摘要
目的建立猿肾病毒40大T抗原（SV40Tag）永生化尿源性干细胞，为组织工程学及再生医学提供稳定的细胞来源。方法利用脂质体（Lipofectamine2000）介导将含SV40Tag基因质粒pMPH-FRT-SV40T及转座酶质粒（transposase）共转染至临床分离的尿源性干细胞中。潮霉素（Hy—gromycin）筛选后阳性细胞克隆并连续传代，观察细胞形态学以及增殖情况，RT-PCR，免疫荧光等检测转染细胞的生物学特性，细胞裸鼠皮下注射检测其安全性。结果筛选获得的阳性克隆扩大培养，即永生化尿源性干细胞（immorterlized urine derived stem cell,iUSC），增殖能力强，能连续传代培养。RT-PCR证实iUSC表达SV40Tag基因，而用翻转重组酶（Flippers recombination enzyme Flp）处理iUSC可敲除RNF-SV40Tag基因而逆转永生化。应用免疫荧光细胞化学技术检测该细胞株表达干细胞表面标志物CD73，CD44，CD90（Thy-1），CD29（Integrin），CD105（Endoglin），CD166（ALCAM），BMPRII，SSEA4，CD117，CD133；BMP9可诱导其分化为成骨细胞，脂肪细胞，具备干细胞多向分化能力；转染细胞2×10^6个／点接种裸鼠，连续观察4周无肿瘤形成。结论脂质体转染技术成功构建SV40Tag永生化尿源性干细胞，该细胞株仍具有干细胞增殖及多向分化潜能，为组织工程学及再生医学的体外研究和发展应用提供了安全稳定的细胞来源。
Objective To establish immortalized urine derived stem cell line with simian vacuolating virus 40 large T antigen （SV40Tag） for providing a source for tissue regeneration. Methods Transfer pMPH-FRT-SV40T with SV40Tag gene and transposase to urine derived stem cell through lipofeetion gene transfer. SV40Tag gene was knocked out by flippers recombination enzyme （Flp）. Hygromycin was used for screening positive clones for cell passaging. Reverse transcriptionpolymerase chain reaction （RT-PCR） was used for detecting the expressions of SV40Tag. CD73, CD44, CD90 （Thy1）, CD29 （ integrin）, CD105 （endoglin）, CD166 （ALCAM）, BMPRII, SSEA4, CD117 and CD133, all major stern cell markers of immunofluorescent cytochemistry. Cellular morphology and growth were observed and analyzed. Results The induction of bone morphogenetic protein 9 （BMP9） had the potential of differentiating into osteoblasts and adipocytes. After the injection of these cells into nude mice, no tumor was formed after 4 weeks. Conclusions An immortalized urine derived stem cell line has been established with SV40Tag. After differentiate to osteoblast and aidpocyte, cells still possess the stem cell potential. This cell line may be used for tissue regeneration.
出处
《中华小儿外科杂志》
CSCD
2016年第3期220-225,共6页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金（81370701）
国家临床重点专科建设项目（国卫办医函[2013]544号）
重庆市博士后资助项目（Xm2014040）
重庆市科委重点实验室专项经费
关键词
干细胞研究
组织工程
再生医学
Stern cell research
Tissue engineering
Regeneration medicine
