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目的利用甲胎蛋白启动子活力筛选人胚肝脏前体细胞克隆。方法经聚合酶链反应(PCR)扩增及酶切后连接,将甲胎蛋白启动子片段构建于pGL3载体中并测序鉴定,将其与pRL-TK质粒共转染到HepG2、A549和HeLa细胞中,通过相对荧光素酶活力分析甲胎蛋白启动子活力的特异性。采用克隆化培养法获得人胚胎肝脏细胞克隆,检测各克隆的甲胎蛋白启动子活力,并应用间接免疫荧光染色方法检测甲胎蛋白表达情况。结果经PCR、酶切分析及DNA序列测定证实pGL3-AFP质粒克隆成功。将其与pRL-TK质粒共转染到表达甲胎蛋白的HepG2细胞和不表达甲胎蛋白的A549、HeLa细胞中,仅在表达甲胎蛋白的HepG2细胞中检测到了较高的甲胎蛋白启动子活力,胚胎肝脏细胞中也检测到了甲胎蛋白启动子活力,表明其中含有表达甲胎蛋白的细胞。利用克隆培养法获得5个胚胎肝脏细胞克隆,将其分别共转染pGL3-AFP和pRL-TK质粒,发现其中1个克隆的甲胎蛋白启动子活力与HepG2细胞接近,且免疫荧光染色结果显示,该克隆细胞甲胎蛋白阳性率为(99.1±0.6)%,表明此克隆为肝脏前体细胞克隆。结论利用甲胎蛋白启动子活力结合克隆培养法可以获得肝脏前体细胞克隆。
OBJECTIVE: To screen human embryonic liver precursor cell clones by using alpha-fetoprotein promoter. Methods After PCR amplification and restriction enzyme digestion, the α-fetoprotein promoter fragment was constructed in pGL3 vector and sequenced. The recombinant plasmid was co-transfected with pRL-TK plasmids into HepG2, A549 and HeLa cells , The specificity of alpha-fetoprotein promoter activity was analyzed by relative luciferase activity. The clone of human embryonic liver cells was obtained by clone culture method. The promoter activity of alpha-fetoprotein of each clone was detected. The expression of alpha-fetoprotein was detected by indirect immunofluorescence staining. Results The plasmid pGL3-AFP was successfully cloned by PCR, restriction analysis and DNA sequencing. Cotransfected with pRL-TK plasmid into HepG2 cells expressing alpha-fetoprotein and A549, HeLa cells not expressing alpha-fetoprotein, higher alpha-fetoprotein activation was detected only in HepG2 cells expressing alpha-fetoprotein AFP activity was also detected in embryonic liver cells, indicating the presence of alpha-fetoprotein-expressing cells. Five clones of embryonic liver cells were cloned and cloned into pGL3-AFP and pRL-TK plasmids, respectively. The cloning of α-fetoprotein promoter was similar to that of HepG2 cells and the result of immunofluorescence staining The positive rate of α-fetoprotein in this clone was (99.1 ± 0.6)%, indicating that this clone was a clone of liver precursor cells. Conclusion Liver precursor cell clones can be obtained through the combination of AFP promoter activity and clonal culture.