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目的研究刚地弓形虫ME49株对体外培养的孕期小鼠胎盘滋养层细胞凋亡的影响。方法制备小鼠胎盘滋养层细胞并分别接种于不同细胞培养板,对照组加入DMEM高糖培养液孵育,实验组加入相同体积不同密度(2×106/ml、4×106/ml、8×106/ml)弓形虫速殖子培养8h后,AnnexinⅤ-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,以蛋白质印迹法分析凋亡相关蛋白Bax、Bcl-2的表达。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升高趋势(P<0.05),呈量效依赖性,弓形虫密度8×106/ml组8h凋亡率最高,为28.37%;荧光显微镜观察,随弓形虫感染密度增加,滋养层细胞凋亡数目增多。蛋白质印迹法检测刚地弓形虫ME49株作用于滋养层细胞8h后实验组的Bax、Bcl-2蛋白表达(与β-actin比值)各为1.24±0.05、1.37±0.03、1.78±0.04与1.15±0.03、1.09±0.05、0.97±0.01,较对照组的比值(1.17±0.06和1.23±0.02)有明显的改变(P<0.05)。结论刚地弓形虫ME49株可促进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表达上调和Bcl-2表达下调有关。
Objective To study the effect of ME49 strain of Toxoplasma gondii on the apoptosis of placental trophoblast cells during pregnancy in vitro. Methods Placental trophoblast cells were prepared and inoculated on different cell culture plates. The control group was cultured in DMEM high glucose medium. The experimental groups were incubated in the same volume of different density (2 × 106 / ml, 4 × 106 / ml, 8 × 106 / ml) Toxoplasma gondii tachyzoites 8h, AnnexinⅤ-FITC / PI staining cells were detected by flow cytometry at each time point after the change of apoptosis rate by Western blot analysis of apoptosis related protein Bax, Bcl-2 expression . Results The apoptosis rate of trophoblast cells after Toxoplasma gondii infection was higher than that of the control group (P <0.05). The apoptosis rate of Toxoplasma gondii at 8 × 106 / ml group was the highest , Which was 28.37%. Fluorescence microscopy showed that the number of trophoblast cells increased with the increase of Toxoplasma infection density. The protein expression of Bcl-2 and β-actin in the experimental group was 1.24 ± 0.05, 1.37 ± 0.03, 1.78 ± 0.04 and 1.15 ± 0.03, 1.09 ± 0.05 and 0.97 ± 0.01, respectively, compared with the control group (1.17 ± 0.06 and 1.23 ± 0.02) (P <0.05). Conclusion ME49 strain of T. gondii can promote normal gestational trophoblast apoptosis in vitro. The mechanism may be related to the up-regulation of Bax and the down-regulation of Bcl-2 in trophoblast cells.