疱疹病毒Ⅱ型感染SH-SY5Y细胞的生物学效应

Cytopathic Effects of SH-SY5Y Cells Infected with Herpes Simplex Virus Type 2

目的:探讨疱疹病毒Ⅱ型(HSV-2)感染人神经母细胞瘤细胞株SH-SY5Y的生物学效应。方法:病毒液接种SH-SY5Y细胞后,用相差和电子显微镜观察感染细胞的形态变化,RT-PCR检测病毒在细胞中的增殖,MTT法检测病毒感染对细胞增殖的影响,流式细胞仪测定感染后的细胞凋亡状况。结果:相差显微镜显示细胞病变,从24～72h,细胞变性、坏死的程度和数量随感染时间延长而增加;电镜结果显示感染24h后,细胞核染色质固缩,出现多核巨细胞,线粒体内嵴紊乱、断裂,出现不同程度的自噬化、溶酶体化、空泡化,并可见大量鹰眼样已包装成熟的病毒颗粒及正在包装的病毒粒子;HSV-2LAT基因RT-PCR扩增表明,病毒能在SH-SY5Y细胞中增殖;凋亡检测显示HSV-2在体外细胞感染中并未使细胞出现凋亡现象;感染后24、48及72h,SH-SY5Y细胞的抑制率分别为11.3%、31.2%和63.1%,与对照组相比均存在显著性差异(P<0.05);分别用0.1、1、10MOI的病毒感染SH-SY5Y细胞,上述不同组在24、48、72h时细胞形态变化基本一致,感染结果相似,各组之间病毒毒力无明显差异(P>0.05)。结论:初步在人神经母细胞瘤细胞株SH-SY5Y中建立了HSV-2感染的细胞模型,并研究了感染对细胞生物性状的影响,为探讨HSV-2的潜伏与激发机制、了解HSV-2的致病机制打下基础。

Objective： To investigate the mechanisms for cytopathic effect of herpes simplex virus type 2（HSV-2） in SHSY5Y cells. Methods： Changes of biological character were observed after HSV-2 was inoculated into SH-SY5Y cells. Phase-contrast and electron microscopy were performed to analyze morphology changes in infected and uninfected groups. RT-PCR was used to detect HSV-2 infection and generation. The cell activity was detected by MTT reduction assay. DNA ladder analysis and flow cytometry were carried out to identify HSV-2-induced apoptosis. Results： The cytopathic effects on SH-SYSY cells were more and more obvious with time lasting from 24 to 72 h post-infection. Results revealed by electron microscope, viral infection induces condensed chromatin within the nuclei of the cells, followed by degenerating of the cellular nuclei. Infected cells formed multinueleated giant cells. Mitochondria crest were become disorder or breakage, followed by spontaneous phagocytosis, lysosome-like and vacuole-like. A lot of ＇eagle eye＇-like mature viron and enclosing viron were seen in cytoplasm. HSV-2 LAT gene was found in the infected group by RT-PCR methods and it showed virus could duplicate in the cells. It was not any apoptosis taken place in SH-SY5Y infected with HSV-2 by DNA ladder analysis and flow cytometry. The virustatic rate of SH-SYSY cells infected with HSV-2 at 24, 48 and 72 h post-infection were respectively 11.3%, 31.2% and 63.1% respectively, and were statistically significant difference compared with control group（P〈0.05）. CPE of HSV-2 infection in SH-SY5Y cells was detected with different MOI of 0.1, 1 and 10. Results indicated CPE was almost the same after 24, 48 and 72 h post-infection in three groups and there were no difference statistically compared with control group（P〉0.05）. Conclusion： We established SH-SY5Y cell model infected with HSV-2 and changes of biological character of cells were studied. The results provided usefulness for study on HSV-2 of latency and reactivation in vitro.