刚地弓形虫RH株对小鼠胎盘滋养细胞凋亡的影响

Effect of Toxoplasma gondii RH strain on apoptosis of mouse placental trophoblastic cells

目的通过研究刚地弓形虫强毒株RH株对体外培养的孕期小鼠胎盘滋养层细胞凋亡的影响,初步探讨不同毒力虫株引起病理妊娠的机制。方法将小鼠胎盘滋养层细胞分别接种于不同细胞培养瓶,对照组加入DMEM培养基孵育,实验组加入相同体积不同数量(2×106/ml、4×106/ml、8×106/ml)弓形虫速殖子培养2、4、8h,Annexin-V-FITC/PI染色细胞后上流式细胞仪检测各个时间点细胞凋亡率变化;荧光显微镜观察细胞形态改变情况;Western blot分析凋亡相关蛋白Bax、Bcl-2的表达或活性。结果流式细胞仪检测弓形虫感染后的滋养层细胞凋亡率较对照组有下降趋势(P<0.05),呈量效依赖性,弓形虫数量8×106/ml实验组8h凋亡率最低,为(15.81±0.19)%;荧光显微镜观察到8h不同数量实验组滋养细胞凋亡形态改变情况,各组均有典型的早期凋亡细胞:细胞膜着绿色,细胞核拒染。随着弓形虫感染数量增加,凋亡细胞数目有所减少。Western blot检测刚地弓形虫RH株作用于滋养层细胞8h后实验组的Bax、Bcl-2蛋白表达较对照组分别有明显的降低与升高(P<0.05)。结论刚地弓形虫RH株可抵抗体外培养的孕期小鼠滋养细胞正常凋亡,其机制可能与滋养细胞Bax表达下调和Bcl-2表达上调有关。

Objective To study the effect of Toxoplasma gondii（T. gondii） RH strain on mouse placental trophoblastic cells in vitro and the mechanism of Toxoplasma gondii RH strain underlying pathological pregnancy. Methods Mouse placental trophoblastic cells were inoculated into different cell culture plates and divided into control group and experimental group. Trophoblastic cells in control group were incubated in DMEM and those in experimental group were incubated with tachyzoites at a concentration of 2×106/ml,4×106/ml,and 8×106/ml for 2,4 and 8 h, respectively. Apoptosis of trophoblastic cells was detected by FCM with Annexin-V-FITC/PI staining. Changes in cell morphology were observed under fluorescence microscope. Expression or activity of apoptosis-related Bax and Bcl-2 proteins in trophoblastic cells was detected by Western blotting. Results FCM showed that the apoptosis rate of trophoblastic cells infected with T. gondii RH strain was decreased in a dose-dependent manner and was lower in experimental group than in control group, with the lowest apoptosis rate of （15.81±0.19）% observed in experimental group 8 h after the concentration of tachyzoites was increased to 8×106/ml. Typical early apoptotic cells were found under fluorescence microscope with their membrane green stained and their nuclei not stained. Western blotting showed that the expression levels of Bcl-2 and Bax proteins was higher in experimental group than in control group（P0.05）. Conclusion T. gondii RH strain can resist apoptosis of mouse placental trophoblastic cells in vitro by down-regulating the Bax expression and up-regulating the Bcl-2 expression.