中药金叶败毒制剂对鼠巨细胞病毒感染后的细胞病变及热休克蛋白70的表达影响

Effect of Jinyebaidu praeparatum on the cytopathic effect and expression of HSP70 in cells infected by murine cytomegalovirus in vitro

目的体外研究中药金叶败毒制剂对小鼠巨细胞病毒感染后的细胞病变及热休克蛋白70(HSP70)表达的影响,为研究金叶败毒(JYBD)抗小鼠巨细胞病毒(MCMV)分子机制打下基础。方法体外培养小鼠胚肺细胞,加入病毒吸附2h,弃病毒液,加入最大无毒浓度TD0金叶败毒,分别作用12、24、72、120h,收获细胞,设对照组;倒置显微镜下观察细胞形态学变化,免疫组织化学方法检测HSP70表达情况。结果细胞病变情况:与病毒对照组比较,金叶败毒组:12～72h细胞胞浆空泡变性程度均减轻,未观察到病变变圆细胞,120h观察到出现病变变圆细胞,部分正常细胞胞浆空泡变性程度减轻。与金叶败毒组比较,GCV组细胞胞浆空泡变性程度与之相当,但120h未观察到病变变圆细胞。金叶败毒组:12hHSP70表达增强,72h继续上升,120h达最高点。与正常细胞对照组比较,各时相HSP70表达强度差异均有统计学意义(P<0.05);与病毒组比较,12、24h2个时相HSP70表达强度差异均有统计学意义(P<0.05)。更昔洛韦组:与金叶败毒作用类似,可增强病毒感染细胞内HSP70表达。结论金叶败毒制剂可抑制MCMV细胞病变作用,还能增强病毒感染细胞HSP70表达,可能是其抗MCMV分子机制。

Objective To investigate the influence of Jinyebaidu praeparatum（JYBD）on cytopathic effect and expression of heat shock proteinT0 （HSP70） in cells infected by murine cytomegalovirus （ MCMV ） in vitro in order to make foundation for the researches about the molecular mechanism of JYBD in resisting MCMV in mice. Methods The murine embryo pneumonocytes were cultured in vitro ,and the MCMV was adsorbed for 2 hours, then virus suspension was abandoned added JYBD praeparatum in the maximum nonvenomous concentration of TD0, effected for 12,24,72,120 hours, respectively, at last, the cells were harvested, and virus control group was set up. The changes of cell morphology were observed under inverted microscope, and the expression of HSPT0 was detected by immunohistochemistry. Results As compared with that in virus control group, the degree of vacuolar degeneration in cell cytoplasma in JYBD group was decreased afterl2 ~ 72h, and cells lesion and rounding were not observed, however, cell lesion and round cell were found after 120h. The degree of vacuolar degeneration of the cytoplasm in ganciclovir group was similar to that in JYBD group, but no cell lesion and round cells were found after 120h. In JYBD group, the expression of HSPT0 after 12 hours ,72h was increased, which reached peak after 120h. There were significant differences in the expression intensity of HSP70 at different time points between normal cells and control group （ P 〈 O. 05 ） , and there were significant differences in the expression intensity of HSPT0 at 12h and 24h between normal cells and virus control group（ P 〈0.05）. The effect in ganciclovir group was similar to that in JYBD group ,which could increase the expression of HSP70 in cells. Conclusion Jinyebaidu praeparatum can inhibit cytopathic effect of MCMV, enhance the expression of HSP70 in cells infected by MCMV, which maybe the molecular mechanism of JYBD in resisting MCMV.