全氟癸酸（PFDA）促进胃上皮细胞增殖的机制研究

发布时间：2019-04-25 18:59

【摘要】：目的研究PFDA、PFOS、PFOA对胃上皮细胞增殖的影响,并探究PFDA影响胃上皮细胞增殖的分子机制,这不仅可以为解析污染环境中胃癌的发生提供新的线索,更促使人类关注环境污染对人类健康造成的危害并采取相应的治理措施。方法1.检测PFDA、PFOS、PFOA对胃上皮细胞增殖情况的影响。以PFDA、PFOS、PFOA处理胃上皮细胞AGS,72h后各组取300个细胞进行克隆形成实验,单克隆生长至肉眼可见时,进行结晶紫染色、计数,检测PFDA、PFOS、PFOA对胃上皮细胞增殖情况的影响。2.细胞凋亡、细胞周期、自噬、衰老以PFDA处理AGS细胞,72h后进行FITC、PI染色,采用流式细胞术检测PFDA对AGS细胞的细胞周期、细胞凋亡的影响。采用Western Blotting在蛋白质水平检测自噬相关蛋白P62、LC3的表达；β-半乳糖苷酶染色研究PFDA对AGS细胞衰老的影响,并探索衰老在PFDA促进AGS细胞增殖中的分子机制。3.生物芯片分析PFDA促进胃上皮细胞增殖可能的靶基因。以PFDA处理AGS细胞,72h后收取细胞,进行表达谱生物芯片分析,并以qRT-PCR方法对显著变化的靶基因在mRNA水平进行验证。4. TCF4、PLA2G2A在PFDA促进AGS细胞增殖的机制中的作用。以PFDA处理AGS细胞24h后,TCF4、PLA2G2A表达质粒分别转染,48h后提取RNA,进行qRT-PCR,检测TCF4、PLA2G2A mRNA的表达,并进行p-半乳糖苷酶染色检测质粒转染后AGS细胞的衰老变化。结果1.克隆形成结果显示,PFDA、PFOS、PFOA都能够促进AGS细胞的增殖,但促进AGS细胞增殖的能力不同,大小顺序依次为PFDAPFOSPFOA。2.流式细胞术结果显示,PFDA对胃上皮细胞的细胞周期没有影响,但能够抑制细胞凋亡,但不明显；同时自噬相关蛋白P62、LC3的表达上调,表明自噬在一定程度上抑制了AGS细胞的增殖。3.基因芯片表达谱结果显示,PFDA处理AGS细胞后,在mRNA水平PLA2G2A、TCF4的表达显著降低。4. qRT-PCR结果表明,PFDA通过TCF4下调PLA2G2A的表达进而抑制细胞的衰老,β-Galactosidase staining结果表明TCF4、PLA2G2A表达质粒转染后AGS衰老的细胞数量显著增加。结论1. PFDA促进胃上皮细胞AGS增殖。2. PFDA通过抑制胃上皮细胞的衰老进而促进细胞增殖。3. PFDA通过TCF4下调PLA2G2A的表达抑制细胞衰老,进而发挥促进细胞增殖的作用。
[Abstract]:Objective to study the effect of PFDA,PFOS,PFOA on the proliferation of gastric epithelial cells and explore the molecular mechanism of PFDA affecting the proliferation of gastric epithelial cells, which can not only provide a new clue for analyzing the occurrence of gastric cancer in polluted environment. It also urges human beings to pay attention to the harm to human health caused by environmental pollution and to take corresponding measures to deal with it. Method 1. The effect of PFDA,PFOS,PFOA on the proliferation of gastric epithelial cells was detected. After the gastric epithelial cells were treated with PFDA,PFOS,PFOA, 300 cells were taken from each group for clone formation test. When the clones were grown to be visible to the naked eye, crystal violet staining was carried out, counting, and detection of PFDA,PFOS, was carried out. Effect of PFOA on proliferation of gastric epithelial cells. Apoptosis, cell cycle, autophagy and senescence of AGS cells were treated with PFDA. FITC,PI staining was performed after 72 hours. The effects of PFDA on cell cycle and apoptosis of AGS cells were detected by flow cytometry. Western Blotting was used to detect the expression of autophagy-associated protein P62 and LC3 at the protein level, 尾-galactosidase staining was used to study the effect of PFDA on the senescence of AGS cells, and the molecular mechanism of senescence in the promotion of AGS cell proliferation by PFDA was explored. Biochip was used to analyze the possible target genes of PFDA promoting the proliferation of gastric epithelial cells. AGS cells were treated with PFDA and collected 72 hours later. The expression profiles of AGS cells were analyzed by biochip analysis. The significantly changed target genes were verified by qRT-PCR at mRNA level. 4. The role of TCF4,PLA2G2A in the mechanism of PFDA promoting the proliferation of AGS cells. AGS cells were treated with PFDA for 24 hours and then transfected with TCF4,PLA2G2A expression plasmid. 48 hours later, RNA, was extracted and qRT-PCR, was used to detect the expression of TCF4,PLA2G2A mRNA, and the senescence changes of AGS cells were detected by pgalactosidase staining. Outcome 1. The results of clone formation showed that PFDA,PFOS,PFOA could promote the proliferation of AGS cells, but the ability to promote the proliferation of AGS cells was different, and the order of size was PFDAPFOSPFOA.2.. The results of flow cytometry showed that PFDA had no effect on the cell cycle of gastric epithelial cells, but could inhibit the apoptosis of gastric epithelial cells, but it was not obvious. At the same time, the expression of autophagy-associated protein P62 and LC3 was up-regulated, indicating that autophagy inhibited the proliferation of AGS cells to a certain extent. The gene chip expression profile showed that the expression of PLA2G2A,TCF4 in AGS cells treated with PFDA decreased significantly at the mRNA level. 4. The results of qRT-PCR showed that PFDA inhibited the senescence of AGS cells by down-regulating the expression of PLA2G2A through TCF4. The results of 尾-Galactosidase staining showed that the number of senescent cells of AGS transfected with TCF4,PLA2G2A expression plasmid increased significantly. Conclusion 1. PFDA promotes the proliferation of AGS in gastric epithelial cells. PFDA promotes cell proliferation by inhibiting the aging of gastric epithelial cells. 3. PFDA inhibits cell senescence by down-regulating the expression of PLA2G2A through TCF4, thus promoting cell proliferation.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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