体外诱导羊膜上皮细胞转分化为雌性生殖细胞的初步实验研究

Preliminary Research on the Differentiation of Human Amniotic Epithelial Cells into Female Germ Cells

目的建立体外分离、培养人羊膜上皮细胞(hAECs)的方法,研究其向雌性生殖细胞转分化的潜能。方法体外分离、培养并鉴定hAECs后,采用添加5%人卵泡液的培养基作为诱导剂,倒置显微镜下观察细胞形态的改变,化学发光免疫技术检测培养基中雌二醇(E2)含量的变化,应用实时荧光定量PCR(RT-PCR)、Western blot等方法检测雌性生殖细胞相关基因及蛋白表达水平的变化。结果体外培养hAECs呈多角形,具有上皮细胞特有的铺路石样外观,并表达其特异性细胞角蛋白19(CK19),同时能表达胚胎干细胞(ESC)特异的转录因子POU5f1转录因子4(Oct-4)。采用添加5%人卵泡液的培养基诱导14d后,对照组大部分细胞仍保持上皮细胞的多角形态,而诱导组细胞融合呈集落样生长。E2含量检测结果显示,对照组无E2生成,而诱导组E2水平从第8d开始增加,至第10d达到高峰,然后下降。RT-PCR、Western blot结果表明,诱导组生殖细胞特异性基因生长分化因子9(GDF9)、无精症缺失基因(DAZL)的mRNA表达水平明显升高(P<0.05),而联会复合体蛋白3(SCP3)的mRNA表达水平无明显升高(P>0.05),DAZL的蛋白表达水平亦明显增加(P<0.05)。结论人卵泡液可在体外诱导hAECs向雌性生殖细胞方向转分化。

Objecitve To establish the method for isolation and culturation of human amniotie epithelial cells （hAECs） in vitro, and to investigate the differentiation potential of hAECs towards germ cells. Methods hAECs were isolated from human term placenta and identified by immunoeytochemistry, hAECs were sequential induced to form germ cells in medium supplemented with 5 % human follicular fluid. Morphological changes were observed by inverted microscope. The oestradiol levels in the spent media were assayed. The expression of germ cell special genes and protein were examined by Real-time PCR and Western blot separately. Results Most of the isolated hAECs were polygon and of typical alabstone-like appearance, could express cytokeratin 19, the specific marker of epithelial cells, as well as oetamer-binding protein 4 （Oct-4）, a specific marker of embryonic stem cells （ESCs）. After induced in medium supplemented with 5% human follicular fluid for 14 days, these stem cells grew as aggregates morphologically. Aliquots of supernatant of culture were collected and were assayed for oestradiol using chemiluminescence immunoassay （CLIA）, although oestradiol was absent in the control group, the levels of the induction group were increased by day 8 and reached the maxima by day 10. Our study showed that hAECs treated with human follicular fluid were able to express germ cell genes, including growth differentiation factor 9（GDFg） and deleted in azoospermia like （DAZL）（P〈0. 05）, but without synaptonemal complex protein 3 （SCP3） （P〉0.05）. The DAZI. protein were also examined （P〈0.05）. Conclusion Germ cells could be induced in vitro from hAECs by human follicular fluid.