【摘要】 目的建立胰高血糖素样多肽2受体(glucagon like peptide-2 receptor,GLP-2R)上调表达的Caco2细胞株。为研究GLP-2肠道保护机制构建体外模型。方法常规扩增抽提GLP-2R／pcDNA 3．1质粒,经酶切、测序鉴定正确后,用脂质体转染法将其转染至Caco2细胞。G418抗性筛选,挑选耐药细胞克隆培养获得稳定细胞株。以HER293细胞、VE细胞、正常Caco2细胞及正常人小肠组织为对照,采用逆转录聚合酶链反应与蛋白质印迹法检测稳定转染细胞中mRNA及其蛋白的表达。结果扩增抽提GLP-2R／pcDNA 3．1质粒后经酶切、测序结果正确。GLP-2R mRNA及其蛋白在HER293细胞及VE细胞中无表达,在正常Caco2细胞中表达微弱,在人小肠组织中有较强表达;转染CLP-2R后,Caco2／GLP-2R(+)细胞中GLP-2R mRNA及其蛋白表达明显增强。结论GLP-2R分布具有相对特异性,正常Caco2细胞中GLP-2R表达较弱;构建的Caco2／GLP-2R(+)细胞模型成立,为深入研究GLP-2的作用机制奠定了良好基础。更多还原

【Abstract】 Objective To establish Caco2 cell line with stable expression of glucagon like peptide-2 receptor(GLP-2R) , in order to establish an in vitro model for the study of protective mechanism of GLP-2 of the intestinal tract. Methods The GLP-2R/pcDNA3. 1 (+) plasmid was verified by restriction endonuclease and sequencing , and then it was transfected into Caco2 cells with lipofectamine. After G418 selection, the clones with stable expression of GLP-2R were obtained by limited dilution cloning and expanding. The mRNA and protein expression of GLP-2R in normal human intestine, Caco2 cells, HER293, VE cells, as well as in transfected Caco2 cells were determined with RT-PCR and Western blot. Results The sequence of GLP-2R/pcDNA 3. 1 plasmid was correct. No expression of GLP-2R mRNA and protein was found in HER293 and VE cells, but weak expression were found in Caco2 cells, and strong expression was found in normal human intestines. The expression of GLP-2R mRNA and protein expression in Caco2/GLP-2R (+) cells were obviously increased after transfection. Conclusion GLP-2R has special distribution. The expression of GLP-2R is weak in normal Caco2 cells. The establishment of Caco2/GLP-2R (+) cellular model is beneficial for the further research of the mechanism of action of GLP-2.更多还原