AIM: To explore the effects of metanephric cell microenvironment on inducing embryonic stem cells ( ESCs ) to differentiate toward renal cells.METHODS : Emhryoid bodies ( EBs ) of D3 mouse emhryonic 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 ohserved in control group.CONCLUSION : Metanephric cell microenvironment promotes ESCs differentiation toward renal cells.%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.
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