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目的探讨酪氨酸激酶受体KIT在小鼠结肠黏膜上皮的表达及作用。方法应用Western blotting和RT-PCR技术检测c-kit基因及KIT蛋白在野生型C57BL/6小鼠结肠黏膜上皮的表达;用免疫荧光染色显示野生型C57BL/6小鼠以及乙基亚硝酸钠(ENU)诱变的c-kit基因点突变纯合子小鼠(Wadsm/m)结肠黏膜上皮KIT阳性细胞的部位;腹腔注射5-溴脱氧尿嘧啶核苷(BrdU)(30 mg/kg)观察对比野生型以及Wadsm/m小鼠结肠黏膜上皮BrdU掺入情况及动态变化,分析KIT在结肠黏膜上皮更新过程的作用。结果 RT-PCR检测结果表明,在野生型小鼠结肠黏膜组织表达c-kit基因,Western blotting证明在肠黏膜组织存在KIT蛋白;免疫荧光染色显示,KIT阳性细胞位于结肠黏膜上皮,主要位于肠腺隐窝基底部,但是Wadsm/m小鼠KIT阳性细胞数以及KIT蛋白表达量较野生型小鼠明显减少;BrdU掺入实验发现,Wadsm/m小鼠远端结肠黏膜上皮BrdU摄入和更新速度较野生型小鼠明显减慢。结论结肠黏膜上皮表达KIT蛋白与结肠黏膜上皮的更新密切相关。
Objective To investigate the expression and role of tyrosine kinase KIT in mouse colonic epithelium. Methods The expression of c-kit gene and KIT protein in colonic mucosa of wild-type C57BL / 6 mice was detected by Western blotting and RT-PCR. Immunofluorescence staining showed that wild-type C57BL / 6 mice and ethyl sodium nitrite ENU) mutated homozygous mouse (Wadsm / m) site of colorectal mucosa KIT positive cells; intraperitoneal injection of 5-bromodeoxyuridine (BrdU) (30 mg / kg) Wild-type and Wadsm / m mouse colon epithelial BrdU incorporation and dynamic changes in the role of KIT in colonic mucosal epithelial renewal process. Results The results of RT-PCR showed that c-kit gene was expressed in the colonic mucosa of the wild-type mice and KIT protein was detected by Western blotting in the intestinal mucosa. Immunofluorescence staining showed that the KIT positive cells were located in the colonic epithelium, However, the number of KIT positive cells and the expression of KIT protein in Wadsm / m mice were significantly decreased compared with those in wild type mice. BrdU incorporation assay showed that BrdU uptake and regeneration rate in Wadsm / m mice distal colonic mucosal epithelium Compared with wild-type mice significantly slowed down. Conclusion The expression of KIT in colonic epithelium is closely related to the renewal of colonic epithelium.