【作者】 李勇； 巩涛； 范立侨； 赵群； 宋振川； 焦志凯； 张志栋； 刘羽；

【Author】 Li Yong Gong Tao Fan Li-Qiao et al. The Third Department of Surgery, The Forth Hospital of Hebei Medical University, Shijiazhuang 050011, China

【机构】 河北医科大学第四医院暨河北省肿瘤医院外科；

【摘要】 目的:近年来研究表明ω-3多不饱和脂肪酸可抑制肿瘤细胞的生长,但其作用机制不甚明确。本研究通过观察ω-3多不饱和脂肪酸制剂二十碳五烯酸(cis-5,8,11,14, 17-eicosapentaenoic acid,EPA)和二十二碳六烯酸(cis- 4,7,10,13,16,19-docosahexaenoic acid,DHA)对人胃癌 BGC-823细胞NF-κB p65的影响,探讨ω-3多不饱和脂肪酸的抗癌机制。方法:将人胃癌BGC-823细胞分对照组、 EPA药物组和DHA药物组,EPA组和DHA组按15 μg／ml、 30 μ g／ml和45 μg／ml药物浓度加药,肿瘤细胞加药继续培养48小时后,用SP-9000免疫细胞化学检测胃癌细胞NF- κ B p65蛋白分布(鼠抗人NF κ B p65单克隆抗体1:100), DAB显色,观察。用Western blot检测胃癌细胞NF—κB p65蛋白情况(鼠抗人NF κ Bp65单克隆抗体1:500),DAB 显色,测定蛋白印迹带的吸光度(A)值。用RT—PCR检测胃癌细胞NF-κ B p65 mRNA表达情况,NF-κ B p65 上游引物5’-GAG GTG TAT TTC ACG GGA CC-3’, 下游引物5’-GAA GTC CAT GTC CGC AAT GG-3’, 扩增片段长度954bp, β-actin上游引物5’-CAG CCA TGT ACG TTG CTA TC-3’,下游引物5’-GGA GTA CTT GCG CTC AGG AG-3’,扩增片段626bp。反应条件如下:NF-κ B p65:37℃、60min,94℃、5min,94℃、 30sec．、59℃、30sec．、72℃、45sec．循环30次,72℃、7min, 4℃,用凝胶成像分析系统对比分析PCR产物电泳条带EB荧光强度(A值),NF—κ B p65 mRNA表达相对值=PKC β II或NF κ Bp65 A值／β-actin A值。应用SPSS13统计软件的单因素方差分析。结果:免疫细胞化学方法染色NF κ B 065在对照组胃癌细胞浆和细胞核中出现棕黄色颗粒, 用EPA或DHA药物后肿瘤细胞浆和细胞核内NF κ B p65 染色明显减弱。用Western blot方法测定对照组肿瘤细胞NF κ B p65蛋白印迹A值为7474．80±302．92,30 μg／ml和 45μg／ml的EPA组肿瘤细胞NF κ B p65蛋白印迹A值分别为2989．93±121．15和1495．08±60．57,30 μg／ml和45 μ g／ml的DHA组肿瘤细胞NF κ B p65蛋白印迹A值分别为2535．04±111．43和1295．74±126．22．药物处理肿瘤细胞后,NF κB p65蛋白印迹A值显著下降(p<0．001),且 NF κ B p65在45 μg／ml组比307g／ml组下降更明显(P <0．001)。用RT-PCR方法测定对照组肿瘤细胞NF κ B p65 mRNA表达值为0．40±0．05,30 μ g／ml和45 μg／ml的 EPA组肿瘤细胞NF κB p65 mRNA表达值分别为0．10± 0．04和0．05±0．03,30 μg／ml和45?g／ml的DHA组NF κ B p65 mRNA表达值分别为0．08±0．03和0．03±0．01, 药物处理细胞后,NF κ B p65 mRNA表达相对值下降(p <0．001)。结论:EPA或DHA可使肿瘤细胞NF κ B p65蛋白表达水平和mRNA基因表达水平下降,进一步抑制胃癌细胞的生长。更多还原

【Abstract】 Objective: To discuss the effect and mechanism of EPA and DHA on NF- k B p65 of gastric cells. Methods: Human gastric BGC-823 cells divided into three groups: control group, EPA group and DHA group. Incubation of human gastric BGC-823 cells with different concentration (15 μ g/ml, 30 μ g/ml and 45 μ g/ml) of EPA and DHA for 48h,then NF- κ B p65 were detected by immuno cytochemistry, Western blot and TR-PCR. Results: Strong staining of NF κ B p65 were located in cytoplasm and nucleus in control group. After treatment with EPA or DHA, staining of NF κ B p65 were decreased. Absorbency of NF κ B p65 in groups of DHA 30 μ g/ml and 45 μ g/ml were 2535.04 ± 111.43 and 1295.74 ± 126.22, respectively. Compared the above with control group (7474.80 ± 302.92 ), NF κ B p65 was decreased ( p <0.001, respectively ). Especially compared 45 μ g/ml with 30 μ g/ml both EPA or DHA was further increased ( p<0.001, respectively ). Absorbency of NF κ B p65 mRNA in groups of DHA 30 μ g/ml and 45 μ g/ml were 0.08 ± 0.03 and 0.03 ± 0.01, respectively. Compared the above with control group (0.40 ± 0.05 ), NF κ B p65 mRNA was decreased (p<0.001, respectively). Conclusion: EPA and DHA can inhibit NF κ B signaling and reduced growth of the cultured cell.更多还原