卵泡液微环境对人羊水干细胞体外分化为雌性生殖细胞的影响

Effect of Follicular Microenvironment on Differentiation of Human Amniotic Fluid Stem Cells into Female Germ Cells in Vitro

目的观察不同直径猪卵泡内卵泡液诱导人羊水干细胞(human amniotic fluid stem cells,hAFSCs)-070607株向雌性生殖细胞分化的效果。探讨卵泡内微环境对体外雌性生殖细胞形成的影响。方法选取不同直径猪卵泡内的卵泡液(S组:直径<3 mm、M组:直径3~6 mm、L组:直径>6 mm),检测卵泡液内雌二醇(E2)和卵泡刺激素(FSH)的含量及骨形态发生蛋白15(BMP15)的表达。将不同分组卵泡液添加到雌性生殖细胞分化体系中,经过5 d的体外诱导培养,观察生殖细胞基因OCT4、FIGLα、GDF9、FSHR和甲基化基因DNMT3B的表达变化。继续用M组卵泡液诱导hAFSCs-070607,观察基因及蛋白表达的变化。结果通过基因、蛋白和三胚层分化的检测,确定hAFSCs-070607具有良好的多能性及分化潜能。3组不同直径的卵泡液内都含有FSH,但不含E2;BMP15的未成熟蛋白在M组中的含量略高于S、L组。M组添加到诱导分化培养基后,E2的产量较高,生殖相关标记基因检测结果发现,OCT4、FIGLα、GDF9、FSHR和DNMT3B基因的表达依次按照大、中、小卵泡组上升。继续用M组卵泡液诱导hAFSCs-070607,诱导10d后,有卵母细胞样细胞(oocyte-like cells,OLCs)和卵泡样结构的出现,经EGFP-BMP15转染后,这些结构显示EGFP表达阳性,表明有OLCs的产生。荧光定量PCR结果证实,生殖细胞形成相关基因OCT4、FIGLα、GDF9、FSHR和表观学基因DNMT3B的表达逐渐呈上升的趋势。经细胞免疫荧光技术检测发现,生殖相关蛋白OCT4、FRAGILIS和SCP3呈阳性表达。结论hAFSCs-070607株人羊水干细胞具有良好的多能性和体外分化潜能,经猪卵泡液体外诱导分化后,可分化为雌性生殖细胞,且M组卵泡液诱导后细胞分泌的E2高于S组和L组。长时间诱导后,生殖细胞相关基因和蛋白上调,并能形成卵泡样结构。

Objective To observe the effect of follicular fluid in different diameter porcine follicles on the differentiation of human amniotic fluid stem cells(hAFSCs)-070607 into female germ cells in vitro.And to investigate the effects of microenvironment in follicles on the formation of female germ cells in vitro.Methods The pluripotency and differentiation potential of hAFSCs-070607 were determined by detection of gene,protein and triple germ layer differentiation.The follicular fluids(diameter<3mm,diameter3~6mm,diameter>6mm)in follicles of different diameters were selected to detect the content of estradiol(E2)and follicle stimulating hormone(FSH)in follicular fluid,and the expression of bone morphogenetic protein15(BMP15).Meanwhile,different groups of follicular fluids were added to the female germ cell differentiation system,and the expression of germ cell genes OCT4,FIGLα,GDF9,FSHR and methylation gene DNMT3B were tested by Real time PCR after 5 days of differentiation.Secondary follicles were selected for further study.The hAFSCs were induced by M group follicular fluid,and the changes of gene and protein expression were observed.Results Through the detection of gene,protein and triple germ layer differentiation,hAFSCs-070607 had good pluripotency and differentiation potential.FSH was contained in all kinds of follicular fluids,but E2 was not.The content of BMP15 immature protein in medium-sized follicular fluid was higher than that of the other two groups.The medium follicular fluid was added to the induced differentiation medium,and the yield of E2 was high.The results of reproductive-related marker gene detection showed that the expression of OCT4,FIGLα,GDF9,FSHR and DNMT3B genes increased in accordance with the large,medium and small follicle groups.Continuing to induce hAFSCs with group M follicular fluid,oocyte-like cells(OLCs)and follicular-like structures were found after 10 days of induction,and these structures showed positive expression of EGFP after transfection with EGFP-BMP15,which indicated that there were oocyte in follicle-like structures.The results of Real time PCR confirmed that the expression of germ cell formation related genes OCT4,FIGLα,GDF9,FSHR and epigenetic gene DNMT3B gradually increased,and the reproductive related proteins OCT4,FRAGILIS and SCP3 were positive by cellular immunofluorescence assay.Conclusion hAFSCs-070607 strain human amniotic fluid stem cells have good pluripotency and differentiation potential in vitro.After induced by porcine follicular fluid,they can differentiate into female germ cells,and the E2 secreted by cells in group M is higher than that of S group.Group L.After prolonged induction,germ cell related genes and proteins are up-regulated and can form a follicular-like structure.