体外扩增大鼠肌源性干细胞的安全性研究  

Safety Experiment of Cultured Muscle-dericed Stem Cell in Vitro

作　　者：张文[1] 李秉枢[1] 李爱斌[1] 曹景云[1] 奚文裕[1] 

机构地区：[1]武汉大学人民医院,湖北武汉430060

出　　处：《武汉大学学报（医学版）》2014年第1期26-31,共6页Medical Journal of Wuhan University

摘　　要：目的:研究经体外扩增的大鼠肌源性干细胞的安全性,为将来肌源性干细胞移植治疗压力性尿失禁的临床应用提供参考。方法:分离培养出大鼠肌源性干细胞,在体外传代培养,对传至第10代(PP6-10)的细胞进行刀豆球蛋白A(ConA)凝聚实验、双层软琼脂培养实验初步分析细胞表面结构和生长特性是否发生恶变;采用免疫细胞化学的方法检测PP6-10的端粒酶活性以及c-myc,p53,c-fos肿瘤相关基因的表达情况;采用裸鼠皮下致瘤实验观察其是否具备致瘤性。结果:体外培养至第10代以内的大鼠肌源性干细胞在培养瓶底分布均匀,贴壁后多为纺锤形或梭形,与原代培养的肌源性干细胞形态相比无明显改变;在不同浓度的ConA溶液中,PP6-10均未产生凝聚反应;将PP6-10置于双层软琼脂中培养12d,未见细胞克隆形成,培养至20d,细胞数量明显减少;通过免疫细胞化学方法检测PP6-10中的c-myc,p53和c-fos等肿瘤相关基因的表达结果均为阴性,PP6-10中的端粒酶逆转录酶(hTERT)的表达结果呈弱阳性;将不同浓度的PP6-10接种于裸鼠皮下,观察4个月后接种部位没有肿块形成,病检其接种部位以及肝脏、肺脏均无异常表现。结论:大鼠肌源性干细胞体外培养至第10代后没有发生突变,不具备体内致瘤性,初步证实了体外传至第10代的大鼠肌源性干细胞的安全性。Objective： To study the security of cultured rats muscle-dericed stem ceils in vitro, in order to provide the security evidence for the muscle-derived stem ceils injection for the treatment of stress uriiaary incontinence. Methods： The muscle-derived stem ceils were acquired from SD rat, and cultured for 10 passages in vitro before experiment. The double layers soft agar test and con- canavalin A（ConA） agglutination test were adopted to observe the surface structures and growth characteristics of the stem cells. The method of immunocytochemistry was used to detect the ex- pression of hTERT and tumor-related genes. The stem ceils were injected in nude mice＇s subcuta- neously to observe the tumorigenicity. Results： The muscle-derived stem ceils which were cul- tured in vitro after 10 passages showed a fusiform shape. There was no agglutination reactions at ConA solution, and no cell clone were found in soft agar. No expression of tumor-related genes P53, c-myc, and e-los were found in the stem cells, but the hTERT were lower expressed in stem support about security evidence for muscle-derived stem cells.

关 键 词：肌源性干细胞 体外培养 致瘤性实验 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]