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.24h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 μ l ) 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 claudin-7 detected by cell immunofluorescence. Results The early apoptosis rate was remarkably higher in the M. SssI ( 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 -Azacytidine ( 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 Methyltransferase.
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