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p57^(kip2)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p57^(kip2)基因启动子区甲基化状态。Ⅱ.将实验组分为12.5μmol·L^(-1)组、12.5μmol·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p57^(kip2)基因启动子区甲基化水平:未加药组(0μmol·L^(-1))p57^(kip2)基因启动子区呈高甲基化状态(88.1%),12.5μmol·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,12.5μmol·L^(-1)组细胞密度仅比对照组略有减少,12.5μmol·L^(-1)组细胞密度比对照组明显稀少。(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,12.5μmol·L^(-1)组比12.5μmol·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p57^(kip2)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�
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 p57^(kip2) gene promoter region.Methods:Ⅰ.The methylation status of p57^(kip2) 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 12.5μmol·L^(-1) groups and 12.5μmol·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 p57^(kip2) gene promoter region in JEC cells:the unmedicated group(0μmol·L^(-1))showed hypermethylation status(88.1%),and the 12.5μmol·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 12.5μmol·L^(-1) group was only slightly lower,and while that of 12.5μmol·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,secretor
作者
周正平
周俊
钟栎
张春英
赵朔
王凤月
ZHOU Zheng-ping;ZHOU Jun;ZHONG Yue;ZHANG Chun-ying;ZHAO Shuo;WANG Feng-yue(Electron Microscopy Laboratory,Zunyi medical University,Zunyi Guizhou 563000;Department of Pathology,Zunyi medical University,Zunyi Guizhou 563000,China)
出处
《电子显微学报》
CAS
CSCD
北大核心
2021年第2期144-150,共7页
Journal of Chinese Electron Microscopy Society
基金
贵州省科技合作计划资助项目[No.黔科合基础(2017)1216]。
