绵羊羊水、胎儿肾脏组织中表达Oct-4细胞的分离培养及其分子学特性研究

Comparative Analysis in Molecular Characteristic of the Oct-4 expressing Cells Isolated from Sheep Amniotic Fluid and Fetal Renal Tissues

试验旨在建立从绵羊羊水、胎儿肾脏组织中分离培养表达Oct-4细胞的新方法,同时,通过实时荧光定量PCR(Real-time PCR)初步探索二者在分子生物学特性方面的联系及差异。采用0.01%胰酶培养液,从同一只受孕中期绵羊的羊水和胎儿肾脏组织中各分离1株细胞。Real-time PCR分析结果表明,从绵羊羊水、胎儿肾脏组织中分离的细胞均表达胚胎干细胞相关标志基因Oct-4、胚胎生殖细胞标志基因SSEA-1、主要组织相容性抗原MHC-Ⅱ、细胞凋亡基因Bax及抑制细胞凋亡基因Bcl-2,不表达MHC-Ⅰ、Nanog及TERT,因此,将羊水中表达Oct-4的细胞暂命名为羊水来源多潜能干细胞(amniotic fluid stem cells,AFSC),胎儿肾脏组织中表达Oct-4的细胞暂命名为肾脏组织干细胞(renal tissue stem cells,RTSC)。相对定量分析结果显示,二者的Oct-4、Bax及Bcl-2基因的相对表达量存在明显差异。绵羊羊水中表达Oct-4的细胞(AFSC)和胎儿肾脏组织中表达Oct-4的细胞(RTSC)体外悬浮培养7d均能形成类胚体。试验成功建立了从绵羊羊水、胎儿肾脏组织中分离培养表达Oct-4细胞的新方法,实时荧光定量PCR分析初步显示绵羊羊水中表达Oct-4的细胞和胎儿肾脏组织中表达Oct-4的细胞具有相同的起源,并在体内处于一种动态的变化之中。

This study aimed at developing a novel technique of isolating the Oct-4 expressing cells from the sheep amniotic fluid and fetal renal tissues, respectively, and comparatively analyzing the difference in the molecular characteristic of these two differently derived cells with Real-time PCR. The results showed that the cells from the sheep amniotic fluid and fetal renal tissue were both positive for Oct-4, embryonic germ cells gene SSEA-1, the main organization compatibility antigen gene MHC-Ⅱ, apoptosis gene Bax, and inhibition gene of apoptosis Bcl-2, negative for the symbol marker of MHC-Ⅰ, Nanog and TERT, but there were marked differences in the relative expression levels of Oct-4， Bax and Bcl-2 genes. Furthermore, AFSC and RTSC were both capable of forming embryoid bodies through the suspension culture in vitro. In conclusion, our study successfully established a new procedure of isolating and culturing Oct-4 expressing cells derived from the sheep amniotic fluid and fetal renal tissues, which might have the same origin.