RNA复制子诱导人牙源性多潜能干细胞的研究  

Generation of human dental induced pluripotent stem cells with RNA replicon

作　　者：谭小兵[1] 徐静舒[1] 郭宇[1] 肖艳[1] 戴青原[2] TAN Xiaobing XU Jingshu GUO Yu XIAO Yan DAI Oingyuan(Department of Cariology and Endodontics, the First People＇ s Hos- pital of Yunnan Province and Engineering, Kunming 650032, Chin)

机构地区：[1]云南省第一人民医院口腔内科,昆明650032 [2]昆明医科大学第一附属医院心内科,昆明650032

出　　处：《口腔医学》2017年第8期698-702,共5页Stomatology

基　　金：国家自然科学基金资助项目(81360161);云南省教育厅基金(2015Y153)

摘　　要：目的研究一种高效安全的人诱导性多潜能干细胞(Induced pluripotent stem cells,i PSCs)重编程新体系,为牙源性组织再生提供安全的干细胞来源。方法体外培养人根尖乳头干细胞(Stem cells from apical papilla,SCAP),Simplicon RNA复制子为载体,人重组玻连蛋白作为底物,将人SCAP重编程为i PSCs。免疫荧光染色检测i PSCs特异性标记物的表达,畸胎瘤形成实验检测i PSCs分化能力,RT-PCR确认其外源性基因序列的沉默。计算人SCAP-i PSCs的重编程效率。结果人SCAPi PSCs具备典型的ES样克隆形态,Oct4、Nanog、Sox2、SSEA-4均为染色阳性。i PSCs植入SCID小鼠体内6周形成畸胎瘤,HE染色显示包含所有三胚层来源组织,RT-PCR显示人SCAP-i PSCs特异性表达干细胞标记物,不再表达外源性基因序列。重编程效率为0.17%~0.20%。结论结果得到的人SCAP-i PSCs安全性能和诱导效率较高,是理想的牙源性组织再生的干细胞来源。Objective To investigate a highly effective, non-integrative reprogramming technique to derive induced pluripotent stem cells （iPSCs） to provide safe stem cells sources for dental tissue regeneration. Methods Human stem cells from apical papilla （SCAP） were primarily cultured and induced into iPSCs on human recombinant vitronectin with Simplicon RNA reprogramming kit. Specific pluripotency markers were detected by immunofluorescence staining. Teratoma was formed to examine the pluripotency of differ- entiation in vivo. Total RNA of iPSCs was isolated and RT-PCR was carried out to verify the loss of exogenous reprogramming factors. Reprogramming efficiency of SCAP-iPSCs was calculated. Results Human SCAP-iPSCs exhibited classical ES-like morphology and positively immunostaining for Oct4, Nanog, Sox2 and SSEA-4. Teratoma formed 6 weeks after injecting iPSCs into SCID mice and HE staining revealed the cell types of all three germ layers ： RT-PCR results suggested hSCAP-iPSCs expressed specific multipotent markers and no more exogenous reprogramming factors. The reprogramming efficiency was 0.17%-0.20%. Conclusions HSCAP-iPSCs derived with highly reprogramming efficiency and safety is the optimal stem cell source for dental tissue regeneration.

关 键 词：人根尖乳头干细胞 RNA 重编程 诱导性多潜能干细胞 

分 类 号：R781.3[医药卫生—口腔医学]