摘要目的 了解宫颈癌组织中Notch基因细胞内区(NICD)蛋白的表达水平及其临床意义,探讨γ分泌酶抑制剂--氮-[氮-(3,5-二氟苯乙酰)-L-丙氨酰]-S-苯基甘氨酸丁酯(DAPT)对宫颈癌细胞系的作用.方法 应用蛋白印迹法检测40份宫颈癌组织及21份正常宫颈组织中NICD蛋白的表达水平.体外培养宫颈癌细胞SiHa、HeLa,加入不同浓度的DAPT,采用四甲基偶氮唑蓝(MTT)比色法检测细胞生长情况;采用流式细胞仪检测细胞的S期细胞比例(SPF)、凋亡细胞百分比和增殖指数(PI);应用蛋白印迹法检测DAPT对SiHa、HeLa细胞中NICD蛋白表达的影响.结果 NICD蛋白的表达水平在宫颈癌组织中明显高于正常宫颈组织(分别为1.237±0.353、0.938±0.105),两者比较,差异有统计学意义(P<0.05).在宫颈癌组织中,低分化者NICD蛋白的表达水平明显高于高～中分化者(分别为1.496±0.540、1.150±0.216),有淋巴结转移者明显高于无淋巴结转移者(分别为1.419±0.532、1.159±0.210),有宫旁转移者明显高于无宫旁转移者(分别为1.718±0.710、1.183±0.258),腺癌明显高于鳞癌(分别为1.463±0.395、1.162±0.187),分别比较,差异均有统计学意义(P<0.05).DAPT作用于SiHa、HeLa细胞后,NICD蛋白的表达水平下降(P<0.05).不同浓度的DAPT作用不同时间能够抑制宫颈癌细胞的增殖、促进其凋亡,且浓度越高出现作用的时间越早.结论 NICD在宫颈癌的发生、发展中起一定的促进作用;DAPT能抑制宫颈癌细胞的增殖,促进宫颈癌细胞的凋亡,其机制可能与抑制NICD的生成有关.

abstractsObjective To study the expression and clinical significance of Notch intracellular domain (NICD) in cervical cancer and the effects of N-[N-(3,5-difluorophenyl)acetyl-L-alanyl]-S-phenyl glycine t-butyl ester (DAPT), a γ-secretase inhibitor on the proliferation and apoptosis of cervical cancer cell lines. Methods Western blot was used to detect the expression of NICD in the tissues of 40 cervical cancers and 21 normal cervix and its relationship with clinical features of cervical cancer was also analyzed. Proliferation of SiHa and HeLa cervical cells was determined by methyl thiazolyl tetrazolium (MTT) assay, cell cycles and apoptosis and index of proliferation were detected by flow cytometry method. The expression of NICD in SiHa and HeLa cells incubated with DAPT was detected by western blot. Results The expression level of NICD in cervical cancers was significantly higher than that of normal cervical tissues (1.237±0.353 vs 0.938±0.105, P<0.05). The NICD expression was higher in cervical cancers with high grade,lymph node involvement and parametrial invasion than that with low-middle grade (1.496±0.540 vs 1.150±0.216), without lymph node involvement (1.419±0.532 vs 1.159±0.210) and no parametrial invasion (1.718±0.710 vs 1.183±0.258), respectively (all P<0.05). The expression of NICD in cervical adenocarcinoma was higher than that of squamous cell cancer (1.463±0.395 vs 1.162±0.187, P<0.05). After SiHa and HeLa cells were incubated with DAPT, NICD expression was significantly lower than that in control (P<0.05). The effects of DAPT inhibited the proliferation and prompted the apoptosis of SiHa and HeLa cells was depended on its concentrations and times. Conclusions NICD may play a key role in the occurrence and progress of cervical cancer. The mechanism of DAPT inhibited the proliferation and prompted the apoptosis of SiHa and HeLa cells may be due to decreased the formation of NICD.