荧光定量PCR检测恶性转化人脐带间充质干细胞中DNA甲基化转移酶的改变  

Quantitative real-time PCR detection of DNA methylation transferase in the malignantly transformed human umbilical cord mesenchymal stem cells

作　　者：陈业增 唐秋灵[1] 陈秋蓉[1] 赖秀蓝[1,2] 邱晓燕[1] 郑泽鑫[1] 李伟中[1] 

机构地区：[1]汕头大学医学院第二附属医院儿科,广东省汕头市515041 [2]汕头大学医学院第二附属医院转化医学中心,广东省汕头市515041

出　　处：《中国组织工程研究》2017年第21期3320-3325,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省医学科学技术研究基金(B2013276)~~

摘　　要：背景:根据体外培养的成体干细胞自发恶性转化现象及肿瘤干细胞理论,人脐带间充质干细胞在体外培养过程中可能产生变异,体内移植后可能存在致瘤性风险。因此,建立健全的体外安全性检测程序,将在干细胞临床应用上起积极推动作用。目的:探讨人脐带间充质干细胞致瘤性机制及DNA甲基化转移酶的表达水平。方法:采用组织块贴壁法分离、培养、扩增出人脐带间充质干细胞,利用3-甲基胆蒽促使人脐带间充质干细胞发生恶性转化,通过形态学观察,裸鼠成瘤实验进行验证,成瘤组织进行病理学鉴定、原代细胞培养,荧光定量PCR检测人脐带间充质干细胞与肿瘤细胞DNA甲基化转移酶表达水平的差异。结果与结论:(1)人脐带间充质干细胞经3-甲基胆蒽处理后形成恶性转化细胞,呈恶性生长,细胞核型呈非整数倍体改变,注入裸鼠体内后能形成恶性肿物;(2)经荧光定量PCR检测,恶性转化后肿瘤细胞(实验组)相比二甲基亚砜培养人脐带间充质干细胞(对照组),其DNA甲基化转移酶的基因表达量明显增高;(3)结果表明,人脐带间充质干细胞在一定条件下能发生恶性转化,包括形态学变化及表观遗传学层面改变,DNA甲基化转移酶可作为预防干细胞移植后成瘤的进一步研究方向。BACKGROUND： Human umbilical cord mesenchymal stem cells（hU C-MSCs） may be mutated during in vitro culture based on the spontaneous malignant transformation of adult stem cells and tumor stem cell theory, and there may be a risk of tumorigenesis after in vivo transplantation. Therefore, to establish and perfect the in vitro safety testing procedures will actively promote the clinical application of stem cells. OBJECTIVE： To investigate the tumorigenic mechanism of h UC-MSCs and the expression level of DNA methyltransferase（DNMTs） in h UC-MSCs. METHODS： Primary h UC-MSCs were isolated and expanded by tissue adherent culture. 3-Methycholanthrene was used to cause the malignant transformation in h UC-MSCs（experimental group）, followed by morphological observation and tumorigenesis experiment in nude mice. Then, the tumor tissues were obtained and identified by pathological examination and primary cell culture, and the levels of DNMTs m RNA in h UC-MSCs treated with 3-methycholanthrene and dimethyl sulfoxide（control group） were detected by real-time RT-PCR and compared. RESULTS AND CONCLUSION： h UC-MSCs treated with 3-methycholanthrene led to malignant transformation, which showed malignant growth and non-integer ploidy changes in the cell nuclei, and formed a malignant tumor in immune-deficient mice after injection. Compared with the control group, the cells in the experimental group showed higher expression of DNMTs m RNA as detected by real-time RT-PCR. To conclude, h UC-MSCs can trigger malignant transformation in the morphology and the epigenetics under certain conditions. DNMTs can be a candidate for prevention against malignant transformation of transplanted stem cells.

关 键 词：脐带 间质干细胞 甲基胆蒽 细胞转化 肿瘤 DNA甲基化 组织工程 干细胞 脐带脐血干细胞 人脐带间充质干细胞 恶性转化 DNA甲基化转移酶 

分 类 号：R394.2[医药卫生—医学遗传学]