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

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.