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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。
Objective To establish a Caco2 cell line with up-regulated expression of glucagon-like peptide-2 receptor (GLP-2R). In vitro model of GLP-2 intestinal protective mechanism was established. Methods The plasmid of GLP-2R / pcDNA 3.1 was amplified by conventional extraction and identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into Caco2 cells by lipofection. G418 resistance screening, selection of resistant cell clonal culture stable cell line. HER293 cells, VE cells, normal Caco2 cells and normal human intestinal tissue as control, reverse transcriptase polymerase chain reaction and Western blotting were used to detect mRNA and protein expression in stable transfected cells. Results Amplification and extraction of GLP-2R / pcDNA 3.1 plasmid by digestion, sequencing results correct. GLP-2R mRNA and its protein were not expressed in HER293 cells and VE cells, but weakly expressed in normal Caco2 cells and strongly expressed in human intestinal tissue. After transfection with CLP-2R, the expression of Caco2 / GLP-2R (+) GLP-2R mRNA and protein expression in cells significantly increased. Conclusion The GLP-2R distribution is relatively specific and the expression of GLP-2R in normal Caco2 cells is weak. The constructed Caco2 / GLP-2R (+) cell model is established, which lays a good foundation for further study on the mechanism of GLP-2.