华支睾吸虫溶血磷脂酶对大鼠肝星状细胞和卵圆细胞的作用

The effects of Lysophospholipase from Clonorchis sinensis on the hepatic stellate cells and oval cells of rat

目的:观察华支睾吸虫溶血磷脂酶(CslysoPLA)重组蛋白体外对大鼠肝星状细胞和卵圆细胞的作用。方法:应用免疫荧光方法,观察CslysoPLA重组蛋白是否可与大鼠肝星状细胞和卵圆细胞膜结合;应用MTT方法,测定CslysoPLA重组蛋白对大鼠肝星状细胞和卵圆细胞的促增殖作用;应用流式细胞术(FCM)检测CslysoPLA重组蛋白对大鼠卵圆细胞细胞周期的影响。结果:CslysoPLA重组蛋白可与大鼠肝星状细胞和卵圆细胞膜结合,MTT方法测定结果表明低浓度重组蛋白对大鼠肝星状细胞和卵圆细胞有一定促增殖作用(P<0.05),20mg/L重组蛋白可促进大鼠卵圆细胞进入G2期,但高浓度重组蛋白可导致大鼠肝星状细胞和卵圆细胞死亡。结论:低浓度的CslysoPLA重组蛋白在体外可刺激大鼠肝星状细胞和卵圆细胞增殖,表明CslysoPLA可能是华支睾吸虫导致胆管上皮增生、腺瘤样病变和肝纤维化的效应分子之一。而高浓度的CslysoPLA重组蛋白可引起肝星状细胞和卵圆细胞死亡,表明CslysoPLA亦可能是华支睾吸虫引起的化学损伤的毒力因子之一。

AIM： To clarify the effects of the recombinant protein of Lysophospholipase from Clonorchis sinensis （CsLysoPLA） on the hepatic stellate cells （HSC） and oval cells of rat. METHODS： Binding of the recombinant Cslyso- PLA protein to the membrane of HSC and oval cells was indentified by immunofluorescent staining. The HSC and oval cells were cultured and treated with the recombinant protein at different doses, and proliferation was quantified by MTT method. Cell cycle analysis was performed by flow cytometry. RESULTS： The recombinant CslysoPLA protein could bind to the membrane of HSC and oval cells. Compared to control, 2 mg/L and 20 mg/L the recombinant protein could promote HSC and oval cells growth （ P 〈 0.05 ）, whereas 200 mg/L the recombinant protein could induce the cells necrosis, which associated with overt plasma membrane disruption. Oval cell number in G2 phase of the recombinant protein 20 mg/L treated group was higher than that of control group. CONCLUSION： In vitro, the recombinant protein could induce HSC and oval cells proliferation at low concentrations （2 mg/L and 20 mg/L）, whereas it also could induce the cells necrosis at high concentration （200 mg/L）. These results suggested that CslysoPLA might play a role in the pathogenicity of C. sinensis.