AGS细胞系中12-LOX的表达及其抑制剂对细胞增殖的影响

Expression of 12-lipoxygenase and its inhibitor's effect on proliferation of human AGS cell line

目的:探讨人胃癌细胞系AGS中12-脂肪氧化酶(12- lipoxygenase,12-LOX)的表达情况及其抑制剂黄芩甙元(baicalein)对AGS细胞增殖的影响. 方法:AGS在含100 mL/L小牛血清+100 kU/L青、链霉素的RPMI1640培养基中常规培养.用RT-PCR 检测12-LOX在AGS中的表达情况,分别以20,40, 80 μmol/L baicalein干预细胞,用四氮唑蓝(thiazolyl blue tetrazolium bromide,MTT)法在24,48,72 h 测量细胞吸光度,检测baicalein对细胞增殖的影响:用倒置显微镜和电子显微镜进行形态观察;通过脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记法(TUNEL)和吖啶橙-溴化乙锭(AO-EB)染色法检测细胞凋亡指数. 结果:AGS细胞中存在12-LOXmRNA表达;MTT显示baicalein大致呈时间、剂量依赖性抑制细胞增殖(各组间P<0.01,除外40 μmol/L 24 h组与48 h组);AGS细胞存活率最高为82.1%,最低为38.4%.倒置显微镜下可见baicalein干预后的细胞皱缩,部分细胞胞体变圆,脱离贴壁状态,成为悬浮细胞;电镜下可见baicalein干预后的细胞出现了凋亡的特征性改变:核染色质边集及凋亡小体形成;AO-EB染色法显示除0μmol/L组不同时间点细胞凋亡指数无显著差异外(P>0.05),其余各干预组细胞凋亡指数均有显著差异(P<0.01);TUNEL染色法显示48 h时,baicalein 80μmol/L细胞调亡指数为38.37±0.36%而0 μmol/L组细胞调亡指数为7.27±0.21%,两组间存在显著差异(P<0.01). 结论:人胃癌细胞AGS中存在12-LOXmRNA表达,其特异性抑制剂baicalein能抑制AGS细胞增殖并促进其凋亡.

AIM： To investigate the expression of 12-1ipoxygenase （12-LOX） and the possible effect of its inhibitor on proliferation of human gastric cancer cell line AGS.METHODS： AGS cells were cultured in RPMI-1640 medium with 100 mL/L fetal bovine serum （FBS）, 100 kU/L penicillin and 100 kU/L streptomycin. 12-LOX mRNA was analyzed by reverse transcription polymerase chain reaction （RT-PCR）. The effect of baicalein on the proliferation of the cells was detected by thiazolyl blue tetrazoliumbromide （MTT） method. The morphological changes of cellular and subcellullar structures were observed under inverted microscope and electron microscopy. AO-EB double and TUNEL staining were used to evaluate the apoptotic index of AGS cells.RESULTS： 12-LOX mRNA was expressed in human AGS cells. At concentrations from 20 to 80 μmol/L, baicaleinin hibited the proliferation of AGS cells in a concentrationand time-dependent manner from 24 to 72 hours. There were significant differences between any other groups （P〈0.01） except 40μmol/L group at 24 h and 48 h. The maximum survival rate of the cells was 82.1% and the minimum one was 38.4%. Morphological changes such as chromatin condensatio, apoptotic bodies were observed after treated with baicalein, and some cells become round and suspended. Significant apoptosis was also induced by baicalein, and the apoptotic indexes detected by AOEB staining showed marked differences between any other groups （P〈0.01） except in 0μlmol/L group. The apoptoticindex detected by TUNEL staining showed marked difference between 80 and 0μmol/L at 48 h （38.37±0.36% vs7.27 ±0.21%, P〈0.01 ）.CONCLUSION： 12-LOX mRNA is expressed in AGS cell line. Baicalein not only promotes proliferation, but also induces apoptosis of human gastric cancer cells in vitro.