妊娠期糖尿病中胎盘外泌体对滋养细胞增殖、细胞周期及凋亡的影响

Effects of exosomes on trophoblast proliferation,cell cycle and apoptosis in placenta of gestational diabetes mellitus

目的:探讨妊娠期糖尿病(GDM)中胎盘外泌体对滋养细胞增殖、细胞周期及细胞凋亡的影响。方法:选取2018年3月至2019年4月间重庆市第七人民医院50例诊断为GDM患者(GDM组),另选取50例正常孕产妇作为对照组。分离纯化GDM组和对照组孕产妇外周血血浆中的胎盘外泌体,通过透射电镜观察外泌体的形态,纳米粒径分析检测外泌体的直径,免疫印迹检测外泌体标志性蛋白CD63、TSG101及胎盘标志性分子胎盘碱性磷酸酶(PLAP)的表达。PKH67染色后荧光共聚焦显微镜观察体外培养滋养细胞系对外泌体的摄取情况;应用MTT实验检测外泌体对滋养细胞增殖能力的影响,流式细胞术检测外泌体对滋养细胞周期的影响,Annexin V-FITC/PI双染色结合流式细胞术检测外泌体对滋养细胞凋亡的影响。结果:成功从外周血中分离获得了胎盘外泌体,形态呈典型的杯状或双凹状,直径为40~120 nm,CD63、TSG101、PLAP表达呈阳性;与对照组相比,GDM组胎盘外泌体以浓度依赖性促进滋养细胞的增殖、细胞周期进展,并抑制滋养细胞的凋亡。结论:GDM中胎盘外泌体可能通过促进滋养细胞的增殖、细胞周期进展,抑制滋养细胞凋亡等参与GDM的发生和发展。

Objective:To investigate the effect of placenta exosomes on trophoblast proliferation,cell cycle and apoptosis in gestational diabetes mellitus(GDM);Methods:Fifty patients diagnosed with GDM(GDM group)were selected from Chongqing Seventh People's Hospital from March 2018 to April 2019,and another 50 normal pregnant were selected as control group.The placenta exosomes in the plasma of pregnant women with GDM and control group were separated and purified.The exosomes were identified by transmission electron microscopy,nanoparticle-tracking analysis and Western blotting to detect morphology,sizes of exosomes and expressions of CD63,TSG101 and placental alkaline phosphatase(PLAP).The uptake by trophoblast was observed by fluorescence confocal microscopy after PKH67 staining.MTT,flow cytometry and Annexin V-FITC/PI were used to detect the effects of exosomes on trophoblast proliferation,cell cycle and apoptosis.Results:Placental exosomes were successfully harvested from peripheral blood,with typical cup shape or double concave shape and diameter of 40-120 nm.Exosome biomarkers CD63,TSG101 and placental PLAP were positively expressed.Compared with control group,GDM could promote trophoblast proliferation,cell cycle progression and inhibit trophoblast apoptosis in a concentration dependent manner.Conclusion:The placenta exosomes may play an important role in the development of gestational diabetes by promoting the proliferation of trophoblasts and cell cycle progression,and inhibiting apoptosis.