全氟癸酸促进胃癌细胞炎症小体激活的机制研究

发布时间：2018-06-05 06:01

本文选题：全氟癸酸 + 胃癌　；参考：《山东大学》2017年硕士论文

【摘要】：背景和目的胃癌是全世界癌症死亡的第二大原因,约占所有侵袭性癌症的10%。虽然对于其发病机制尚未完全了解,但值得注意的是,已经证实胃癌与环境污染的正相关性,胃癌的病例和死亡总数随着中国广泛的人口变化和持续增加的环境污染而增加。全氟癸酸(PFDA)是一种高毒性的全氟化脂肪酸(PFCA),一种持久的环境污染物。PFDA存在于空气,食品和水中,特别是在中国,在北京周围的雪地中检测到139ng/L的PFDA。PFDA摄入主要来自食物和饮用水,并且它可以在人体血液和器官中积累,其血清清除半衰期持续数年。已报道PFDA处理可以使大鼠出现严重的体重减轻,心动过缓,低体温和降低的血清甲状腺激素水平。尽管如此,然而到目前为止,很少知道PFDA对人体的毒性作用和其对恶性肿瘤的促进作用。通过评估全氟癸酸(PFDA)对胃癌细胞AGS中炎症小体的激活和炎症调节的影响,研究AGS在PFDA处理后炎症小体NLRP3激活促进IL-1β和IL18分泌的分子机制。我们希望进一步研究慢性炎症在胃恶性肿瘤的发展中所起到的重要作用,以及环境污染物对于人类健康和生活的影响。研究方法选取状态良好的胃癌细胞AGS,铺于无菌的细胞培养六孔板中。以特定浓度的PFDA(75μM)处理六孔板中的AGS细胞,对照组加入等体积DMSO,以用作下一步实验。1.实时PCR实验使用Trizol提取PFDA处理后的AGS细胞总RNA,按逆转录试剂盒说明进行逆转录后,用RT-PCR测量相关基因在mRNA水平的变化。2.ELISA 实验PFDA处理72h后收集细胞培养液上清,离心处理后立即用相应的ELISA试剂盒测定其浓度。3.Western blot 实验取PFDA处理后的AGS细胞六孔板,加入蛋白裂解液RIPA(含PMSF)提取其总蛋白,用BCA法测定其蛋白浓度后,进行后续实验。4.siRNA干扰实验用siRNA转染试剂将已设计好的cIAP2的siRNA和control siRNA转入胃癌细胞AGS中,其余步骤与RT-PCR实验相同。5.HE染色实验以25 mg/kg/d的剂量在C57BL/6小鼠的饮用水中添加PFDA,对照组小鼠饮水中则加入等量DMSO,两周后统一取出小鼠胃组织进行HE染色和观察。结果当添加到细胞培养物中时,与对照细胞相比,PFDA显著刺激IL-1β和IL18分泌,并且提高其mRNA水平的表达。通过RT-PCR和Western blot我们发现NLRP3的上调与IL-1β和IL-18产生的促进相关。然后分析cIAP1/2,c-Re1和p52的表达变化,结果证明在PFDA处理后导致的炎症小体活性增强促使所有测试基因中mRNA表达提高。使用cIAP2 siRNA和NFκB报告基因的测定提供了这些基因参与PFDA诱导的炎症小体组装的额外的证据。此外,在PFDA处理的小鼠的胃组织中检测到IL-1β和IL-18的分泌增加,在PFDA处理的小鼠胃组织中也观察到上皮细胞的无序排列和炎症细胞浸润。结论1.PFDA可以诱导胃癌细胞AGS中炎症小体NLRP3的激活。2.PFDA处理后,胃癌细胞AGS通过炎症小体NLRP3激活Caspase-1,进而诱导IL-1β和IL-18分泌增加。3.PFDA调节人类细胞的炎症小体组装和炎症形成。
[Abstract]:Background and objective gastric cancer is the second leading cause of cancer death worldwide, accounting for about 10 percent of all aggressive cancers. Although the pathogenesis of gastric cancer has not been fully understood, it is worth noting that the positive correlation between gastric cancer and environmental pollution has been confirmed, and the total number of cases and deaths of gastric cancer has increased with the extensive population changes and increasing environmental pollution in China. Perfluorodecanoic acid (PFDAA) is a highly toxic perfluorinated fatty acid (PFCA), a persistent environmental pollutant. PFDA is found in air, food and water, especially in China, where PFDA.PFDA intake of 139ng/L is detected in snow around Beijing mainly from food and drinking water. And it accumulates in the body's blood and organs, and its serum-clearing half-life lasts several years. It has been reported that PFDA treatment can cause severe weight loss, bradycardia, hypothermia and decreased serum thyroid hormone levels in rats. However, so far little is known about the toxicity of PFDA to humans and its role in promoting malignant tumors. To investigate the effect of perfluorodecanoic acid (PFDAA) on the activation and regulation of inflammatory corpuscles in gastric cancer cell line AGS, the molecular mechanism of NLRP3 activation of inflammatory corpuscles and IL18 secretion after PFDA treatment was studied. We hope to further study the important role of chronic inflammation in the development of gastric malignant tumors and the impact of environmental pollutants on human health and life. Methods AGSs of gastric cancer cells in good condition were selected and laid in the six hole plates of aseptic cell culture. The AGS cells were treated with PFDA(75 渭 M at a specific concentration, and the control group was treated with the same volume of DMSO for the next experiment. 1. In real-time PCR experiment, Trizol was used to extract the total RNAs of AGS cells treated with PFDA. After reverse transcription was carried out according to the reverse transcription kit instructions, RT-PCR was used to measure the changes of related genes at mRNA level. 2. The supernatant of cell culture medium was collected after PFDA treatment with Elisa for 72 hours. After centrifugation, the concentration of AGS cells was determined by the corresponding ELISA kit. 3. Western blot assay was used to extract the total protein from AGS cells treated with PFDA. The total protein was extracted by adding protein lysate RIPA (containing PMSF), and the protein concentration was determined by BCA method. The siRNA and control siRNA of the designed cIAP2 were transfected into the gastric cancer cell line AGS with siRNA transfection reagent. The other steps were the same as the RT-PCR test. 5. The drinking water of C57BL/6 mice was added by HE staining at a dose of 25 mg/kg/d, while that of the control mice was added with the same amount of DMSO.Two weeks later, the gastric tissues of the mice were taken out for HE staining and observed. Results compared with the control cells, PFDA significantly stimulated the secretion of IL-1 尾 and IL18 and increased the expression of mRNA. Through RT-PCR and Western blot, we found that the up-regulation of NLRP3 is related to the promotion of IL-1 尾 and IL-18 production. The expression of c-Re1 and p52 in 1 / 2 of cIAP1 / 2 was analyzed. The results showed that the increased activity of inflammatory corpuscles induced by PFDA increased the expression of mRNA in all the tested genes. The use of cIAP2 siRNA and NF 魏 B reporter genes provides additional evidence of the involvement of these genes in the assembly of inflammatory corpuscles induced by PFDA. In addition, the increased secretion of IL-1 尾 and IL-18 was detected in the gastric tissues of mice treated with PFDA, and the disordered arrangement of epithelial cells and the infiltration of inflammatory cells were also observed in the gastric tissues of mice treated with PFDA. Conclusion 1.PFDA can induce the activation of inflammatory body NLRP3 in gastric cancer AGS. 2. After treated with PFDA, AGS activates Caspase-1 through inflammatory body NLRP3, and then induces the increase of IL-1 尾 and IL-18 secretion. 3. PFDA regulates the inflammatory body assembly and inflammatory formation of human cells.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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