人结直肠癌组织细胞衰老减慢、糖代谢增强

发布时间：2018-05-24 02:07

本文选题：结直肠癌 + p16　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:本文通过检测人结直肠癌组织及肿瘤细胞中p16、p21、衰老相关β-半乳糖苷酶(SA-β-Gal)衰老标志物以及糖代谢途径关键酶的表达变化,试图探讨结直肠癌衰老活性及其与糖代谢重编程的关系。方法:1人结直肠癌标本:收集河北医科大学第四医院外二科人结直肠癌患者63例,取其手术切除标本,以距肿瘤10 cm以外的正常肠粘膜组织为对照组。石蜡切片用于免疫组织化学染色检测p16和p21的表达。冰冻切片用于检测细胞SA-β-Gal活性。2人结肠癌细胞株:TNF-α处理的人正常结直肠粘膜细胞系FHC细胞及三种人结肠癌细胞系HCT116、HT29、CACO2细胞,进行MTT分析,以及SA-β-Gal染色。3葡萄糖-6-磷酸脱氢酶(G6PD)和丙酮酸激酶-2(PKM2)表达分析:提取结直肠癌新鲜组织或细胞总蛋白,用Western blot方法检测G6PD和PKM2的表达;提取总RNA,逆转录后进行实时荧光定量聚合酶链式反应,检测G6PD、PKM2 mRNA水平。4己糖激酶(HK)活性测定:按试剂盒说明书操作。结果:1人结直肠癌肿瘤组织中p16和p21表达下调,SA-β-Gal活性降低免疫组织化学染色结果显示,人结直肠癌肿瘤组织中衰老相关蛋白p16、p21表达低于癌旁正常组织(P0.05);并且肿瘤组织衰老相关标志物SA-β-Gal染色强度低于癌旁正常组织(P0.05)。2 TNF-α诱导人结肠癌细胞SA-β-Gal活性降低,活力增强使用TNF-α对人结直肠癌细胞株HCT116、HT-29、Caco-2和正常结直肠粘膜细胞株FHC刺激72h,而后进行SA-β-Gal染色及MTT分析。结果显示,相同刺激条件下,HCT116、HT-29和Caco-2三种肿瘤细胞SA-β-Gal活性低于FHC细胞(P0.05);MTT分析显示,TNF-α诱导三种肿瘤细胞活力较FHC细胞升高(P0.05)。3人结直肠癌肿瘤组织中G6PD、PKM2表达升高qRT-PCR和Western blot结果显示,人结直肠癌肿瘤组织中G6PD、PKM2 mRNA和蛋白表达均高于癌旁正常组织(P0.05)。4人结直肠癌细胞中G6PD、PKM2和HK表达及活性升高Western blot结果显示,HCT116、HT-29和Caco-2三种人结肠癌肿瘤细胞中G6PD、PKM2蛋白表达高于FHC正常结直肠粘膜细胞(P0.05);细胞免疫荧光结果显示,人结肠癌肿瘤细胞中PKM2蛋白表达量高于正常结直肠粘膜细胞(P0.05);并且三种肿瘤细胞中己糖激酶(HK)活性明显高于正常结直肠粘膜细胞(P0.05)。结论:1人结直肠癌组织细胞中p16、p21蛋白表达下调,SA-β-Gal活性降低,细胞活力升高;2人结直肠癌组织细胞中,糖酵解途径关键酶G6PD、PKM2和HK的表达及活性升高;3人结直肠癌组织细胞具有更强的抗衰老生存能力,糖酵解代谢增强。
[Abstract]:Objective: to detect the expression of p16 p21, senescence associated 尾 -galactosidase SA- 尾 -galactosidase and the key enzymes of glucose metabolism pathway in human colorectal cancer tissues and tumor cells. The purpose of this study was to explore the relationship between the aging activity of colorectal cancer and the reprogramming of glucose metabolism. Methods one human colorectal cancer specimen was collected from 63 patients with colorectal cancer in the second department of the fourth Hospital of Hebei Medical University. The surgical specimens were removed and the normal intestinal mucosa (10 cm away from the tumor) was taken as the control group. Paraffin sections were used to detect the expression of p16 and p21 by immunohistochemical staining. Frozen sections were used to detect the activity of SA- 尾 -Gal. 2 Human colon cancer cell line: TNF- 伪 treated human normal colorectal mucosal cell line FHC and three human colon cancer cell lines HCT116 and HT29CACO _ 2. MTT analysis was performed. And SA- 尾 -Gal staining for the expression of G6PD3 glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (2PKM2): total protein was extracted from fresh tissues or cells of colorectal cancer and the expression of G6PD and PKM2 was detected by Western blot method. Total RNAs were extracted, real-time fluorescence quantitative polymerase chain reaction was performed after reverse transcription, and the activity of G6PD-PKM2 mRNA was determined. Results the down-regulated expression of p16 and p21 in human colorectal cancer tissues decreased the activity of SA- 尾 -Gal by immunohistochemical staining. The expression of senescence-associated protein p16p21 in human colorectal cancer tissues was lower than that in adjacent normal tissues, and the staining intensity of SA- 尾 -Gal was lower than that of P0.052.TNF- 伪 induced decrease of SA- 尾 -Gal activity in human colon cancer cells. TNF- 伪 was used to stimulate human colorectal cancer cell line HCT116HT-29Caco-2 and normal colorectal mucosal cell line FHC for 72 h, then SA- 尾 -Gal staining and MTT analysis were performed. The results showed that the activity of SA- 尾 -Gal in three kinds of tumor cells, HCT116, HT-29 and Caco-2, was lower than that of FHC cells. The results showed that TNF- 伪 increased the expression of G6PD-PKM2 in human colorectal cancer cells, and the expression of G6PD-PKM2 was higher than that of FHC cells. Expression of G6PD-PKM2 and protein in Human Colorectal Cancer tissue is higher than that in Human Colorectal Cancer adjacent normal tissue P0.05.4 the expression and activity of G6PD-PKM2 and HK in Human Colorectal Cancer cells increased Western blot results showed that the expression of G6PD-PKM2 protein in three kinds of human colon cancer tumor cells, HCT116, HT-29 and Caco-2, was higher than that in normal tissues of human colorectal cancer. It was higher than FHC normal colorectal mucosal cells (P0.05G), and the results of cellular immunofluorescence showed that, The expression of PKM2 protein in human colon cancer tumor cells was higher than that in normal colorectal mucosal cells, and the activity of hexokinase in three kinds of tumor cells was significantly higher than that in normal colorectal mucosal cells. Conclusion the expression of p16 p21 protein in human colorectal cancer tissue cells was down-regulated and the activity of SA- 尾 -Gal was decreased, and the cell viability was increased in two human colorectal cancer tissue cells. The expression and activity of PKM2 and HK, the key enzymes of glycolytic pathway, were increased. 3 human colorectal cancer tissue cells had stronger anti-aging survival ability and enhanced glycolytic metabolism.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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