小鼠拟胚体中定型内胚层的比例对小肠吸收细胞分化的促进作用

Differentiation of intestinal absorptive cells derived from mouse embryonic bodies can be promoted by inducing the differentiation of def initive endoderm in vivo

目的:探讨悬浮状态下小鼠拟胚体中定型内胚层的分化比例对其体内小肠吸收细胞分化的影响.方法:利用标志分子检测不同阶段胚体中定型内胚层的分化及ActivinA对其分化的促进作用.随后,接种含高比例定型内胚层的胚体至裸鼠皮下培育移植瘤,研究移植瘤中吸收细胞的分化.结果:培养5d的胚体中定型内胚层的比例较1、3、7d高(Gsc:0.9809±0.1001vs0.5435±0.0821,0.5525±0.0786,0.2234±0.0425;Tm4sf2:0.9231±0.1121vs0.0017±0.0007,0.0176±0.0058,0.6542±0.0742;Gpc1:0.8639±0.1098vs0.5882±0.1027,0.7112±0.0956,0.4239±0.0874,均P<0.05),50μg/L的ActivinA可使其分化比例进一步升高(均P<0.05).诱导组胚体细胞所形成的皮下移植瘤中,小肠吸收细胞标志分子的表达量显著高于对照组(均P<0.05),且含有较多表达Fabp2的细胞团及腺样结构.结论:ActivinA可通过提高悬浮状态下小鼠拟胚体中定型内胚层的分化比例促进其小肠吸收细胞的体内分化.

AIM： To investigate the effect of inducing the differentiation of definitive endoderm derived from mouse embryonic bodies （EBs） cultured bythe hanging drop method in promoting the differentiation of intestinal absorptive cells in vivo. METHODS： The differentiation of definitive endoderm during EBs formation derived from mouse ES-E14TG2a embryonic stem cells （ESC） and the role of Activin A in promoting its differentiation were monitored by detecting its markers by RT-PCR and fluorescence-activated cell sorting. Subsequently, the EBs with high proportion of definitive endoderm were hypodermi- cally engrafted into the back of NOD/SCID mice to form grafts. The markers for small intestinal absorptive cells, including SI, LPH, and Fabp2, were detected in these grafts by quantitative RT- PCR and immunohistochemistry. RESULTS： The marker genes for definitive endoderm were more highly expressed in the 5-day EBs than in other stages of EBs （Gsc： 0.9809 ± 0.1001 vs 0.5435 ± 0.0821, 0.5525 ± 0.0786, 0.2234 ± 0.0425; Tm4sf2： 0.9231 ± 0.1121 vs 0.0017 ± 0.0007, 0.0176 ± 0.0058, 0.6542 ± 0.0742; Gpc1： 0.8639 ± 0.1098 vs 0.5882 ± 0.1027, 0.7112 ± 0.0956, 0.4239 ± 0.0874, all P 0.05）. The percentage of definitive endoderm cells in the 5-day EBs in- duced with 50 μg/L Activin A （SF-A group） was signif icantly higher than that in controls （all P 0.05）. SI and LPH mRNA expression in the grafts from the SF-A group was significantly higher than that in control groups （all P 0.05）. Immu- nohistochemical analysis revealed that Fabp2 was expressed in some immature cells without specific structure or adenoid structures in the grafts from the SF-A group. CONCLUSION： The differentiation of def initive endoderm derived from mouse ESC could be induced with 50 μg/L Activin A in EBs cultured by the hanging drop method. Increasing the proportion of def initive endoderm in EBs promotes the differentiation of intestinal absorptive cells in vivo.