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

发布时间：2019-06-02 05:29

【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。
[Abstract]:Background: according to the spontaneous malignant transformation of adult stem cells cultured in vitro and the theory of tumor stem cells, human umbilical cord mesenchymal stem cells may mutate in vitro, and there may be tumorigenic risk after in vivo transplantation. Therefore, the establishment of a sound safety detection procedure in vitro will play an active role in the clinical application of stem cells. Objective: to investigate the tumorigenic mechanism of human umbilical cord mesenchymal stem cells and the expression of DNA methyltransferase. Methods: human umbilical cord mesenchymal stem cells were isolated, cultured and expanded by tissue block adherent method. 3-methylcholanthracene was used to promote the malignant transformation of human umbilical cord mesenchymal stem cells, which were verified by morphological observation and nude mice tumorigenesis experiment. Pathological identification of tumorigenic tissues, primary cell culture and fluorescence quantitative PCR were used to detect the expression of DNA methyltransferase between human umbilical cord mesenchymal stem cells and tumor cells. Results and conclusion: (1) Human umbilical cord mesenchymal stem cells were treated with 3-methylcholanthracene to form malignant transformed cells with malignant growth and aninteger ploidy change in karyotype. Malignant masses could be formed after injection into nude mice. (2) the expression of DNA methyltransferase gene in malignant transformed tumor cells (experimental group) was significantly higher than that in human umbilical cord mesenchymal stem cells cultured with dimethyl sulfoxide (control group) by fluorescence quantitative PCR. (3) the results showed that human umbilical cord mesenchymal stem cells could undergo malignant transformation under certain conditions, including morphological changes and epigenetic changes. DNA methyltransferase can be used as a further research direction to prevent tumor formation after stem cell transplantation.
【作者单位】：汕头大学医学院第二附属医院儿科;汕头大学医学院第二附属医院转化医学中心;
【基金】：广东省医学科学技术研究基金(B2013276)~~
【分类号】：R457.7

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