利用甲胎蛋白启动子活力筛选人胚胎肝脏前体细胞  

Selecting human fetal hepatic progenitor cells based on α-fetoprotein promoter activities

作　　者：王萍[1,2] 刘保清[1] 李卫红[1] 董凌月[1] 尤红[2] 贾继东[2] 安威[1] 张海燕[1] 

机构地区：[1]首都医科大学细胞生物学系,北京市肝脏保护与再生调节重点实验室,北京100069 [2]首都医科大学附属北京友谊医院肝病研究中心,北京100050

出　　处：《解剖学报》2011年第1期45-49,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(30971472);北京市自然科学基金资助项目(5102013)

摘　　要：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 select hepatic progenitor cell clones from human fetal liver cells （FLCs） based on α- fetoprotein （AFP） promoter activities. Methods AFP promoter was amplified from human fetal liver genome by polymerase chain reaction （PCR） and cloned into pGL3-basic vector, forming the pGL3-AFP in a direct orientation for the promoters to drive firefly luciferase expression. To analyze the specificity of the AFP promoter, the pGL3-AFP and pRL-TK plasmids were co-transfected into HepG2, A549, and HeLa cells; the latter two cells do not express AFP. In order to obtain the homogenous cell populations, the proliferating colonies were isolated from clonal cultures of FLCs, and the AFP promoter activity of each clone was analyzed in each well of the cell lysates after co-transfection of pGL3-AFP and pRL-TK plasmids for 48hours. Immunofluorescence was used to confirm the AFP expression in each clone. Results The results of PCR, restriction enzyme cutting and DNA sequencing confirmed that the pGL3-AFP had been constructed successfully. After co-transfection of the pGL3-AFP and pRL-TK plasmids, the AFP promoter activity was detected in HepG2 cells, but was weakly detected in A549 and HeLa cells, indicating that the AFP promoter could drive downstream firefly luciferase gene expression in AFP-expressing cells. Although the promoter activity in FLCs was lower than that in HepG2 cells, it was higher than that in AFP-non-expressing A549 and HeLa cells, further indicating that there were AFP-expressing ceils among the FLCs. Five proliferating cell colonies were generated from clonal cultures of the FLCs, and the AFP promoter activity could be detected in one of the colonies. Immunofluorescence results revealed that the percentage of AFP positive ceils was （99.1± 0. 6 ）% in this clone, indicating a hepatic progenitor cells clone. Conclusion The homogenous hepatic progenitor cell clones could be selected by AFP promoter activity.

关 键 词：肝脏前体细胞 细胞克隆 甲胎蛋白 启动子 免疫荧光 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]