可诱导表达BMP4的正常来源及隐睾特异性iPSCs的建立及分化研究

Establishment and differentiation of iPSCs derived from normal and cryptorchidism with inducible expression of BMP4

目的建立可诱导表达骨形态发生蛋白质4(bone morphogenetic protein 4,BMP4)的正常来源及隐睾特异性诱导多能干细胞(induced pluripotent stem cells,iPSCs),比较两者体外定向分化的差异,为研究部分隐睾患儿成年后不育的原因提供具有人类遗传背景的体外研究模型。方法①建立可诱导表达BMP4的正常来源及隐睾特异性iPSCs,将正常来源iPSCs作为对照组,隐睾特异性iPSCs作为隐睾组;利用多西环素(doxycycline,Dox)进行诱导,筛选出BMP4表达量最高的细胞株;②将外源性添加BMP4的正常来源iPSCs作为外源性添加BMP4组(外源组),将Dox内源诱导BMP4表达的正常来源iPSCs作为Dox内源诱导BMP4表达组(内源组),此两组共同作为实验组;在细胞形态和基因表达水平比较实验组两组间BMP4因子诱导效果的差异;③通过Dox诱导BMP4的表达、双氢睾酮(dihydrotestosterone,DHT)、全反式维甲酸(all trans retinoid acid,ATRA)组成的三步法诱导分化方案,逐步诱导人的正常来源及隐睾特异性iPSCs向生精细胞方向分化,在基因及蛋白水平进行检测,比较两株细胞在分化过程中的差异。结果①经Dox诱导后,在正常来源及隐睾特异性iPSCs两株细胞中均可检测到BMP4表达明显升高、同时多能性基因OCT4的表达均呈明显下降趋势,与d0相比差异具有统计学意义,其中对照组的3#细胞株与隐睾组的5#细胞株在Dox诱导后BMP4表达量最高;②与外源组相比,内源组的细胞株形态变化更为均一、同步,BMP4的基因表达量更高,两组间的差异具有统计学意义;③在向生精细胞诱导分化过程中,对照组及隐睾组细胞的DAZL和PRDM1的表达量在两组之间的差异均无统计学意义;隐睾组的VASA、SYCP3、ACROSIN和TEKT1的表达量于诱导d14和d21时均明显低于对照组,两组之间的差异具有统计学意义。免疫染色结果显示:DAZL的阳性率在对照组和隐睾组细胞间的差异无统计学意义;隐睾组的VASA和SYCP3的阳性率于诱导d14和d21时均明显低于对照组,ACROSIN的阳性率于诱导d21时明显低于对照组,差异均具有统计学意义。结论本研究为部分隐睾患儿成年后不育的原因提供了具有人类遗传背景的体外研究模型,为未来的进一步研究奠定了良好基础。

Objective To establish normal source and cryptorchidism specific induced pluripotent stem cells(iPSCs)capable of inducing the expression of bone morphogenetic protein 4(BMP4)and compare the differences in directional differentiation between the two in vitro,providing an in vitro research model with human genetic background for elucidating the causes of infertility in some children with cryptorchidism in adulthood.Methods Normal source and cryptorchidism specific iPSCs were established with inducible expression of BMP4.Normal source iPSCs were selected as control group while cryptorchidism specific iPSCs as cryptorchidism group;with doxycycline(Dox)for induction,cellline was selected with the highest expression of BMP4;With normal source iPSCs with exogenous addition of BMP4 as exogenous addition BMP4 group(exogenous group)and normal source iPSCs with Dox-induced BMP4 as Dox-induced BMP4 endogenous group(endogenous group),these two groups served jointly as experimental group;the difference of BMP4 factor induction effect between these two groups in experimental group was compared at the levels of cellular morphology and gene expression;the differentiation protocol consisting of BMP4 expression,dihydrotestosterone(DHT),all-trans retinoic acid(ATRA)gradually induced human normal source and cryptorchidism specific iPSCs to differentiate into spermatogenic cells and the differences in differentiation process between two strains were compared at the levels of gene and protein.Results After an induction of Dox,the expression of BMP4 was markedly up-regulated and the expression of pluripotency gene OCT4 declined obviously in two cell lines of normal source and cryptorchidism specific iPSCs and the difference was statistically significant compared with d0.The expression level of BMP4 peaked in 3#cell line of control group and 5#cell line of cryptorchidism group after an induction of Dox;Compared with exogenous group,the morphological changes of endogenous group were more homogeneous and synchronous and the gene expression level of BMP4 was higher and inter-group difference was statistically significant;During the induction and differentiation of spermatogenic cells,the expression levels of DAZL and PRDM1 were not significantly different between two groups;the expression levels of VASA,SYCP3,ACROSIN and TEKT1 were significantly lower in cryptorchidism group than those in control group at Day 14/21 post-induction and the inter-group difference was statistically significant.The results of immunostaining revealed no significant difference in positive rate of DAZL between control and cryptorchidism groups;positive rates of VASA and SYCP3 were significantly lower in cryptorchidism group than those in control group on at Day 14/21 post-induction and the positive rates of ACROSIN were significantly lower than those in control group at Day 21 post-induction and the differences were statistically significant.Conclusions This study provides an in vitro research model with human genetic background for elucidating the causes of infertility in some children with cryptorchidism in adulthood and lays a solid foundation for further researches.