后肾细胞微环境诱导胚胎干细胞向肾系细胞分化的实验研究  

Metanephric cell microenvironment induces embryonic stem cells to differentiate toward renal cells

作　　者：汪泱[1] 张立行[1] 王共先[1] 谢安[1] 娄远蕾[1] 阮琼芳[1] 崔苏萍[1] 杨阳[1] 

机构地区：[1]南昌大学泌尿外科研究所,江西南昌330006

出　　处：《中国病理生理杂志》2011年第1期129-133,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30960385);江西省自然科学基金资助项目(No.2007GZY1470)

摘　　要：目的:探讨胚胎后肾细胞微环境诱导胚胎干细胞(ESCs)分化为肾系细胞的作用。方法:小鼠D3系胚胎干细胞通过悬滴法制备拟胚体(EBs),将EBs细胞与取自孕12.5 d小鼠胚胎的后肾细胞通过间接共培养以诱导其分化,即为共培养诱导组,设EBs细胞自然分化为对照组;采用免疫荧光染色法检测共培养第3、5、7 d后的EBs细胞Pax2、WT-1蛋白表达情况,通过逆转录PCR法检测诱导3 d后的EBs细胞Pax2、WT-1、Lim1、Sall1、Emx2、GDNF、Wnt4、BMP7、Nephl、Nephrin、KSP和CD24 mRNA的表达情况。结果:EBs细胞在诱导的第3 d即出现了全部肾发育相关基因的表达,其中共培养组Pax2、WT-1、Emx2、GDNF、Nephl、Nephrin、KSP和CD24的表达强于对照组;免疫荧光结果显示在诱导3 d后的EBs细胞中可观察到Pax2阳性细胞,阳性细胞数在共培养第5、7 d增多;诱导5 d后可观察到WT-1阳性细胞,对照组未见Pax2或WT-1蛋白阳性细胞出现。结论:后肾细胞微环境可促进ESCs分化为肾系细胞。AIM：To explore the effects of metanephric cell microenvironment on inducing embryonic stem cells（ESCs） to differentiate toward renal cells.METHODS：Embryoid bodies（EBs） of D3 mouse embryonic stem cells were prepared by hanging drop culture,and the EBs were co-cultured indirectly with metanephric cells derived from E12.5 d mouse embryo.The EBs cell with spontaneous differentiation was used as the control.The proteins of Pax2 and WT-1 were analyzed by immunofluorescence assay.The mRNA expression of Pax2,WT-1,Lim1,Sall1,Emx2,GDNF,Wnt4,BMP7,Nephl,Nephrin,KSP and CD24 genes was detected by RT-PCR.RESULTS：The genes related to kidney development were expressed in the EBs cells after co-culture on day 3,and the mRNA expression of Pax2,WT-1,Emx2,GDNF,Nephl,Nephrin,KSP and CD24 was stronger than those in control group.Pax2 positive cells were found on day 3 in the co-cultured EBs cells,and the positive cells increased on day 5 and day 7.WT-1 protein positive cells were found in the co-cultured EBs cells on day 5.No Pax2 or WT-1 positive cell was observed in control group.CONCLUSION：Metanephric cell microenvironment promotes ESCs differentiation toward renal cells.

关 键 词：胚胎干细胞 后肾细胞 肾系细胞 

分 类 号：R363[医药卫生—病理学]