基带连续。 (2)GM130和VP1的免疫荧光双标结果显示:正常细胞GM130免疫荧光呈带状分布,位于近核区;而自病毒感染3 h后,GM130的荧光在病毒感染细胞中出现散点状分布。 (3)GRASP65和VP1的免疫荧光双标结果显示:正常细胞GRASP65免疫荧光呈带状分布,位于近核区;而自病毒感染3 h后,GRASP65的荧光开始出现较明显的弥散分布。 2、Western blot结果显示:GM130的表达量自病毒感染后逐渐下降,至病毒感染18 h后未检测出GM130的表达;GRASP65的表达量变化趋势与GM130基本一致,但病毒感染12 h后即未检测出其表达;VP1的蛋白表达量自病毒感染开始即持续上升,6 h后一直维持在较高水平。 3、流式细胞仪检测结果显示: (1)对胸苷双阻断法同步在G1/S期的细胞,释放0 h、3 h、6 h和9 h后,正常细胞组G1期的细胞比例分别是:89.14%、65.77%、14.14%和13.11%;而病毒感染细胞组G1期细胞比例分别是:91.08%、84.41%、81.81%和81.74%。 (2)经诺考达唑(Nocodazole)作用后,G2期的细胞比例由非同步化状态时的4.62%上升到40%以上,表明诺考达唑已将部分细胞同步化G2/M期;同步化释放0 h、3 h、6 h和9 h后,正常细胞组G2期细胞比例分别是:42.99%、51.93%、37.97%和21.35%;而病毒感染细胞组G2期细胞比例分别是:61.25%、43.34%、29.37%和27.62%,与此同时G1期细胞比例分别是:8.48%、28.01%、28.10%和33.46%。结论: 1、CVB3感染引起Hela细胞高尔基带断裂,高尔基体结构受到影响。这可能是CVB3感染导致宿主细胞病变效应的机制之一。 2、CVB3感染下调了GM130和GRASP65的表达。GM130和GRASP65的蛋白表达下降可能是高尔基体结构破坏的机制之一。 3、CVB3感染G1/S期细胞后,细胞周期主要停滞在G1期,但CVB3并不阻滞G2期细胞。
[Abstract]:Objective: to investigate the effects of Coxsackievirus B3 (CVB3) on Golgi body and cell cycle of Hela (Henrietta packs strain of cancer cells) cells, and to provide a basis for the study of the pathogenesis of CVB3. Methods: Hela cell lines were used as sensitive host cells. The location of VP1GM130 (Golgi matrix protein 130) and GRASP65 (Golgi reassembly stacking protein 65) at 0 h, 1.5 h, 3 h, 4.5 h and 6 h after infection were detected by immunofluorescence double labeling. 2. The cells were collected at the time points of 0 h, 1.5 h, 3 h, 4.5 h, 7.5 h, 9 h, 10.5 h, 12 h, 13.5 h, 15 h, 16.5 h, 18 h, 21 h and 24 h of CVB3 infection, respectively. The expression of GM130 and GRASP65 was detected by Western Blot after total protein quantification. 3, the time points of release of the cells from G1 / S phase or G2 / M phase were 0 h ~ 3 h ~ 6 h and 9 h, respectively, and the expression of GM130 and GRASP65 protein was detected by Western blot. The difference of cell cycle between CVB 3 infection group and normal cell group was detected by flow cytometry. Results: 1. The results of immunofluorescence double labeling showed: (1) the immunofluorescence double labeling results of GM130 and GRASP65 showed that there was obvious co-localization between GM130 and GRASP65, and the Golgi body of normal Hela cells was located in the proximal nucleus. (2) the immunofluorescence double labeling of GM130 and VP1 showed that the GM130 immunofluorescence of normal cells was zonal and located in the proximal nucleus; The fluorescence of GM130 was scattered in the infected cells 3 hours after the virus infection. (3) the immunofluorescence double labeling results of GRASP65 and VP1 showed that the immunofluorescence of GM130 in normal cells was banded and located in the proximal nuclear region. However, the fluorescence distribution of GRASP65 began to appear obviously after 3 h of virus infection. 2 the results of Western blot showed that the expression of GM130 decreased gradually after virus infection. No GM130 expression was detected at 18 h after infection, and the trend of GM130 expression was basically the same as that of GM130. However, the expression of VP1 protein was not detected 12 h after infection. The expression of VP1 protein was maintained at a relatively high level after 6 h of continuous increase from the beginning of virus infection. 3. The results of flow cytometry showed that: (1) the cells in G 1 / S phase were synchronized by double blocking of thymidine. 3 h and 9 h after 0 h release. The percentage of G1 phase cells in normal cell group was 14.14% and 13.11%, respectively, while that in virus infected cell group was 84.41% and 81.74%, respectively. (2) the proportion of cells in G2 phase after treatment with Nocodazole was non-synchronous. When 4.62% rose to more than 40%, The results showed that Nordazol had synchronized G2 / M phase of some cells, and released 0 h ~ 3 h ~ 6 h and 9 h after release of G _ 2 / M phase. The proportion of G2 phase cells in normal cells group was 51.933% and 21.35%, respectively, while in virus infected cells group, the proportion of G2 phase cells was 29.37% and 27.62%, respectively, while the G1 phase cell proportion was 28.0128.10% and 33.4646%, respectively. Conclusion: 1 the Golgi band of Hela cells was broken and the Golgi body structure was affected by CVB3 infection. This may be one of the mechanisms by which CVB3 infection induces the host cytopathic effect. 2 the down-regulation of the expression of GM130 and GRASP65. GM130 and GRASP65 may be one of the mechanisms of the destruction of Golgi body structure. 3After the infection of G1 / S phase cells with CVB3, Cell cycle was mainly arrested in G 1 phase, but CVB 3 did not block G 2 phase cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373

【相似文献】