本文選題：結(jié)直腸癌 + p16�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:本文通過檢測(cè)人結(jié)直腸癌組織及腫瘤細(xì)胞中p16、p21、衰老相關(guān)β-半乳糖苷酶(SA-β-Gal)衰老標(biāo)志物以及糖代謝途徑關(guān)鍵酶的表達(dá)變化,試圖探討結(jié)直腸癌衰老活性及其與糖代謝重編程的關(guān)系。方法:1人結(jié)直腸癌標(biāo)本:收集河北醫(yī)科大學(xué)第四醫(yī)院外二科人結(jié)直腸癌患者63例,取其手術(shù)切除標(biāo)本,以距腫瘤10 cm以外的正常腸粘膜組織為對(duì)照組。石蠟切片用于免疫組織化學(xué)染色檢測(cè)p16和p21的表達(dá)。冰凍切片用于檢測(cè)細(xì)胞SA-β-Gal活性。2人結(jié)腸癌細(xì)胞株:TNF-α處理的人正常結(jié)直腸粘膜細(xì)胞系FHC細(xì)胞及三種人結(jié)腸癌細(xì)胞系HCT116、HT29、CACO2細(xì)胞,進(jìn)行MTT分析,以及SA-β-Gal染色。3葡萄糖-6-磷酸脫氫酶(G6PD)和丙酮酸激酶-2(PKM2)表達(dá)分析:提取結(jié)直腸癌新鮮組織或細(xì)胞總蛋白,用Western blot方法檢測(cè)G6PD和PKM2的表達(dá);提取總RNA,逆轉(zhuǎn)錄后進(jìn)行實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng),檢測(cè)G6PD、PKM2 mRNA水平。4己糖激酶(HK)活性測(cè)定:按試劑盒說明書操作。結(jié)果:1人結(jié)直腸癌腫瘤組織中p16和p21表達(dá)下調(diào),SA-β-Gal活性降低免疫組織化學(xué)染色結(jié)果顯示,人結(jié)直腸癌腫瘤組織中衰老相關(guān)蛋白p16、p21表達(dá)低于癌旁正常組織(P0.05);并且腫瘤組織衰老相關(guān)標(biāo)志物SA-β-Gal染色強(qiáng)度低于癌旁正常組織(P0.05)。2 TNF-α誘導(dǎo)人結(jié)腸癌細(xì)胞SA-β-Gal活性降低,活力增強(qiáng)使用TNF-α對(duì)人結(jié)直腸癌細(xì)胞株HCT116、HT-29、Caco-2和正常結(jié)直腸粘膜細(xì)胞株FHC刺激72h,而后進(jìn)行SA-β-Gal染色及MTT分析。結(jié)果顯示,相同刺激條件下,HCT116、HT-29和Caco-2三種腫瘤細(xì)胞SA-β-Gal活性低于FHC細(xì)胞(P0.05);MTT分析顯示,TNF-α誘導(dǎo)三種腫瘤細(xì)胞活力較FHC細(xì)胞升高(P0.05)。3人結(jié)直腸癌腫瘤組織中G6PD、PKM2表達(dá)升高qRT-PCR和Western blot結(jié)果顯示,人結(jié)直腸癌腫瘤組織中G6PD、PKM2 mRNA和蛋白表達(dá)均高于癌旁正常組織(P0.05)。4人結(jié)直腸癌細(xì)胞中G6PD、PKM2和HK表達(dá)及活性升高Western blot結(jié)果顯示,HCT116、HT-29和Caco-2三種人結(jié)腸癌腫瘤細(xì)胞中G6PD、PKM2蛋白表達(dá)高于FHC正常結(jié)直腸粘膜細(xì)胞(P0.05);細(xì)胞免疫熒光結(jié)果顯示,人結(jié)腸癌腫瘤細(xì)胞中PKM2蛋白表達(dá)量高于正常結(jié)直腸粘膜細(xì)胞(P0.05);并且三種腫瘤細(xì)胞中己糖激酶(HK)活性明顯高于正常結(jié)直腸粘膜細(xì)胞(P0.05)。結(jié)論:1人結(jié)直腸癌組織細(xì)胞中p16、p21蛋白表達(dá)下調(diào),SA-β-Gal活性降低,細(xì)胞活力升高;2人結(jié)直腸癌組織細(xì)胞中,糖酵解途徑關(guān)鍵酶G6PD、PKM2和HK的表達(dá)及活性升高;3人結(jié)直腸癌組織細(xì)胞具有更強(qiáng)的抗衰老生存能力,糖酵解代謝增強(qiáng)。
[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- 偽 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- 偽 induced decrease of SA- 尾 -Gal activity in human colon cancer cells. TNF- 偽 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- 偽 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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34

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本文編號(hào)：1927271