本文選題：全氟癸酸 + 胃癌��； 參考：《山東大學》2017年碩士論文

【摘要】：背景和目的胃癌是全世界癌癥死亡的第二大原因,約占所有侵襲性癌癥的10%。雖然對于其發(fā)病機制尚未完全了解,但值得注意的是,已經證實胃癌與環(huán)境污染的正相關性,胃癌的病例和死亡總數(shù)隨著中國廣泛的人口變化和持續(xù)增加的環(huán)境污染而增加。全氟癸酸(PFDA)是一種高毒性的全氟化脂肪酸(PFCA),一種持久的環(huán)境污染物。PFDA存在于空氣,食品和水中,特別是在中國,在北京周圍的雪地中檢測到139ng/L的PFDA。PFDA攝入主要來自食物和飲用水,并且它可以在人體血液和器官中積累,其血清清除半衰期持續(xù)數(shù)年。已報道PFDA處理可以使大鼠出現(xiàn)嚴重的體重減輕,心動過緩,低體溫和降低的血清甲狀腺激素水平。盡管如此,然而到目前為止,很少知道PFDA對人體的毒性作用和其對惡性腫瘤的促進作用。通過評估全氟癸酸(PFDA)對胃癌細胞AGS中炎癥小體的激活和炎癥調節(jié)的影響,研究AGS在PFDA處理后炎癥小體NLRP3激活促進IL-1β和IL18分泌的分子機制。我們希望進一步研究慢性炎癥在胃惡性腫瘤的發(fā)展中所起到的重要作用,以及環(huán)境污染物對于人類健康和生活的影響。研究方法選取狀態(tài)良好的胃癌細胞AGS,鋪于無菌的細胞培養(yǎng)六孔板中。以特定濃度的PFDA(75μM)處理六孔板中的AGS細胞,對照組加入等體積DMSO,以用作下一步實驗。1.實時PCR實驗使用Trizol提取PFDA處理后的AGS細胞總RNA,按逆轉錄試劑盒說明進行逆轉錄后,用RT-PCR測量相關基因在mRNA水平的變化。2.ELISA 實驗PFDA處理72h后收集細胞培養(yǎng)液上清,離心處理后立即用相應的ELISA試劑盒測定其濃度。3.Western blot 實驗取PFDA處理后的AGS細胞六孔板,加入蛋白裂解液RIPA(含PMSF)提取其總蛋白,用BCA法測定其蛋白濃度后,進行后續(xù)實驗。4.siRNA干擾實驗用siRNA轉染試劑將已設計好的cIAP2的siRNA和control siRNA轉入胃癌細胞AGS中,其余步驟與RT-PCR實驗相同。5.HE染色實驗以25 mg/kg/d的劑量在C57BL/6小鼠的飲用水中添加PFDA,對照組小鼠飲水中則加入等量DMSO,兩周后統(tǒng)一取出小鼠胃組織進行HE染色和觀察。結果當添加到細胞培養(yǎng)物中時,與對照細胞相比,PFDA顯著刺激IL-1β和IL18分泌,并且提高其mRNA水平的表達。通過RT-PCR和Western blot我們發(fā)現(xiàn)NLRP3的上調與IL-1β和IL-18產生的促進相關。然后分析cIAP1/2,c-Re1和p52的表達變化,結果證明在PFDA處理后導致的炎癥小體活性增強促使所有測試基因中mRNA表達提高。使用cIAP2 siRNA和NFκB報告基因的測定提供了這些基因參與PFDA誘導的炎癥小體組裝的額外的證據(jù)。此外,在PFDA處理的小鼠的胃組織中檢測到IL-1β和IL-18的分泌增加,在PFDA處理的小鼠胃組織中也觀察到上皮細胞的無序排列和炎癥細胞浸潤。結論1.PFDA可以誘導胃癌細胞AGS中炎癥小體NLRP3的激活。2.PFDA處理后,胃癌細胞AGS通過炎癥小體NLRP3激活Caspase-1,進而誘導IL-1β和IL-18分泌增加。3.PFDA調節(jié)人類細胞的炎癥小體組裝和炎癥形成。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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本文編號：1980886