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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 safety of rat myogenic stem cells expanded in vitro and to provide references for the future clinical application of myogenic stem cell transplantation in the treatment of stress urinary incontinence. Methods: Rat myogenic stem cells were isolated and cultured in vitro. ConA agglutination test was performed on the cells transfected to passage 10 (PP6-10), and the primary soft agar culture experiment Cell surface structure and growth characteristics of malignant transformation; using immunocytochemistry to detect the telomerase activity of PP6-10 and c-myc, p53, c-fos tumor-related gene expression; nude mice subcutaneous tumor experiment Whether it has tumorigenicity. RESULTS: Rat myogenic stem cells cultured in vitro to the tenth passage were evenly distributed on the bottom of the culture flask. Most of the myogenic stem cells cultured in vitro were spindle or fusiform in shape and had no obvious changes compared with the primary cultured myogenic stem cells. In different concentrations of ConA solution, PP6-10 did not produce agglutination reaction; PP6-10 placed in double soft agar culture 12d, no cell clone formation, cultured to 20d, the number of cells significantly reduced by immunocytochemistry The results showed that the expression of c-myc, p53 and c-fos in PP6-10 was negative and the expression of telomerase reverse transcriptase (hTERT) in PP6-10 was weakly positive. The concentration of PP6-10 was inoculated subcutaneously in nude mice. After 4 months of inoculation, there was no lump formation in the inoculated area. The inoculation site of the pathological examination and the liver and lung were not abnormally observed. CONCLUSION: Rat myogenic stem cells cultured in vitro to the tenth generation did not mutate, do not have in vivo tumorigenicity, initially confirmed in vitro to the tenth generation of rat myogenic stem cell safety.