Cdx2反转录病毒载体的构建及转染膀胱上皮细胞的研究

摘要

BACKGROUND:Caudal type homeobox transcription factor 2 plays an important role in the development of epithelium in digestive tract, especial y the smal intestine and colon. OBJECTIVE:To construct the retroviral expression vector of pLNCX2-caudal type homeobox transcription factor 2, and to observe the effect of in vitro transfection of pLNCX2-caudal type homeobox transcription factor 2 in intestinal metaplasia. METHODS:Gene recombinant technology was employed to clone human caudal type homeobox transcription factor 2 gene to the retroviral expression vector of pLNCX2, then identified with enzyme digestion and sequencing and packed to the PA317 cells. The plasmids were transfect in urothelium cells, real-time PCR and western blot were used to detect the expressions of caudal type homeobox transcription factor 2, vil in and liverintestin-cadherin at the protein and mRNA levels. RESULTS AND CONCLUSION:The retroviral vector pLNCX2-caudal type homeobox transcription factor 2 was successful y constructed. The levels of caudal type homeobox transcription factor 2 protein and mRNA expressions in bladder urothelium celltransfected with pLNCX2-caudal type homeobox transcription factor 2 were higher than that in control. And the over-expression of caudal type homeobox transcription factor 2 could up-regulate the levels of vil in and liverintestin-cadherin expressions. Interestingly, specific changes of intestine-like cells were seen in the bladder urothelium cells transfected by pLNCX2-caudal type homeobox transcription factor 2. Over-expression of caudal type homeobox transcription factor 2 can activate the caudal type homeobox transcription factor 2 of urothelium cells and induce intestinal epithelial differentiation, thus inducing the development of cystitis glanduaris.%背景：尾侧型同源转录因子2在消化道尤其是小肠与结肠上皮的发育中起到关键作用。　　目的：构建尾侧型同源转录因子2反转录病毒表达载体pLNCX2-Cdx2，观察pLNCX2-Cdx2体外转染对人膀胱上皮细胞发生肠上皮化生的作用。　　方法：应用基因重组技术将人尾侧型同源转录因子2cDNA 克隆到反转录病毒载体 pLXSN2,酶切鉴定，包装到PA317细胞中。转染人膀胱尿路上皮细胞，应用荧光PCR及Western blot检测尾侧型同源转录因子2以及绒毛蛋白、LI-cadherin的表达情况。　　结果与结论：pLNCX2-Cdx2成功构建；细胞转染后尾侧型同源转录因子2的mRNA及蛋白水平表达明显增加；尾侧型同源转录因子2的过表达明显激活了绒毛蛋白与LI-cadherin的表达；转染后细胞可出现肠细胞样特征改变。结果提示体外尾侧型同源转录因子2过表达可使膀胱上皮细胞发生尾侧型同源转录因子2激活并产生肠上皮分化，从而诱导腺性膀胱炎发生。