鞘磷脂酶在柯萨奇病毒B3感染心肌细胞中的作用

Role of Sphingomyelinases in Coxsackievirus B_3 infection of cardiomyocytes

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 study the role of sphingomyelinases in Coxsackievirus B3(CVB3)infection of cardiomyocytes via observing the changes of sphingomyelinase activity and mRNA expression post infection and its macroscopic relation with the progeny virus RNA synthesis and titer in cultured cardiomyocytes.Methods After different time courses of CVB3 infection as indicated,sphingomyelinase activity was detected by thin layer chromatography(TLC).Both sphingomyelinase mRNA expression level and CVB3 RNA level were assessed using Real time-PCR.And the CVB3 in the supernatant was titrated as well.Cardiomyocytes phenotype was oberserved observed with Annexin V-FITC,PI double staining.Normal controls were set and three wells were prepared respectively at different timepoint.Results Comparing with normal controls,the acid sphingomyelinase activity in CVB3 infected cardiomyocytes rised 4h p.i.and 12h p.i.respectively and its mRNA expression showed an increase tendency 2h p.i.Neutral Mg2+-independent sphingomyelinase activity increased 4h p.i.and decreased 12h p.i.with an elevated mRNA expression level 20h p.i.Both neutral Mg2+-dependent sphingomyelinase activity and its mRNA expression achieved no significantly changes after 4h and 12h CVB3 infection respectively.Virus RNA synthesis in the cardiomyocytes got an upward tendency 4h p.i.and showed a magnificent increase 8h p.i.CVB3 titer rised 2h p.i.in the supernatant.Experiments were repeated three times.Early apoptosis and necrosis of cardiomyocytes rised 2h p.i.and 4 h p.i.Necrosis of cardiomyocytes significantly increased 24h p.i.Conclusion Neutral Mg2+-independent sphingomyelinase and acid sphingomyelinase might play an important role in CVB3 invasion process and its proliferation in cardiomyocytes while neutral Mg2+-dependent sphingomyelinase wasn’t involved.