人滋养层干细胞的分离培养与鉴定

ISOLATION,CULTURE,AND IDENTIFICATION OF HUMAN TROPHOBLAST STEM CELLS

目的建立可在体外培养的人滋养层干细胞系(hTSCs),为研究胎盘的发育机制提供新的模型。方法取临沂市妇幼保健院生殖医学科患者捐赠的发育正常的5~6 d囊胚,利用延时培养技术分离获取hTSCs,进行细胞核型分析,利用免疫荧光技术和实时荧光定量PCR(RT-qPCR)技术检测hTSCs标记物的表达,并对hTSCs的侵袭能力、分化能力以及内分泌功能进行检测。结果本实验成功建立4株hTSCs,其均具有正常细胞核型。免疫荧光技术检测显示,第8代hTSCs中转录因子活化蛋白2C(TFAP2C)、细胞角蛋白7(CK7)、转录因子GATA结合蛋白3(GATA-3)稳定表达;RT-qPCR检测结果显示,第8、16代hTSCs中CK7、GATA3、TFAP2C、TEAD4基因的相对表达量无显著性差异(P>0.05);分化组第2、4、6天穿过trasnswell微孔的细胞数量均多于未分化组(F=53.31~234.55,P<0.05);第4、6天分化组穿过trasnswell微孔的细胞表达GCM1的比例均高于未分化组(F=50.25,131.13,P<0.05);分化组中CK7、SDC-1、β-hCG和ITGA5基因的相对表达量均显著高于未分化组(t=-6.78~-2.51,P<0.05);分化组第2、4、6天培养基中雌二醇、孕酮浓度均高于未分化组(F=25.06~225.62,P<0.05)。结论本研究成功分离获取的hTSCs具有正常核型,均表达hTSCs特异标记物,其侵袭能力、分化能力及分泌功能等与人类早期胎盘滋养层细胞相似。这将为进一步研究人类滋养层细胞的发育和功能提供有力的工具。

Objective To establish human trophoblast stem cell(hTSC)lines that can be cultured in vitro,and to provide a new model for the study of placental development.Methods The normal blastocysts of 5-6 d were collected from the patients who were treated in Department of Reproductive Medicine,Linyi Maternal and Child Health Hospital,and the karyotype of cells was analyzed.Immunofluorescence assay and quantitative real-time PCR were used to measure the expression of hTSC mar-kers,and hTSCs were tested in terms of invasion ability,differentiation capacity,and secretory function.Results Four hTSC cell lines with normal karyotype were successfully established in this experiment.Immunofluorescence assay showed stable expression of TFAP2C,CK7,and GATA3 in the 8th-generation hTSCs,and quantitative real-time PCR showed that there were no significant differences in the relative expression levels of CK 7,GATA 3,TFAP 2 C,and TEAD 4 genes between the 8th-generation hTSCs and the 16th-generation hTSCs(P>0.05).Compared with the undifferentiated group,the differentiated group had a signi-ficantly higher number of cells passing through Transwell micropores on days 2,4,and 6(F=53.31-234.55,P<0.05)and a significantly higher proportion of cells expressing GCM1 among such cells on days 4 and 6(F=50.25,131.13,P<0.05).Compared with the undifferentiated group,the differentiated group had significantly higher relative expression levels of CK 7,SDC-1,β-hCG,and ITGA 5 genes(t=-6.78--2.51,P<0.05),as well as significantly higher concentrations of estradiol and progesterone in culture medim on days 2,4,and 6(F=25.06-225.62,P<0.05).Conclusion The hTSCs successfully isolated and obtained in this study have normal karyotypes and express the specific markers of hTSCs,and they are similar to early human placental trophoblast cells in terms of invasion ability,differentiation capacity,and secretory function.This will provide a powerful tool for further research on the development and function of human trophoblast cells.