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目的 探讨野生型 p53(wtp53)基因及其相关调控基因转录、翻译水平的变化与肠道干细胞分化的关系。方法 利用逆转录 多聚酶链反应 (RT PCR)和免疫组织化学技术 ,以细胞分化标志肝脂肪酸结合蛋白 (L Fabp)mRNA水平为动态参照 ,分别检测大鼠胚胎期E1 4d ,E1 7d,E1 9d和幼鼠期P1d,P2d,P7d,P2 8d及成年大鼠小肠细胞内p53mRNA、p2 1mRNA及其蛋白表达。 结果 从胚胎E1 9d至成年大鼠 ,RT PCR能够检测到小肠细胞内L Fabp的mRNA ,其中P2d,L Fabp的mRNA含量最高 ,证实这个期间为肠道干细胞高分化阶段。从胚胎到成年大鼠 ,小肠细胞p2 1mRNA及其蛋白表达均为阴性 ,p53mRNA水平无明显变化。然而从E1 4d～P2d ,小肠上皮细胞p53蛋白信号呈逐渐增强趋势。P7d以后至成年期 ,p53蛋白信号为阴性。结论 在肠道干细胞迅速分化期 ,p53蛋白高表达可能是因为其半衰期延长 ,最终导致蛋白量累积的结果 ,而与转录水平无关 ,提示蛋白的稳定性对于p53调节肠道干细胞分化期基因的稳定性十分重要 ,另外 ,p53蛋白调节肠道干细胞的分化机制与p2 1途径无关
Objective To investigate the relationship between transcription and translation of wild type p53 (wtp53) gene and its related regulatory genes and the differentiation of intestinal stem cells. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression of L Fabp mRNA in rat embryos at 4 days, 4 days, 7 days and 9 days respectively The P1m, P2d, P7d and P2 ~ (8d) pups of young rats and the expression of p53 mRNA and p21mRNA in intestinal cells of adult rats. Results From embryo E1 9d to adult rat, mRNA of L Fabp in small intestine was detected by RT-PCR. Among them, mRNA of P2d and L Fabp was the highest, confirming that this period was the stage of intestinal stem cell differentiation. From embryos to adult rats, the expression of p21 mRNA and protein in small intestine cells was negative, but there was no significant change in p53 mRNA level. However, from 4 days to 2 days, the p53 protein in intestinal epithelial cells gradually increased. After P7d to adulthood, p53 protein signal is negative. Conclusions During the rapid differentiation of intestinal stem cells, the high expression of p53 protein may be the result of the prolongation of its half-life and finally the accumulation of protein, but not the transcriptional level, suggesting that the stability of p53 protein may regulate the stability of p53 gene in the differentiation of intestinal stem cells In addition, the p53 protein regulates the differentiation of intestinal stem cells independent of the p2 1 pathway