目的 探究塞來昔布通過上調(diào)Cyclin D1基因甲基化水平對(duì)食管癌細(xì)胞增殖和凋亡的作用及機(jī)制。方法 將細(xì)胞分為對(duì)照、轉(zhuǎn)染和塞來昔布組，分別轉(zhuǎn)染陰性對(duì)照載體、Cyclin D1過表達(dá)載體和Cyclin D1轉(zhuǎn)染后給予60 μmol/L塞來昔布。采用MTT法、流式細(xì)胞術(shù)分別檢測細(xì)胞增殖、細(xì)胞周期和細(xì)胞凋亡率的變化，Western blotting法檢測細(xì)胞周期和細(xì)胞凋亡相關(guān)蛋白表達(dá)水平；甲基化特異性PCR（MS-PCR）、qRT-PCR法用于檢測Cyclin D1甲基化特異性擴(kuò)增片段和Cyclin D1 mRNA表達(dá)水平。結(jié)果 塞來昔布能夠抑制Cyclin D1誘導(dǎo)的細(xì)胞體外增殖，阻滯細(xì)胞周期向S期轉(zhuǎn)化，并促進(jìn)食管癌細(xì)胞凋亡；MS-PCR結(jié)果顯示塞來昔布能夠上調(diào)Cyclin D1基因甲基化水平，在轉(zhuǎn)錄水平抑制細(xì)胞內(nèi)Cyclin D1 mRNA的表達(dá)。結(jié)論 塞來昔布能夠通過上調(diào)Cyclin D1基因甲基化水平發(fā)揮抑制食管癌細(xì)胞增殖、促進(jìn)其體外凋亡的作用。

Objective To investigate the effects and mechanism of celecoxib on the cell proliferation and apoptosis by regulating methylation status of Cyclin D1 in esophageal carcinoma cells. Methods Cells were divided into control group, transfected group and celecoxib group. Cells in the control group were transfected with negative control vector while cells in the transfected group and celecoxib group were transfected with Cyclin D1-overexpression vector. The celecoxib group was treated with 60 μmol/L celecoxib solution after transfection. MTT and flow cytometry were used to detect the cell proliferation capacity, cell cycle distribution, and cell apoptotic rates in vitro. Western blotting was used to measure the expression levels of related proteins. MS-PCR and qRT-PCR were used to detect the Cyclin D1 promoter region methylation level and Cyclin D1 mRNA expression level. Results Celecoxib can inhibit cell proliferation in vitro, which was promoted by Cyclin D1 overexpression, block cell cycle transition into S phase and promote cell apoptosis. MS-PCR showed that celecoxib could up-regulate the methylation level of Cyclin D1 gene and suppressed Cyclin D1 mRNA expression at transcriptional level. Conclusion Celecoxib can inhibit cell proliferation and promote apoptotic rate of esophageal cancer cells through up-regulating the methylation level of Cyclin D1.

R966

河南省科技計(jì)劃發(fā)展項(xiàng)目（202102310408）