CpG甲基转移酶诱导人结肠癌细胞凋亡的实验研究  

CpG methyltransferase induces the apoptosis of human colorectal cancer cell line HT-29

作　　者：王文辉[1] 汪芳裕[1] 沈芸竹[1] 王颖超[2] 袁俊芳[2] 

机构地区：[1]南京大学医学院临床学院(南京军区南京总医院)消化内科,南京医学硕士研究生210002 [2]南京大学医学院临床学院(南京军区南京总医院)内分泌科,南京210002

出　　处：《医学研究生学报》2012年第9期905-908,共4页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81070289)

摘　　要：目的紧密连接蛋白(claudin)-7在结肠癌中表达上调,并与肿瘤的发生转移密切相关。文中探讨CpG甲基转移酶(CpG methyltransferase)又称M.SssI在体外对人结肠癌细胞HT-29细胞的凋亡诱导和增殖的抑制作用及对紧密连接蛋白-7表达的影响。方法分别用50U/ml M.SssI为过甲基化组,10μmol/L氮杂胞苷(5-azacytidine)为甲基化酶抑制组干预培养HT-29细胞,只加PBS为对照组。24 h后用流式细胞术观察3组细胞的凋亡情况。应用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)比色法检测各组细胞的增殖能力。用细胞免疫荧光技术观察紧密连接蛋白-7的细胞学定位和表达程度在3组细胞中的差异。结果过甲基化组细胞早期凋亡率明显高于PBS对照组,P<0.05,而甲基化酶抑制组早期凋亡率低于对照组,差异无统计学意义,P>0.05。M.SssI显著抑制HT-29细胞增殖,P<0.05,抑制率达32.1%,而甲基化酶抑制剂5-氮杂胞苷可促进细胞增殖,但与对照组比较差异无统计学意义,P>0.05。与对照组和甲基化酶抑制组相比,紧密连接蛋白-7在过甲基化组表达明显下降,P<0.05,而5-氮杂胞苷组中紧密连接蛋白-7的表达量较对照组表达明显增强,P<0.05。结论CpG甲基转移酶可诱导HT-29细胞的凋亡、抑制增殖,可能与CpG甲基转移酶促使紧密连接蛋白-7基因启动子发生过甲基化,导致相应蛋白表达下降有关。Objective Claudin-7 is up-regulated in colorectal cancer tissue and plays an important role in tumorigenesis and metastasis. This study was to investigate the effects of CpG Methyltransferase （M. SssI） on the apoptosis and proliferation of human colorectal cancer cell line HT-29 and the expression of claudin-7 in vitro. Methods HT-29 cells were divided into three groups to be treated with M. SssI （50 U/ml）, PBS （2 μl） and 5-Azacytidine （ 10 μmol/L） respectively. After 24 hours, the apoptosis of the cells was measured by flow cytometry, their proliferation determined by MTT assay, and the localization and expression of clandin-7 detected by cell immunofluorescence. Results The early apoptosis rate was remarkably higher in the M. Sssl （P 〈 0.05） but lower in the 5-Azacytidine （P 〉 0.05 ） than in the PBS group. M. SssI inhibited the proliferation of the HT-29 cells by 32.1% （P 〈 0.05 ） while 5-Azacytidine promoted it （P 〉 0.05 ）. The expression level of claudin-7 was significantly lower in the M. SssI （ P 〈 0.05 ） but markedly higher in the 5-Azacytidiue （P 〈 0.05） than in the PBS group. Conclusion M. SssI promotes the apoptosis and inhibits the proliferation of HT-29 cells, which can be attributed to the high methylation of the gene promoter and decreased expression of claudin-7 induced by CpG Methyhransferase.

关 键 词：CpG甲基转移酶 HT-29细胞 细胞凋亡 紧密连接蛋白-7 

分 类 号：R735.35[医药卫生—肿瘤]