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目的研究作为参与调节细胞凋亡的关键蛋白之一的胱天蛋白酶募集域蛋白9(CARD9)在胃癌中的表达及作用机制。方法收集2012年3月至2014年3月深圳市龙岗区第二人民医院及第四人民医院收治的90例中晚期胃癌患者的胃癌组织和癌旁组织,采用Western blot法和免疫组织化学法检测其胃癌组织和癌旁组织中CARD9表达情况;在37℃,5%CO2条件下常规培养胃癌细胞株BGC-823,分别转染无意义干扰RNA[绿色荧光蛋白(GFP)]作对照(C GFP)和含于Sh Card 9 pool中的短发夹RNA(ShRNA)后,通过三种方法作如下检测:用四氮唑盐比色法(MTT比色法)检测抑制CARD9作用后对胃癌细胞增殖的影响;用流式细胞术分析抑制CARD9作用后细胞周期变化及细胞凋亡情况;用Western blot法检测抑制CARD9对抑制性κB激酶α(IKK-α)及磷酸化IKK-α(p-IKK-α)表达的影响。结果胃癌组织中CARD9表达水平高于癌旁组织,同时阳性率高于癌旁组织(70.0%vs36.0%,χ2=20.58,P<0.01)。胃癌组织中IKKα蛋白水平低于癌旁组织,p-IKKα水平高于癌旁组织。抑制CARD9后,胃癌细胞中p-IKKα水平降低,而IKKα水平升高;转染ShRNA的胃癌细胞增殖能力高于转染C GFP的细胞,并且抑制CARD9后细胞凋亡程度低于转染C GFP的胃癌细胞。结论 CARD9可能通过促进IKKα的磷酸化激活抑制胃癌细胞的增殖,继而促进胃癌细胞的凋亡。
Objective To investigate the expression and mechanism of CARD9, one of the key proteins involved in the regulation of apoptosis, in gastric cancer. Methods Totally 90 gastric cancer and para-cancerous tissues were collected from the Second People’s Hospital of Longgang District and the Fourth People’s Hospital of Longgang District from March 2012 to March 2014. Western blot and immunohistochemistry The expression of CARD9 in gastric cancer tissues and adjacent normal tissues was detected. The gastric cancer cell line BGC-823 was cultured in the condition of 37 ℃, 5% CO2, and transfected with non-sense interfering RNA [GFP] ) And short hairpin RNA (ShRNA) contained in Sh Card 9 pool were detected by three methods as follows: Tetrazolium salt colorimetry (MTT colorimetric assay) was used to detect the effect of CARD9 on proliferation of gastric cancer cells . The cell cycle and apoptosis of CARD9 cells treated with CARD9 were analyzed by flow cytometry. The inhibitory effect of CARD9 on the expression of IKK-α and p-IKK-α -α) expression. Results The positive expression rate of CARD9 in gastric cancer tissues was higher than that in paracancer tissues (70.0% vs 36.0%, χ2 = 20.58, P <0.01). The level of IKKα in gastric cancer tissues was lower than that in paracancerous tissues, and the level of p-IKKα was higher than that in paracancerous tissues. After CARD9 was inhibited, the level of p-IKKα in gastric cancer cells was decreased and the level of IKKα was increased. The proliferation of gastric cancer cells transfected with ShRNA was higher than that in C-GFP transfected cells, and the degree of apoptosis was lower than that of transfected C GFP Of gastric cancer cells. Conclusion CARD9 may inhibit the proliferation of gastric cancer cells by promoting the phosphorylation of IKKα, and then promote the apoptosis of gastric cancer cells.