小鼠巨细胞病毒诱导热休克蛋白70异常表达和细胞凋亡的体外实验研究
发布时间：2018-09-08 06:58
【摘要】： 第一部分小鼠巨细胞病毒致细胞病变作用的体外实验研究 目的从细胞形态学角度研究鼠巨细胞病毒(Mouce Cytomegalovirus, MCMV)感染鼠胚肺成纤维细胞引起细胞的病理改变。 方法常规体外培养小鼠胚肺成纤维细胞株NIH3T3细胞,将MCMV Smith株按100TCID50/0.1ml接种NIH3T3细胞,每天倒置显微镜下观察感染细胞的形态学变化,扫描电镜观察感染细胞超微结构改变及感染细胞中的病毒颗粒。 结果倒置显微镜下可观察到感染细胞增大、变圆,细胞核变大;电镜结果可见胞浆内有较多空泡、脂滴,线粒体肿胀、嵴断裂、空泡变性,板层状高尔基复合体明显增多,内质网明显扩张;胞核内见较多病毒颗粒。 结论MCMV感染后小鼠成纤维细胞株NIH3T3细胞出现增大、变圆等病理改变,超微结构发生明显变化,感染MCMV68h的细胞核中可见病毒颗粒。 第二部分热休克蛋白70异常表达与小鼠巨细胞病毒致细胞凋亡的相关性研究 目的探讨小鼠巨细胞病毒(Mouce Cytomegalovirus ,MCMV)感染小鼠胚肺成纤维细胞时热休克蛋白70( heat shock protein 70, HSP70)的表达和细胞凋亡的关系。 方法常规体外培养小鼠胚肺成纤维细胞株NIH3T3细胞,将MCMV Smith株按100TCID50/0.1ml接种NIH3T3细胞,经免疫荧光染色法观察感染细胞和正常细胞中HSP70的表达部位,流式细胞仪双标检测感染细胞和正常细胞中HSP70的表达密度和细胞凋亡。 结果免疫荧光染色可观察到病毒感染组细胞内见强的阳性信号,病毒作用24h以胞浆中阳性信号为主,作用72h以细胞核内阳性信号为主。流式细胞仪检测结果见病毒感染组细胞内HSP70表达水平均较正常细胞对照组增高,感染后72～96h HSP70表达最高;MCMV感染组细胞凋亡发生率较同时相正常细胞对照组显著增加(P0.05)。 结论MCMV感染可诱导细胞表达HSP70, HSP70的表达部位与病毒定位并增殖的部位一致,其高峰表达时间亦与细胞病变程度和病毒大量复制的时间一致;MCMV感染可诱导NIH3T3细胞凋亡增加, HSP70可抑制感染细胞凋亡且具有明显的细胞保护作用。 第三部分中药金叶败毒对小鼠巨细胞病毒诱导的热休克蛋白70异常表达和细胞凋亡的影响 目的研究金叶败毒对小鼠巨细胞病毒(Mouce Cytomegalovirus ,MCMV)感染小鼠胚肺成纤维细胞所致的热休克蛋白70( heat shock protein 70, HSP70)异常表达及细胞凋亡的影响,探讨金叶败毒抗CMV的作用机制。 方法常规体外培养小鼠胚肺成纤维细胞株NIH3T3细胞,将MCMV Smith株按100TCID50/0.1ml接种NIH3T3细胞,更昔洛韦(GCV)和金叶败毒分别作用于MCMV感染的小鼠成纤维细胞株NIH3T3细胞,经流式细胞仪双标检测更昔洛韦处理的感染细胞、金叶败毒处理的感染细胞、感染细胞和正常细胞中HSP70的表达密度和细胞凋亡。 结果流式细胞仪检测结果见病毒感染组、药物处理组(金叶败毒组和更昔洛韦组)细胞内HSP70表达水平均较正常细胞对照组增高,感染后72-96h HSP70表达最高;MCMV感染组细胞凋亡发生率较同时相正常细胞对照组显著增加(P0.05),药物处理组细胞凋亡发生率亦较同时相正常细胞对照组显著增加(P0.05));但药物处理组和同时相MCMV感染组细胞比较,细胞凋亡发生率显著降低(P0.05)。 结论MCMV感染可诱导细胞表达HSP70, HSP70的表达部位与病毒定位并增殖的部位一致,其高峰表达时间亦与细胞病变程度和病毒大量复制的时间一致,金叶败毒制剂可下调MCMV感染所致的HSP70异常表达,这可能与金叶败毒的抗病毒作用相关;MCMV感染可诱导NIH3T3细胞凋亡增加,金叶败毒制剂和更昔洛韦均可抑制感染细胞凋亡而具有明显的抗病毒作用。
[Abstract]:The first part is the cytopathic effect of murine cytomegalovirus in vitro.
Objective To study the pathological changes of mouse embryonic lung fibroblasts infected with Mouce Cytomegalovirus (MCMV).
Methods NIH3T3 cells were cultured in vitro. MCMV Smith strain was inoculated into NIH3T3 cells according to 100TCID50/0.1ml. Morphological changes of infected cells were observed under inverted microscope every day. Ultrastructural changes of infected cells and virus particles in infected cells were observed by scanning electron microscope.
Results Under the inverted microscope, the infected cells were enlarged, rounded and the nucleus enlarged, and the electron microscopic results showed that there were more vacuoles, lipid droplets, mitochondria swelling, cristae rupture, vacuole degeneration, lamellar Golgi complex increased and endoplasmic reticulum dilated obviously in the cytoplasm.
Conclusion The pathological changes such as enlargement and roundness of NIH3T3 cells were observed after MCMV infection in mice. The ultrastructure of NIH3T3 cells changed obviously. Virus particles were found in the nucleus of the cells infected with MCMV for 68 hours.
The second part is the correlation between the abnormal expression of heat shock protein 70 and the apoptosis of murine cytomegalovirus.
Objective To investigate the relationship between the expression of heat shock protein 70 (HSP70) and cell apoptosis in mouse embryonic lung fibroblasts infected with mouse cytomegalovirus (MCMV).
Methods NIH3T3 cells were cultured in vitro. MCMV Smith strain was inoculated into NIH3T3 cells according to 100TCID 50/0.1ml. The expression of HSP70 in infected cells and normal cells was observed by immunofluorescence staining. The expression density and apoptosis of HSP70 in infected cells and normal cells were detected by flow cytometry.
Results Immunofluorescence staining showed that there were strong positive signals in the cells of the virus infected group. The positive signals were mainly in cytoplasm at 24 h and in nucleus at 72 h. The expression of HSP70 in the cells of the virus infected group was higher than that of the control group by flow cytometry. The expression of HSP70 in the cells of the virus infected group was higher than that of the normal cells at 72-96 h after infection. The rate of apoptosis in MCMV infection group was significantly higher than that in normal control group (P0.05).
Conclusion MCMV infection can induce the expression of HSP70 in cells, the expression site of HSP70 is consistent with the location and proliferation of the virus, and the peak expression time is also consistent with the degree of cytopathy and the time of viral replication; MCMV infection can induce the apoptosis of NIH3T3 cells, and HSP70 can inhibit the apoptosis of NIH3T3 cells and has obvious cytoprotective effect.
The third part: the effect of Jinye Baidu on the abnormal expression of HSP70 and cell apoptosis induced by cytomegalovirus in mice
Objective To study the effect of Golden Leaf Sepsis Virus (GPV) on the abnormal expression of heat shock protein 70 (HSP70) and apoptosis of mouse embryonic lung fibroblasts induced by Mouce Cytomegalovirus (MCMV) infection, and to explore the anti-CMV mechanism of GPV.
Methods Mouse embryonic lung fibroblasts NIH3T3 cells were cultured in vitro. MCMV Smith strain was inoculated into NIH3T3 cells according to 100TCID 50/0.1ml. Ganciclovir (GCV) and Golden Leaf Sepsis were used to treat NIH3T3 cells infected by MCMV respectively. The infected cells were detected by flow cytometry and treated with Golden Leaf Sepsis respectively. The expression density and apoptosis of HSP70 in infected cells, infected cells and normal cells.
Results The results of flow cytometry showed that the expression of HSP70 in the virus-infected group, the drug-treated group (Jinye detoxification group and ganciclovir group) was higher than that in the control group, and the expression of HSP70 was the highest at 72-96 hours after infection. The incidence of cell apoptosis in the MCMV-infected group was significantly higher than that in the control group (P 0.05). The incidence of cell apoptosis was also significantly higher than that of the control group (P 0.05), but the incidence of cell apoptosis was significantly lower in the drug treatment group than in the MCMV infection group (P 0.05).
Conclusion MCMV infection can induce the expression of HSP70 in cells. The expression of HSP70 is consistent with the location of virus localization and proliferation, and its peak expression time is consistent with the degree of cytopathy and the time of viral replication. Jinye Baidu preparation can down-regulate the abnormal expression of HSP70 caused by MCMV infection, which may be related to the antiviral effect of Jinye Baidu. MCMV infection can induce the apoptosis of NIH3T3 cells. Both Jinye Baidu preparation and ganciclovir can inhibit the apoptosis of NIH3T3 cells and have obvious antiviral effect.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373
本文编号：2229715
[Abstract]:The first part is the cytopathic effect of murine cytomegalovirus in vitro.
Objective To study the pathological changes of mouse embryonic lung fibroblasts infected with Mouce Cytomegalovirus (MCMV).
Methods NIH3T3 cells were cultured in vitro. MCMV Smith strain was inoculated into NIH3T3 cells according to 100TCID50/0.1ml. Morphological changes of infected cells were observed under inverted microscope every day. Ultrastructural changes of infected cells and virus particles in infected cells were observed by scanning electron microscope.
Results Under the inverted microscope, the infected cells were enlarged, rounded and the nucleus enlarged, and the electron microscopic results showed that there were more vacuoles, lipid droplets, mitochondria swelling, cristae rupture, vacuole degeneration, lamellar Golgi complex increased and endoplasmic reticulum dilated obviously in the cytoplasm.
Conclusion The pathological changes such as enlargement and roundness of NIH3T3 cells were observed after MCMV infection in mice. The ultrastructure of NIH3T3 cells changed obviously. Virus particles were found in the nucleus of the cells infected with MCMV for 68 hours.
The second part is the correlation between the abnormal expression of heat shock protein 70 and the apoptosis of murine cytomegalovirus.
Objective To investigate the relationship between the expression of heat shock protein 70 (HSP70) and cell apoptosis in mouse embryonic lung fibroblasts infected with mouse cytomegalovirus (MCMV).
Methods NIH3T3 cells were cultured in vitro. MCMV Smith strain was inoculated into NIH3T3 cells according to 100TCID 50/0.1ml. The expression of HSP70 in infected cells and normal cells was observed by immunofluorescence staining. The expression density and apoptosis of HSP70 in infected cells and normal cells were detected by flow cytometry.
Results Immunofluorescence staining showed that there were strong positive signals in the cells of the virus infected group. The positive signals were mainly in cytoplasm at 24 h and in nucleus at 72 h. The expression of HSP70 in the cells of the virus infected group was higher than that of the control group by flow cytometry. The expression of HSP70 in the cells of the virus infected group was higher than that of the normal cells at 72-96 h after infection. The rate of apoptosis in MCMV infection group was significantly higher than that in normal control group (P0.05).
Conclusion MCMV infection can induce the expression of HSP70 in cells, the expression site of HSP70 is consistent with the location and proliferation of the virus, and the peak expression time is also consistent with the degree of cytopathy and the time of viral replication; MCMV infection can induce the apoptosis of NIH3T3 cells, and HSP70 can inhibit the apoptosis of NIH3T3 cells and has obvious cytoprotective effect.
The third part: the effect of Jinye Baidu on the abnormal expression of HSP70 and cell apoptosis induced by cytomegalovirus in mice
Objective To study the effect of Golden Leaf Sepsis Virus (GPV) on the abnormal expression of heat shock protein 70 (HSP70) and apoptosis of mouse embryonic lung fibroblasts induced by Mouce Cytomegalovirus (MCMV) infection, and to explore the anti-CMV mechanism of GPV.
Methods Mouse embryonic lung fibroblasts NIH3T3 cells were cultured in vitro. MCMV Smith strain was inoculated into NIH3T3 cells according to 100TCID 50/0.1ml. Ganciclovir (GCV) and Golden Leaf Sepsis were used to treat NIH3T3 cells infected by MCMV respectively. The infected cells were detected by flow cytometry and treated with Golden Leaf Sepsis respectively. The expression density and apoptosis of HSP70 in infected cells, infected cells and normal cells.
Results The results of flow cytometry showed that the expression of HSP70 in the virus-infected group, the drug-treated group (Jinye detoxification group and ganciclovir group) was higher than that in the control group, and the expression of HSP70 was the highest at 72-96 hours after infection. The incidence of cell apoptosis in the MCMV-infected group was significantly higher than that in the control group (P 0.05). The incidence of cell apoptosis was also significantly higher than that of the control group (P 0.05), but the incidence of cell apoptosis was significantly lower in the drug treatment group than in the MCMV infection group (P 0.05).
Conclusion MCMV infection can induce the expression of HSP70 in cells. The expression of HSP70 is consistent with the location of virus localization and proliferation, and its peak expression time is consistent with the degree of cytopathy and the time of viral replication. Jinye Baidu preparation can down-regulate the abnormal expression of HSP70 caused by MCMV infection, which may be related to the antiviral effect of Jinye Baidu. MCMV infection can induce the apoptosis of NIH3T3 cells. Both Jinye Baidu preparation and ganciclovir can inhibit the apoptosis of NIH3T3 cells and have obvious antiviral effect.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373
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