【摘要】： 第一部分小鼠巨細(xì)胞病毒致細(xì)胞病變作用的體外實驗研究 目的從細(xì)胞形態(tài)學(xué)角度研究鼠巨細(xì)胞病毒(Mouce Cytomegalovirus, MCMV)感染鼠胚肺成纖維細(xì)胞引起細(xì)胞的病理改變。 方法常規(guī)體外培養(yǎng)小鼠胚肺成纖維細(xì)胞株NIH3T3細(xì)胞,將MCMV Smith株按100TCID50/0.1ml接種NIH3T3細(xì)胞,每天倒置顯微鏡下觀察感染細(xì)胞的形態(tài)學(xué)變化,掃描電鏡觀察感染細(xì)胞超微結(jié)構(gòu)改變及感染細(xì)胞中的病毒顆粒。 結(jié)果倒置顯微鏡下可觀察到感染細(xì)胞增大、變圓,細(xì)胞核變大;電鏡結(jié)果可見胞漿內(nèi)有較多空泡、脂滴,線粒體腫脹、嵴斷裂、空泡變性,板層狀高爾基復(fù)合體明顯增多,內(nèi)質(zhì)網(wǎng)明顯擴張;胞核內(nèi)見較多病毒顆粒。 結(jié)論MCMV感染后小鼠成纖維細(xì)胞株NIH3T3細(xì)胞出現(xiàn)增大、變圓等病理改變,超微結(jié)構(gòu)發(fā)生明顯變化,感染MCMV68h的細(xì)胞核中可見病毒顆粒。 第二部分熱休克蛋白70異常表達(dá)與小鼠巨細(xì)胞病毒致細(xì)胞凋亡的相關(guān)性研究 目的探討小鼠巨細(xì)胞病毒(Mouce Cytomegalovirus ,MCMV)感染小鼠胚肺成纖維細(xì)胞時熱休克蛋白70( heat shock protein 70, HSP70)的表達(dá)和細(xì)胞凋亡的關(guān)系。 方法常規(guī)體外培養(yǎng)小鼠胚肺成纖維細(xì)胞株NIH3T3細(xì)胞,將MCMV Smith株按100TCID50/0.1ml接種NIH3T3細(xì)胞,經(jīng)免疫熒光染色法觀察感染細(xì)胞和正常細(xì)胞中HSP70的表達(dá)部位,流式細(xì)胞儀雙標(biāo)檢測感染細(xì)胞和正常細(xì)胞中HSP70的表達(dá)密度和細(xì)胞凋亡。 結(jié)果免疫熒光染色可觀察到病毒感染組細(xì)胞內(nèi)見強的陽性信號,病毒作用24h以胞漿中陽性信號為主,作用72h以細(xì)胞核內(nèi)陽性信號為主。流式細(xì)胞儀檢測結(jié)果見病毒感染組細(xì)胞內(nèi)HSP70表達(dá)水平均較正常細(xì)胞對照組增高,感染后72～96h HSP70表達(dá)最高;MCMV感染組細(xì)胞凋亡發(fā)生率較同時相正常細(xì)胞對照組顯著增加(P0.05)。 結(jié)論MCMV感染可誘導(dǎo)細(xì)胞表達(dá)HSP70, HSP70的表達(dá)部位與病毒定位并增殖的部位一致,其高峰表達(dá)時間亦與細(xì)胞病變程度和病毒大量復(fù)制的時間一致;MCMV感染可誘導(dǎo)NIH3T3細(xì)胞凋亡增加, HSP70可抑制感染細(xì)胞凋亡且具有明顯的細(xì)胞保護(hù)作用。 第三部分中藥金葉敗毒對小鼠巨細(xì)胞病毒誘導(dǎo)的熱休克蛋白70異常表達(dá)和細(xì)胞凋亡的影響 目的研究金葉敗毒對小鼠巨細(xì)胞病毒(Mouce Cytomegalovirus ,MCMV)感染小鼠胚肺成纖維細(xì)胞所致的熱休克蛋白70( heat shock protein 70, HSP70)異常表達(dá)及細(xì)胞凋亡的影響,探討金葉敗毒抗CMV的作用機制。 方法常規(guī)體外培養(yǎng)小鼠胚肺成纖維細(xì)胞株NIH3T3細(xì)胞,將MCMV Smith株按100TCID50/0.1ml接種NIH3T3細(xì)胞,更昔洛韋(GCV)和金葉敗毒分別作用于MCMV感染的小鼠成纖維細(xì)胞株NIH3T3細(xì)胞,經(jīng)流式細(xì)胞儀雙標(biāo)檢測更昔洛韋處理的感染細(xì)胞、金葉敗毒處理的感染細(xì)胞、感染細(xì)胞和正常細(xì)胞中HSP70的表達(dá)密度和細(xì)胞凋亡。 結(jié)果流式細(xì)胞儀檢測結(jié)果見病毒感染組、藥物處理組(金葉敗毒組和更昔洛韋組)細(xì)胞內(nèi)HSP70表達(dá)水平均較正常細(xì)胞對照組增高,感染后72-96h HSP70表達(dá)最高;MCMV感染組細(xì)胞凋亡發(fā)生率較同時相正常細(xì)胞對照組顯著增加(P0.05),藥物處理組細(xì)胞凋亡發(fā)生率亦較同時相正常細(xì)胞對照組顯著增加(P0.05));但藥物處理組和同時相MCMV感染組細(xì)胞比較,細(xì)胞凋亡發(fā)生率顯著降低(P0.05)。 結(jié)論MCMV感染可誘導(dǎo)細(xì)胞表達(dá)HSP70, HSP70的表達(dá)部位與病毒定位并增殖的部位一致,其高峰表達(dá)時間亦與細(xì)胞病變程度和病毒大量復(fù)制的時間一致,金葉敗毒制劑可下調(diào)MCMV感染所致的HSP70異常表達(dá),這可能與金葉敗毒的抗病毒作用相關(guān);MCMV感染可誘導(dǎo)NIH3T3細(xì)胞凋亡增加,金葉敗毒制劑和更昔洛韋均可抑制感染細(xì)胞凋亡而具有明顯的抗病毒作用。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 崔文華,陳素華,王昕榮,劉海智,鄧群蓉;小鼠巨細(xì)胞病毒感染誘導(dǎo)熱休克蛋白70表達(dá)的體外實驗研究[J];山東醫(yī)藥;2005年14期

2 陳素華,聞良珍,馮玲,曾萬江,王志新,凌霞珍;胎盤感染人巨細(xì)胞病毒與胎兒生長發(fā)育關(guān)系探討[J];中國實用婦科與產(chǎn)科雜志;1996年05期

3 李紅,董永綏,方峰,李革;巨細(xì)胞病毒感染對細(xì)胞凋亡及bcl-2和fas基因表達(dá)的影響初探[J];中華兒科雜志;1998年11期

4 姜宏,聞良珍,凌霞珍,陳素華;中藥“熱毒清”對人巨細(xì)胞病毒抑制作用的實驗與臨床研究[J];中華實驗和臨床病毒學(xué)雜志;1999年02期

5 陳素華,熊錦文,邢瑋,趙婕,劉海智,王昕榮;中藥金葉敗毒防治巨細(xì)胞病毒宮內(nèi)感染的動物實驗初探[J];中華檢驗醫(yī)學(xué)雜志;2003年04期

6 張菁,江德華,浦佩玉;熱耐受對鼠腦海馬缺血后細(xì)胞凋亡的保護(hù)[J];中國神經(jīng)精神疾病雜志;1999年04期

7 邢瑋,聞良珍,董繼華,江漢珍,李鳴真;熱毒清抗人巨細(xì)胞病毒作用的臨床及實驗研究[J];中國中西醫(yī)結(jié)合雜志;2000年04期

，

本文編號：2229715