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

doi:10.3969/j.issn.2095-4344.2017.21.007

Abstract

Abstract:

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-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 hUC-MSCs and the expression level of DNA methyltransferase (DNMTs) in hUC-MSCs.
METHODS: Primary hUC-MSCs were isolated and expanded by tissue adherent culture. 3-Methycholanthrene was used to cause the malignant transformation in hUC-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 mRNA in hUC-MSCs treated with 3-methycholanthrene and dimethyl sulfoxide (control group) were detected by real-time RT-PCR and compared.
RESULTS AND CONCLUSION: hUC-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 mRNA as detected by real-time RT-PCR. To conclude, hUC-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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cells, Methylcholanthrene, Cell Transformation, Neoplastic, DNA Methylation, Tissue Engineering

CLC Number:

R394.2

Chen Ye-zeng, Tang Qiu-ling, Chen Qiu-rong, Lai Xiu-lan, Qiu Xiao-yan, Zheng Ze-xin, Li Wei-zhong. Quantitative real-time PCR detection of DNA methylation transferase in the malignantly transformed human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3320-3325.

Figures/Tables 1

2.1 人脐带间充质干细胞和恶性转化的人脐带间充质干细胞的生物学特征采用组织块贴壁法进行原代培养5-7 d，部分组织块贴壁，周围可见大量细小成纤维样细胞爬出，传代后人脐带间充质干细胞呈成纤维细胞样贴壁生长，第1代细胞生长较慢，继续传代后细胞生长速度变快。当细胞达90%左右融合时，可将细胞按梯度冷冻法将细胞冻存于-80 ℃冰箱内，3个月内这些细胞复苏后的存活率至少达50%左右。此方法所得细胞即为人脐带间充质干细胞。课题组前期研究已证实人脐带间充质干细胞具有间充质干细胞的表面标志，CD10、CD13、CD29、CD44、CD90、CD166、CD106、SH2(CD105)及人淋巴细胞位点1抗原(HLA-1)均阳性，其中CD106、SH2(CD105)及HLA-1表达低于骨髓间充质干细胞，表明人脐带间充质干细胞具有更原始的间充质干细胞群；CD14、CD33、CD56、CD31、CD34、CD45及人淋巴细胞位点DR抗原(HIA-DR)均阴性[18]。 9批实验组经3-甲基胆蒽诱导后的人脐带间充质干细胞中，8批细胞10代(约30 d)至20代(最长约60 d)陆续凋亡，仅1批在经过32代传代后细胞生长分裂速度较前加快，传代效率明显提高。镜下观察出现细胞聚团现象，细胞出现相互重叠，甚至向三维空间发展，形成堆积的细胞团块。这些表明细胞在恶性诱导后出现了细胞特性改变，增长速度、自我更新性能都产生变化(图1A)。而对照组加入二甲基亚砜培养的人脐带间充质干细胞经过长期传代培养，特别是在第8，9代之后，细胞逐渐出现老化，表现为细胞状态、生长分裂速度、传代效率等缓慢降低，再经过数次传代后逐渐出现生长停滞、凋亡现象(图1B)。 2.2 恶性转化的人脐带间充质干细胞成瘤后肿瘤细胞生物学特征将以上恶性转化人脐带间充质干细胞注射裸鼠皮下，约2个月后注射部位出现包块(图2)，病理解剖呈鱼肉状。病理切片(苏木精-伊红染色)可见大量核分裂相，异型性高(图3)。课题组前期研究显示其免疫组织化学中各抗体结果：Ki-67(+)，结合苏木精-伊红染色结果，推断为恶性肿瘤细胞，恶性程度高；Vim(+)，CK(±)和EMA(-)，提示细胞间叶来源，肉瘤可能性大；NF(+)和NES(+)，提示细胞神经来源可能性大，不排除低分化癌细胞向神经细胞发展。肿瘤细胞进行常染色体G显带分析，可见其染色体形态呈人染色体形态，染色体数量波动在61-69，呈多倍体，具有某些肿瘤细胞染色体特征[17]。 2.3 肿瘤细胞原代培养结果取部分肿瘤组织行原代培养，原代细胞很快爬出，细胞生长分裂迅速(图4A)，细胞传代到生长至90%融合度所需时间缩短为两三天，而且由于生长速度快，肿瘤细胞在培养瓶中培养时极易出现细胞聚团现象，细胞形态、生长状态均具有肿瘤细胞特性(图4B)。 2.4 荧光定量RT-PCR检测人脐带间充质干细胞、肿瘤细胞DNMTs的表达 2.4.1 总RNA 提取及纯度鉴定取80%-90%融合度的二甲基亚砜对照培养的第3代人脐带间充质干细胞，以及肿瘤组织培养传代第3代细胞，用Takara RNAiso Plus提取总RNA。提取后采用核酸蛋白分析仪测定RNA浓度和纯度，测吸光度值，所有样本RNA提取物A260/A280在1.8-2.0之间，表明提取的总RNA纯度较高，蛋白质及酚去除较干净。 2.4.2 熔解曲线分析每次实时定量PCR反应结束后，可得到扩增样本的熔解曲线。每个样本的熔解曲线只产生一个熔解峰，全部样本的熔解峰非常接近，熔解峰所在位置的温度为该 PCR 产物片段的熔解温度，表明只产生1种特异性扩增产物(图5)。每个样品的目的基因浓度除以其管家基因的浓度，即为样品基因校正后的相对含量。 2.4.3 荧光定量PCR结果统计分析经荧光定量PCR检测，肿瘤组织传代培养第3代细胞(实验组)相比二甲基亚砜培养的第3代人脐带间充质干细胞(对照组)，其DNMT1、DNMT3a、DNMT3b的基因表达量均明显增高，其中以DNMT1表达量增高最为明显。这些结果均表明人脐带间充质干细胞的恶性转化伴随着3种甲基转移酶DNMT1、DNMT3a、DNMT3b的表达增加(图6)。DNMT1平均呈4.90倍升高，DNMT3a平均呈2.46倍升高，DNMT3b平均呈1.96倍升高，经t 检验，P均< 0.01(< 0.000 1，0.002，0.003)。单因素方差分析得到的结果是P值< 0.000 1，表明3者差异有显著性意义，再次采用Bartlett’s test对恶性转化细胞中3种甲基化转移酶的mRNA表达差异进行两两比较，结果显示DNMT1与DNMT3a、DNMT3b比较差异有显著性意义(P < 0.000 1)，而DNMT3a与DNMT3b的表达差异无显著性意义(P=0.2)。因此可以证明，在人脐带间充质干细胞恶性转化后，其3个甲基化转移酶DNMT1、DNMT3a、DNMT3b的mRNA表达均较二甲基亚砜培养的人脐带间充质干细胞明显升高，其中DNMT1的mRNA表达比DNMT3a、DNMT3b的表达升高量更为显著。"

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