胎盘来源外泌体对胎盘血管内皮细胞钙敏感受体和凋亡的影响

Effect of placenta-derived exosomes on calcium-sensitive receptors and apoptosis of umbilical vein endothelial cells

目的 探讨胎盘来源外泌体对胎盘血管内皮细胞钙敏感受体(Ca SR)和凋亡的影响。方法 通过Exo Quick提取并分离得到妊娠期高血压孕妇和正常孕妇胎盘外泌体,采用蛋白质免疫印迹(Western blot)法检测外泌体标志性蛋白CD63和TSG101表达的以鉴定外泌体,电镜观察外泌体直径大小;单独常规培养的人脐静脉内皮细胞作为对照组,与妊娠期高血压孕妇胎盘来源外泌体共培养的人脐静脉内皮细胞作为处理组,与正常孕妇胎盘来源外泌体共培养的人脐静脉内皮细胞作为阴性对照组,培养48 h;采用Western blot检测Ca SR及凋亡相关蛋白Bax及Bcl-2的表达,TUNEL染色检测内皮细胞的凋亡。结果 成功分离得到40~100 nm胎盘来源的外泌体,其高表达CD63及TSG101;培养48 h时,阴性对照组Ca SR、Bax和Bcl-2蛋白表达与对照组相比,差异无统计学意义(P>0.05);与阴性对照组相比,处理组Ca SR和Bax表达水平、TUNEL阳性细胞率明显升高,差异有统计学意义(P<0.01)。结论 妊娠高血压孕妇来源外泌体能够促进内皮细胞Ca SR表达,诱导内皮细胞凋亡。

Objective To investigate the effect of placenta-derived exosomes on calcium sensitive receptors(CaSR) and apoptosis of placental vascular endothelial cells.Methods ExoQuick kit was used to isolate placental exosomes from pregnant women with hypertension during pregnancy and normal pregnant women.Western blot was used to detect the expression levels of exosomal marker proteins CD63 and TSG101 to verify exosomes.The diameter sizes of isolated exosome were evaluated using electron microscope.Cultured human umbilical vein endothelial cells(HUVECs) were used as a control group.HUVECs co-cultured with exosomes isolated from pregnant women with hypertension for48 h were used as the treatment group,while HUVES co-cultured with isolated exosomes from normal pregnant women for 48 h as a negative control group.Western blot was used to detect the expression of CaSR and apoptosis-related proteins Bax and Bcl-2.TUNEL staining was used to detect HUVEC apoptosis.Results The diameters of the exosomes isolated from placenta were around 40 ~ 100 nm.Isolated exosomes expressed high levels of CD63 and TSG101.At 48 h after co-culture,the expression of CaSR,Bax,and Bcl-2 in the negative control group was not significantly different from those in control group(P>0.05).Compared with the negative control group,the expression levels of CaSR,Bax and the rate of TUNEL positive cells were significantly increased in the treatment group(P<0.01).Conclusion Exosomes isolated from pregnant women with pregnancy-induced hypertension can upregulate CaSR expression and induce HUVEC apoptosis.