【摘要】：前列腺癌是常見的男性泌尿系腫瘤,在美國其發(fā)病率已經(jīng)超過肺癌,成為第一位危害男性健康的腫瘤。晚期前列腺癌常因發(fā)生遠處轉(zhuǎn)移并容易產(chǎn)生化學(xué)耐藥和雄激素抵抗而發(fā)展為去勢抵抗性前列腺癌。異黏蛋白(metadherin,MTDH)是一個與腫瘤發(fā)生、發(fā)展密切相關(guān)的癌基因,參與調(diào)節(jié)腫瘤細胞增殖、侵襲轉(zhuǎn)移、血管生成、抵抗凋亡和化學(xué)耐藥等生物學(xué)行為,其在前列腺癌組織中的表達明顯高于前列腺增生和正常前列腺組織。腫瘤中不僅存在基因異常,同時還具有能量代謝異常,偏好有氧糖酵解方式產(chǎn)生能量是腫瘤的一個重要特征。但在前列腺癌中異常表達的MTDH是否與腫瘤細胞糖酵解有關(guān)尚不是十分清楚。因此,本研究擬利用藥物抑制MTDH的表達和小干擾RNA技術(shù)(small interfering RNA,si RNA)下調(diào)MTDH的表達,觀察其對前列腺癌細胞糖酵解的影響及其分子作用機制,為MTDH作為前列腺癌的潛在治療靶點提供理論和實驗依據(jù)。隱丹參酮抑制MTDH表達對前列腺癌DU145細胞中糖酵解的影響目的:探討隱丹參酮抑制MTDH表達對前列腺癌DU145細胞中糖酵解的影響。方法:不同濃度隱丹參酮作用DU145細胞不同時間后,MTT檢測各組細胞的增殖情況;葡萄糖和乳酸檢測試劑盒分別檢測各組細胞葡萄糖消耗值及乳酸分泌值;Western blot和逆轉(zhuǎn)錄PCR(RT-PCR)檢測各組細胞中MTDH和丙酮酸激酶M2(PKM2)的蛋白和m RNA表達情況。結(jié)果:隱丹參酮能夠抑制DU145細胞的增殖,并且呈明顯時間和劑量依賴性(P0.05)。隨著隱丹參酮濃度的增加,DU145細胞對葡萄糖的消耗值減少,乳酸分泌值也下降(P0.05)。MTDH和PKM2蛋白的表達可隨隱丹參酮濃度的增加及作用時間的延長逐漸下調(diào),而且隨著作用時間的延長,MTDH和PKM2 m RNA的表達量也逐漸下降(P0.05)。結(jié)論:隱丹參酮能夠抑制前列腺癌DU145細胞的增殖及其糖酵解水平,其可能機制是通過下調(diào)MTDH和PKM2的表達,進而抑制細胞的糖酵解途徑,最終抑制腫瘤細胞的增殖。sh RNA下調(diào)MTDH表達對前列腺癌DU145細胞中糖酵解的影響目的:探討利用shRNA下調(diào)MTDH表達后對前列腺癌DU145細胞中糖酵解的影響。方法:建立穩(wěn)定沉默MTDH的前列腺癌細胞株和空載體細胞株,倒置顯微鏡下觀察細胞形態(tài)的改變,細胞免疫組化、Western blot和逆轉(zhuǎn)錄PCR技術(shù)檢測干擾效果;MTT檢測MTDH基因下調(diào)對DU145細胞增殖的影響;葡萄糖和乳酸檢測試劑盒分別檢測MTDH基因下調(diào)后細胞葡萄糖消耗值和乳酸分泌值;Western blot檢測MTDH基因下調(diào)后細胞中AKT、p-AKT、PKM2蛋白的表達情況,逆轉(zhuǎn)錄PCR檢測PKM2 m RNA的表達水平。結(jié)果:MTDH基因下調(diào)后,細胞形態(tài)變圓、間距變大,細胞的增殖能力顯著下降(P0.05)。細胞對葡萄糖消耗和乳酸分泌也明顯減少(P0.05)。P-AKT和PKM2蛋白的表達下降,而AKT蛋白的表達無明顯改變,且PKM2 m RNA的表達也相對下調(diào)(P0.05)。結(jié)論:MTDH基因表達下調(diào)可抑制前列腺癌DU145細胞的增殖和糖酵解水平,并且我們發(fā)現(xiàn)下調(diào)MTDH基因表達可以抑制PI3K/AKT信號通路,下調(diào)PKM2的表達,從而抑制前列腺癌DU145的糖酵解途徑。
[Abstract]:Prostate cancer is a common male urinary tract tumor, whose incidence has exceeded lung cancer in the United States, becoming the first tumor to risk male health. Advanced prostate cancer is often developed as castration-resistant prostate cancer due to distant metastasis and susceptibility to chemical resistance and androgen resistance. metadherin (mtdh) is an oncogene closely related to tumorigenesis and development, and is involved in regulating the biological behaviors of tumor cell proliferation, invasion and metastasis, angiogenesis, resistance to apoptosis and chemical resistance, Its expression in prostate cancer tissue is significantly higher than that of prostate hyperplasia and normal prostate tissue. There is not only gene abnormality in the tumor, but also the abnormal metabolism of energy metabolism. It is an important feature of the tumor to generate energy by preference for aerobic glycolysis. However, it is not very clear whether MTDH abnormally expressed in prostate cancer is related to tumor cell glycolysis. Therefore, it is proposed to use drugs to inhibit MTDH expression and small interfering RNA (si RNA) down-regulate the expression of MTDH, observe its effect on the glycolysis of prostate cancer cells and its molecular mechanism, and provide theoretical and experimental basis for the potential therapeutic targets of MTDH as prostate cancer. Objective: To investigate the effect of cryptotanshinone on glycolysis in DU145 cells of prostate cancer by inhibiting the expression of MTDH in prostate cancer DU145 cells. Methods: After different concentrations of cryptotanshinone on DU145 cells, MTT was used to detect the proliferation of each group, and glucose and lactic acid detection kits were used to detect glucose consumption and lactic acid secretion in each group. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of MTDH and pyruvate kinase M2 (PKM2) in each group. Results: Cryptotanshinone could inhibit the proliferation of DU145 cells, and showed significant time and dose-dependent (P <0.05). With the increase of cryptotanshinone concentration, the depletion of glucose in DU145 cells decreased and the secretion of lactic acid decreased (P0.05). The expression of MTDH and PKM2 protein could decrease gradually with the increase of cryptotanshinone concentration and the prolongation of action time, and with the prolongation of action time, The expression of MTDH and PKM2 mRNA decreased gradually (P 0.05). Conclusion: cryptotanshinone can inhibit the proliferation of prostate cancer DU145 cells and its sugar level. The possible mechanism is to decrease the expression of MTDH and PKM2, so as to inhibit the glycolytic pathway of the cells and finally inhibit the proliferation of tumor cells. Effect of sh RNA down-regulation of MTDH expression on glycolysis in prostate cancer DU145 cells: To investigate the effect of MTDH expression on glycolysis in prostate cancer DU145 cells. Methods: To establish a stable silencing MTDH prostate cancer cell line and an empty vector cell line, observe the changes of cell morphology, cell immunohistochemistry, Western blot and reverse transcription polymerase chain reaction (RT-PCR) under an inverted microscope, and detect the effect of MTDH gene down-regulation on the proliferation of DU145 cells. The expression of AKT, p-AKT and PKM2 protein was detected by Western blot, and the expression level of PKM2 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results: After the down-regulation of the MTDH gene, the cell morphology became round, the interval became big, and the proliferation ability of the cells decreased significantly (P0.05). There was no significant change in the expression of AKT and PKM2 protein, but the expression of PKM2 mRNA was also decreased (P <0.05). Conclusion: The down-regulation of MTDH gene can inhibit the proliferation and glycolysis of prostate cancer DU145 cells, and we find that the down-regulation of MTDH gene expression can inhibit the expression of p38/ AKT signaling pathway and down-regulate the expression of PKM2, so as to inhibit the glycolysis pathway of prostate cancer DU145.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.25

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1 鄧年豐;馮云枝;;舌鱗狀細胞癌組織中EphA7和MTDH表達及其臨床病理意義[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2011年12期

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本文編號：2270223