HLA-G基因敲除人滋养层干细胞模型构建  

Construction of human trophoblast stem cell model with HLA-G knockout

作　　者：王思东 程延飞 曹丹丹 崔馨元 庄白妹 张博伦 姚元庆 WANG Si-dong;CHENG Yan-fei;CAO Dan-dan;CUI Xin-yuan;ZHUANG Bai-mei;ZHANG Bo-lun;YAO Yuan-qing(Graduate School of the Chinese PLA General Hospital,Beijing 100853;Department of Obstetrics&Gynecology,the Seventh Medical Center,Chinese PLA General Hospital,Beijing 100010;Shenzhen Key Laboratory of Fertility Regulation,Reproductive Medicine Center,the University of Hong Kong-Shenzhen Hospital,Shenzhen 518053;School of Medicine,Nankai University,Tianjin 300071)

机构地区：[1]中国人民解放军总医院研究生院,北京100853 [2]中国人民解放军总医院第七医学中心妇产科,北京100010 [3]深圳市生育调控重点实验室,香港大学-深圳医院生殖医学中心,深圳518053 [4]南开大学医学院,天津300071

出　　处：《生殖医学杂志》2023年第8期1216-1222,共7页Journal of Reproductive Medicine

基　　金：国家自然科学基金面上项目(82271704);国家高技术研究发展计划(2015AA020402)。

摘　　要：目的构建人白细胞抗原G(HLA-G)基因敲除的人滋养层干细胞(TSC)模型,初步探究HLA-G对TSC向绒毛外滋养层细胞(EVT)分化的影响。方法利用CRISPR/Cas9基因编辑技术敲除人TSC的HLA-G基因,将TSC向EVT细胞分化,采用免疫荧光染色及实时定量聚合酶链式反应(qPCR)方法观察HLA-G基因敲除对其标志基因表达的影响。结果经测序、免疫荧光染色及蛋白质印记法(WB)验证,成功获得了HLA-G基因完全敲除的人TSC细胞系。HLA-G^(-/-)-TSC可诱导分化为EVT样细胞,HLA-G^(-/-)-EVT的标志基因表达量与野生型相比无显著性差异(P>0.05)。结论本研究成功构建了稳定的HLA-G基因敲除人TSC模型,且HLA-G并不明显影响EVT的分化过程。Objective:To construct the human trophoblast stem cell(TSC)model with HLA-G knockout,and preliminarily investigate the effect of HLA-G on the differentiation of TSC into extravillous trophoblast(EVT).Methods:The HLA-G gene of human TSC was knocked out by using CRISPR/Cas9 gene editing technology,and then the differentiation of TSC into EVT were inducted.Immunofluorescence staining and qPCR were performed to observe the effect of HLA-G knockout on the expression of their marker genes.Results:After sequencing,immunofluorescence staining and Western blot verification,the human TSC cell line with complete HLA-G gene knockout was successfully obtained.HLA-G^(-/-)-TSC can be differentiate into EVT-like cells.There was no significant difference in the expression of EVT marker genes compared to wild type(P>0.05).Conclusions:Human trophoblast stem cell model with HLA-G knockout is successfully constructed.HLA-G knockout does not significantly affect the EVT differentiation process.

关 键 词：人白细胞抗原G 人滋养层干细胞 绒毛外滋养细胞 CRISPR/Cas9 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]