5-氮杂-2'-脱氧胞苷对人子宫内膜癌JEC细胞生长及形态影响的研究
周正平，周 俊，钟 栎，张春英，赵 朔，王凤月5-氮杂-2'-脱氧胞苷对人子宫内膜癌JEC细胞生长及形态影响的研究
周正平，周 俊，钟 栎，张春英，赵 朔，王凤月
（遵义医科大学1.电镜室；2.病理学教研室，贵州 遵义 563000）
摘 要 目的：通过DNA甲基化转移酶抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对人子宫内膜样癌JEC细胞（中分化）中p57kip2基因启动子区去甲基化干预，研究5-Aza-CdR对JEC细胞生长抑制及形态改变的影响。方法：Ⅰ.用亚硫酸氢盐测序PCR(BSP)法检测不同浓度5-Aza-CdR干预后JEC细胞中p57kip2基因启动子区甲基化状态。Ⅱ.将实验组分为12.5 μmol·L-1组、12.5+ μmol·L-1组（48h换液时追加一次5-Aza-CdR），对照组不加任何浓度的5-Aza-CdR：（1）干预24 h、72 h，倒置显微镜下观察各组细胞的生长情况。（2）干预72 h，电镜观察各组细胞的超微结构改变情况。结果：Ⅰ.JEC细胞中p57kip2基因启动子区甲基化水平：未加药组（0 μmol·L-1）p57kip2基因启动子区呈高甲基化状态（88.1%），12.5 μmol·L-1组总体甲基化率最低（76.2%）。Ⅱ.JEC细胞的生长情况及形态改变情况：（1）光镜：对照组JEC细胞呈多角形或不规则形，梁索状、巢团状、小巢状或散在分布，局部形成微腺泡样结构。随着培养时间的延长，细胞密度逐渐增加，微腺泡样结构更加明显。与对照组比较，干预24 h后，两实验组细胞密度均有所减少；干预72 h后，12.5 μmol·L-1组细胞密度仅比对照组略有减少，12.5+ μmol·L-1组细胞密度明显比对照组稀少。（2）电镜：对照组JEC细胞呈不规则形，表面可见短小的微绒毛；胞质内可见线粒体、粗面内质网、分泌泡、脂滴和自噬溶酶体等结构；胞核不规则形，以常染色质为主，可见篮网状核仁，偶见核分裂像及凋亡的细胞。干预72 h后，两实验组细胞表面微绒毛均较对照组增多，胞质内分泌泡、自噬溶酶体增多，扩张的内质网腔内充满合成的蛋白质，未见线粒体肿胀，12.5+ μmol·L-1组比12.5 μmol·L-1组的结构改变更明显。结论：5-Aza-CdR可下调人子宫内膜样癌JEC细胞中抑癌基因p57kip2启动子区甲基化水平，从而发挥其抑制JEC细胞生长与增殖的作用，还可使细胞向较好的分化方向转化；浓度为12.5 μmol·L-1的5-Aza-CdR对JEC细胞不具备细胞毒性作用，追加用药比单纯一次用药对JEC细胞生长与增殖的抑制的效果更好。
关键词 子宫内膜癌；子宫内膜样癌；5-氮杂-2'-脱氧胞苷；JEC细胞；甲基化；p57kip2
中图分类号：R737.33；Q336 文献标识码：A doi:10.3969/j.issn.1000-6281.2021.02.008
Effects of 5-Aza-2'-deoxycytidineon cell growth and morphology of JEC cells inhumanendometrioid carcinoma
ZHOU Zheng-ping1, ZHOU Jun2, ZHONG Yue2, ZHANG Chun-ying2, ZHAO Shuo1,WANG
Feng-yue1
（1. Electron Microscopy Laboratory, Zunyi medical University, Zunyi Guizhou 563000;2.Department of Pathology, Zunyi medical University, Zunyi Guizhou 563000,China）
Abstract Objective: To investigate the effects of DNA methylation transferase inhibitor 5-aza-2'-deoxycytidin(5-Aza-CdR) on the growth inhibition and morphological changes of human endometrioid carcinoma JEC cells (moderately differentiated) by 5-Aza-CDR on the demethylation of p57kip2 gene promoter region. Methods: Ⅰ.The methylation status of p57kip2 gene promoter region in JEC cells was detected by bisulfite sequencing PCR (BSP) after different concentrations of 5- Aza-CdR intervention;Ⅱ. The experimental group was divided into 12.5 μmol·L-1 groups and 12.5+ μmol·L-1 (5- Aza-CdR was added once at 48 hours for Replacement of liquid), control group without any concentration of 5 - Aza - CdR: (1) After 24 hours and 72 hours of intervention, to observe the growth of cells in each group under inverted microscope. (2) After 72 hours of intervention, the ultrastructural changes of each group were observed by electron microscopy. Results: I. The methylation level p57kip2 gene promoter region in JEC cells: the unmedicated group (0 μmol·L-1) showed hypermethylation status (88.1%),and the 12.5 μmol·L-1 group's overall methylation rate is the lowest (76.2%).Ⅱ.Growth and morphological changes of JEC cells：(1) Observation inverted light microscopy: JEC cells in the control group were polygonal or irregular in shape, with a beam cord-like, nest-like, small nest-like or scattered distribution, and a micro-bubble-like structure was formed in the local. With the prolongation of culture time, the cell density gradually increased, and the micro-glandular-like structure became more obvious. Compared with the control group, after 24 hours of intervention, the cell density of both experimental groups decreased. After 72 hours of intervention, the cell density of 12.5 μmol·L-1 group was only slightly lower, and while that of 12.5+ μmol·L-1 group was was obviously scarce. (2) Observation unde electron microscopy: JEC cells in the control group showed irregular shape with short microvilli on the surface. Mitochondria, rough endoplasmic reticulum, secretory vesicles, lipid droplets, autophagic lysosomes and other structures were observed in the cytoplasm.The nucleus were irregular shape, mostly euchromatin, with basket reticular nucleoli. Occasionally, mitotic images and apoptosis are seen. After 72 hours of intervention, the cell surface microvilli of the two experimental groups were increased compared with the control group. In the cytoplasm, the secretory vesicles and autophagy lysosomes increase, and the expanded endoplasmic reticulum cavity is filled with synthetic proteins. There was no obvious mitochondrial swelling. These structural changes were more obvious in 12.5+μmol·L-1 group than in 12.5 μmol·L-1 group. Conclusion：5'- Aza-CdR can down-regulate the methylation level of tumor suppressor gene p57kip2 promoter region in human endometrioid carcinoma JEC cells, thus exert its function of inhibiting the growth and proliferation of JEC cells, and also make the cells transform to a better direction of differentiation.5-Aza-CDR at 12.5 μmol·L-1 was not cytotoxic to JEC cells. Addition of drugs is more effective in inhibiting JEC cell growth and proliferation than a single dose.
Keywords endometrial carcinoma；endometrioid carcinoma；5-Aza-CdR；JEC cells；methylation；p57kip2
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