复发性流产中活性维生素D3与维生素D3受体的表达及其对滋养细胞增殖与侵袭的调控和机制研究

Expression of active Vitamin D3 and Vitamin D3 receptor in recurrent abortion and their regulation and mechanism on proliferation and invasion of trophoblast cells

目的:通过检测复发性流产(RSA)中活性维生素D(VD)与维生素D受体(VDR)的表达,初步研究其相关作用和分子机制。方法:免疫组化(IHC)与RT-PCR法检测20例RSA患者(RSA组)与20例自愿选择终止妊娠的正常对照女性(对照组)胎盘绒毛组织中VD关键代谢酶25-羟基维生素D3-1α-羟化酶(CYP27B1)与VDR的表达,Western blot法检测两组绒毛组织中自噬关键蛋白LC3-Ⅱ与LC3-Ⅰ比值和p62蛋白表达水平。体外培养滋养细胞系HTR8/SVneo,利用脂质体转染技术将沉默VDR的siRNA(si-VDR)与阴性对照siRNA(si-NC)转染入细胞,RT-PCR与Western blot法检测转染效率。按处理条件不同,将滋养细胞分为4组si-NC组,活性VD处理转染si-NC或si-VDR的si-NC+VD组、si-VDR+VD组,联合使用VD与自噬抑制剂3-MA处理的si-NC+VD+3-MA组。MTT与EdU法检测各组滋养细胞的增殖能力;Transwell实验检测各组滋养细胞侵袭能力;透射电镜观察各组滋养细胞中自噬囊泡数;免疫荧光检测细胞中LC3B表达水平;Western blot实验检测LC3-Ⅱ/LC3-Ⅰ比值和p62蛋白表达;Western blot实验明确各组滋养细胞中AMPK/mTOR途径的AMPK(p-AMPK/AMPK)与mTOR(p-mTOR/mTOR)磷酸化水平。结果:与对照组相比,RSA组胎盘绒毛组织中CYP27B1与VDR表达和自噬水平均明显降低(P<0.05或P<0.01);与转染si-NC的滋养细胞相比,转染si-VDR能明显下调VDR的mRNA与蛋白表达(均P<0.01);与si-NC组相比,si-NC+VD组的细胞增殖、侵袭和自噬能力均显著增加(P<0.05),AMPK(p-AMPK/AMPK)磷酸化水平明显升高(P<0.05),mTOR(p-mTOR/mTOR)的磷酸化水平显著降低(P<0.05)。si-VDR+VD组上述细胞功能或检测指标无明显改变(P>0.05),而si-NC+VD+3-MA组均明显降低(P<0.05)。与si-NC+VD组相比,si-VDR+VD组与si-NC+VD+3-MA组的上述细胞功能改变均明显降低(P<0.05),但AMPK(p-AMPK/AMPK)磷酸化水平显著降低(P<0.05),而mTOR(p-mTOR/mTOR)磷酸化水平显著增加(P<0.05)。结论:RSA患者胎盘绒毛组织中VD的代谢与VDR表达和自噬水平均较正常妊娠女性显著降低,而外源性予以活性VD能通过AMPK/mTOR信号通路调控滋养细胞的自噬,从而促进其增殖与侵袭功能。

Objective:To detect the expression of active Vitamin D(VD)and its receptor VDR in recurrent abortion(RSA),and its related effect and molecular mechanism.Methods:Immunohistochemistry(IHC)and RT-PCR were used to detect the expressions of 25-hydroxyvitamin D3-1α-hydroxylase(CYP27B1)and VDR in placental villus tissues of 20 RSA patients(RSA group)and 20 normal control women(Ctrl group)who had voluntarily terminated pregnancy.The ratio of autophagy key protein LC3-Ⅱto LC3-Ⅰand the expression level of p62 protein in villi of participants in the above two groups were detected by Western blot assay.Human trophoblasts cell line HTR8/SVneo was cultured in vitro,and VDR-silenced siRNA(si-VDR)and negative control siRNA(si-NC)were transfected into the cells by liposome transfection technology.The transfection efficiency was detected by RT-PCR and Western blot.According to different treatment conditions,trophoblast cells were divided into four groups:si-NC group,the si-NC+VD group transfected with si-NC or si-VDR was treated with active VD,si-NC+VD+3-MA group treated with VD combined with autophagy inhibitor 3-MA.MTT and EdU were used to detect the proliferation of trophoblast cells in each group.The invasion ability of trophoblast cells in each group was detected by Transwell assay.The number of autophagy vesicles in trophoblast cells was observed by transmission electron microscopy,the expression level of LC3B was detected by immunofluorescence,and the ratio of LC3-Ⅱand LC3-Ⅰand the expression of p62 protein were detected by Western blot assay.Western blot was used to determine the phosphorylation levels of AMPK(p-AMPK/AMPK)and mTOR(p-mTOR/mTOR)in the AMPK/mTOR pathway in trophoblast cells of each group.Results:Compared with Ctrl group,the expressions of CYP27B1 and VDR and the level of autophagy in placental villus of RSA group were significantly decreased(P<0.05 or P<0.01).Compared with the trophoblast cells transfected with si-NC group,the mRNA and protein expression of VDR could be significantly down-regulated after transfection with si-VDR(P<0.01).Compared with si-NC group,cell proliferation,invasion and autophagy in si-NC+VD group were significantly increased(P<0.05),and phosphorylation levels of AMPK(p-AMPK/AMPK)and mTOR(p-mTOR/mTOR)were significantly increased.There were no significant changes in the above cell functions or detection indexes in si-VDR+VD group(P>0.05),while those in si-NC+VD+3-MA group were significantly decreased(P<0.05).Meanwhile,compared with si-NC+VD group,the above cell function changes in si-VDR+VD group and si-NC+VD+3-MA group were significantly decreased(P<0.05),the phosphorylation levels of AMPK and mTOR were significantly increased(P<0.05).Conclusions:VD metabolism,VDR expression and autophagy level in placental villi of RSA patients were significantly decreased compared with normal pregnant women,and exogenous active VD could regulate autophagy of trophoblast cells through AMPK/mTOR signaling pathway,thereby promoting their proliferation and invasion.