论文部分内容阅读
目的检测柯萨奇病毒B3(CVB3)感染心肌细胞后鞘磷脂酶活性的变化、鞘磷脂酶mRNA、CVB3RNA的表达、病毒滴度的变化及心肌细胞表型的变化。初步探讨鞘磷脂酶在CVB3感染的心肌细胞中的作用。方法 CVB3感染心肌细胞后不同时间点,用薄层色谱法(TLC)检测鞘磷脂酶活性;用Real time-PCR检测心肌细胞中鞘磷脂酶mRNA与CVB3RNA的表达;同时取细胞培养上清液检测CVB3滴度。用Annexin V-FITC,PI双染法观察心肌细胞表型变化。设正常心肌细胞对照组,每个时间点设三个复孔。结果病毒感染组与正常心肌细胞相比,酸性鞘磷脂酶在4h、12h时活性有升高,其mRNA表达在2h时有升高趋势(P<0.05);中性Mg2+非依赖性鞘磷脂酶活性在4h时有明显升高,在12h时活性下降,而其mRNA表达在20h时升高(P<0.05);中性Mg2+依赖性鞘磷脂酶活性于病毒感染后4h、12h无明显变化,其mRNA表达在各组间也无明显变化。心肌细胞内病毒RNA表达在4h时有升高趋势,8h后开始明显升高(P<0.01)。CVB3病毒滴度在2h时明显升高(P<0.05)。心肌细胞在病毒感染2h后早期凋亡及坏死开始增加,4h时凋亡坏死增加更明显,到24h时坏死心肌细胞明显增多。以上实验均重复3次。结论中性Mg2+非依赖性鞘磷脂酶与酸性鞘磷脂酶在CVB3侵入心肌细胞及病毒的扩散增殖过程中可能起重要作用;而中性Mg2+依赖性鞘磷脂酶在此过程中可能不起作用。
Objective To detect the changes of sphingomyelinase activity, the expression of sphingomyelinase mRNA and CVB3RNA, the virus titer and the changes of cardiomyocyte phenotypes after coxsackievirus B3 (CVB3) infection. Preliminary study of the role of sphingomyelinase in CVB3 infected cardiomyocytes. Methods The cardiomyocytes were infected with CVB3 at different time points, and the activity of sphingomyelinase was detected by TLC. The expression of sphingomyelinase mRNA and CVB3 mRNA in cardiomyocytes was detected by Real time-PCR. The cell culture supernatants CVB3 titer. Cardiomyocyte phenotype changes were observed by Annexin V-FITC and PI double staining. Set normal control group of cardiomyocytes, each time point set three complex hole. Results Compared with normal cardiomyocytes, the activity of sphingomyelinase increased at 4h and 12h, and the mRNA expression increased at 2h (P <0.05). Neutral Mg2 + -dependent sphingomyelinase The activity increased at 4h and decreased at 12h, while the mRNA expression increased at 20h (P <0.05). Neutral Mg2 + -dependent sphingomyelinase activity did not change significantly at 4h and 12h after virus infection, The mRNA expression did not change significantly among the groups. The expression of viral RNA in cardiomyocytes increased at 4h and began to increase at 8h (P <0.01). The titer of CVB3 virus was significantly increased at 2h (P <0.05). Early apoptosis and necrosis of cardiomyocytes began to increase 2 h after virus infection, and apoptosis and necrosis increased more obviously at 4 h, and the number of necrotic cardiomyocytes increased obviously at 24h. The above experiments were repeated 3 times. Conclusion Neutral Mg2 + -dependent sphingomyelinase and sphingomyelinase may play an important role in the proliferation and proliferation of CVB3-infected cardiomyocytes and viruses. Neutral Mg2 + -dependent sphingomyelinase may not play a role in this process.