青蒿琥酯对HSC-T6细胞PKC蛋白细胞内转位的影响

Effect of Artesunate on Intracellular Translocation of PKC Protein in HSC-T6 Cell Line

目的:探讨青蒿琥酯抗肝纤维化的作用机理。方法:将培养的肝星形细胞(HSC)-T6细胞分为阴性对照组和15mg/L青蒿琥酯组、60mg/L青蒿琥酯组、120mg/L青蒿琥酯组、240mg/L青蒿琥酯组,阴性对照组用培养液代替青蒿琥酯,其它组分别加入15mg/L、60mg/L、120mg/L、240mg/L浓度的青蒿琥酯,培养2h。通过青蒿琥酯对体外培养的HSC-T6细胞进行干预,采用免疫荧光的技术从信号转导通路的途径检测细胞因子蛋白激酶(PKC)的转位情况。结果:阴性对照组HSC-T6细胞为典型的梭形,形态正常,细胞表面可见细长的突起,没有荧光显示。15mg/L青蒿琥酯组细胞形态基本正常,见有细长的突起,但表面有些发泡,荧光显示在胞浆中;青蒿琥酯60mg/L组细胞突起变短变少,表面有些发泡,荧光显示在胞浆中;120mg/L青蒿琥酯组细胞发泡更加明显,突起变短,由梭形渐变成椭圆形,胞浆较多颗粒,荧光显示在胞浆中;240mg/L青蒿琥酯组细胞数目较少,细胞明显皱缩,荧光显示在胞浆中。结论:经过青蒿琥酯作用后,免疫荧光检测显示,未发现PKC在HSC-T6细胞内出现转位,绿色荧光显示区域主要是在胞浆内,细胞膜上没有明显荧光。青蒿琥酯作用于细胞时可能阻断了血小板源性生长因子(PDGF)/PKC的细胞内信号转导,这可能是其抑制HSC细胞增殖的重要机制之一。

Objective:To approach the possible anti-hepatic fibrosis mechanism of Artesunate.Methods:Hepatic stellate cells(HSC)-T6 cells were divided into the negative control group,15mg/L Artesunate group,60mg/L Artesunate group,120mg/L Artesunate group,and 240mg/L Artesunate group.The negative control group was cultured with culture fluid,and the other Artesunate groups were cultured with 15mg/L,60mg/L,120mg/L and 240mg/L Artesunate,respectively.The culture lasted 2 hours.Translocation of protein kinase C(PKC)of HSC-T6 cells was detected by immunofluorescence technique through the signal transduction channel.Results:The cells of the negative control group were fusiform in normal shape,and had slender processes on the surface without fluorescence display.The cells of 15mg/L Artesunate group were normal in shape,and had some foam on the surface,with fluorescence display in the endochylema.The cells of 60mg/L Artesunate group had short and less processes,and had some foam on the surface,with fluorescence display in the endochylema.The cells of 120mg/L Artesunate group became into oval and had shorter processes and much more foams on the surface,with more grain display in the endochylema.The cells of 240mg/L Artesunate group were less in number and shrinked,with fluorescence display in the endochylema.Conclusion:After culturing with Artesunate,the green fluorescence displays only in the endochylema but not on the cell membrane,showing no intracellular translocation of PKC protein.Artesunate can block the signal pathway of PDGF/PKC,which may be one of its important mechanisms of inhibiting the proliferation of HSC-T6 cells.