本文關(guān)鍵詞： 乳腺癌 化療耐藥 阿霉素 葡萄糖轉(zhuǎn)運蛋白1 耐藥逆轉(zhuǎn)　出處：《南京大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的:觀察人乳腺癌耐阿霉素細(xì)胞株相對于其親本細(xì)胞株的葡萄糖攝取率、GLUT1的變化,探究利用藥物抑制人乳腺癌耐藥細(xì)胞株GLUT1后對細(xì)胞株對阿霉素敏感性的變化,并嘗試探索其作用機制。背景:乳腺癌已經(jīng)成為當(dāng)今全世界女性惡性腫瘤中新發(fā)病率和死亡率最高的疾病。包括乳腺癌患者在內(nèi),約有90%的癌癥病人治療失敗應(yīng)當(dāng)歸因于化療耐藥。癌細(xì)胞有氧糖酵解效應(yīng)是公認(rèn)的癌細(xì)胞的最重要區(qū)別于正常組織來源細(xì)胞的特征之一,其能量的產(chǎn)生相對正常細(xì)胞需要消耗更大量的葡萄糖。許多研究揭示了高葡萄糖消耗及GLUTs的表達與惡性腫瘤的高組織學(xué)級別之間具有相關(guān)性,并且與臨床預(yù)后相對較差密切關(guān)聯(lián);眾多研究直接表明有氧糖酵解和GLUTs的過表達與惡性腫瘤的化療耐藥、放療耐受存在相關(guān)性。方法:運用濃度梯度遞增誘導(dǎo)法構(gòu)建人乳腺癌耐阿霉素細(xì)胞株MCF-7/ADR;分別評價阿霉素濃度梯度、WWZB117濃度梯度作用72h下耐藥株與親本株的IC50,檢測兩細(xì)胞株自然生長狀態(tài)下72h的葡萄糖消耗,分析兩細(xì)胞株的葡萄糖消耗率、生長速度,比較耐藥株和親本株的GLUT1表達差異。根據(jù)72h的WZB117單藥IC50,選擇低毒性的WZB117藥物濃度聯(lián)合阿霉素濃度梯度,處理耐阿霉素細(xì)胞株72h,測定聯(lián)合用藥情況下的阿霉素IC50變化情況。將耐藥細(xì)胞株分別于0.5 μg/ml阿霉素單藥、10μMWZB117單藥、0.5μg/ml阿霉素和10μMWZB117聯(lián)合用藥單獨處理72h,同陰性無藥物處理組比較細(xì)胞株的凋亡率、遷移能力、生長能力、葡萄糖攝取率、乳酸產(chǎn)生量的差異;并分析以上四組細(xì)胞于48h處理時長時的AMPK/mTOR信號通路和BCL-2蛋白家族的變化情況。結(jié)果:1、濃度梯度遞增誘導(dǎo)法構(gòu)建人乳腺癌耐阿霉素細(xì)胞株MCF-7/ADR阿霉素的IC50 為(21.88 ± 1.0)μ g/ml,MCF-7 為(0.29 ± 0.1)μ g/ml,RI 為 75.44;耐藥細(xì)胞株的第2、3d生長速度、葡萄糖攝取率高于親本細(xì)胞株(p0.05);耐藥細(xì)胞株的GLUT1表達水平也顯著高于親本細(xì)胞株。2、72h 條件下 WZB117 作用于 MCF-7/ADR 的 IC50 為 257.04 ± 3.4μ M;5μM 的 WZB117 聯(lián)合阿霉素 72h 的 IC50 為 8.07 ± 0.9μg/ml,10μM 的 WZB117聯(lián)合阿霉素72h的IC50為1.18 ± 0.8 μ g/ml,RF分別為2.71和18.54,使耐藥性顯著逆轉(zhuǎn)(P0.01)。聯(lián)合應(yīng)用兩種藥物能夠顯著增加癌細(xì)胞的凋亡率(P0.01);阿霉素單藥組、聯(lián)合用藥組的細(xì)胞增殖速度均顯著低于陰性對照組(P0.01),但阿霉素單藥組于3d時的生長速度明顯提升、高于聯(lián)合用藥組(P0.01);WZB117單藥組、聯(lián)合用藥組較長作用時間下(2周)的肉眼可見細(xì)胞集落數(shù)目均顯著小于陰性對照組、阿霉素單藥組(P0.01);阿霉素的對耐藥株遷移能力的抑制作用要顯著強于WZB117(P0.05),但兩種藥物聯(lián)合應(yīng)用時細(xì)胞遷移抑制能力并未表現(xiàn)出進一步增強。3、72h的WZB117單藥或聯(lián)合用藥作用下,MCF-7/ADR的葡萄糖消耗量均顯著低于陰性對照組(P0.05);阿霉素單藥對癌細(xì)胞的葡萄糖消耗并未顯示出顯著影響;但是阿霉素單藥作用于MCF-7/ADR則會顯著增加細(xì)胞的乳酸產(chǎn)生量(P0.01),聯(lián)合阿霉素和WZB117則會顯著抑制乳酸的產(chǎn)生(P0.01)。處理48h時,WZB117單藥組、聯(lián)合用藥組的AMPK磷酸化活化狀態(tài)含量升高,而其下游信號分子mTOR的磷酸化失活狀態(tài)隨之升高;單獨阿霉素處理并未表現(xiàn)出類似現(xiàn)象;阿霉素、WZB117單藥組和聯(lián)合用藥組均導(dǎo)致BAX轉(zhuǎn)位至線粒體上,但是WZB117單藥、聯(lián)合用藥組的促進BAX轉(zhuǎn)移至線粒體的能力要強于阿霉素單藥組,細(xì)胞內(nèi)BCL-2蛋白總體表達水平并未因兩種藥物的處理而出現(xiàn)顯著改變。結(jié)論:1、濃度梯度遞增誘導(dǎo)法構(gòu)建的人乳腺癌耐阿霉素細(xì)胞株MCF-7/ADR較其親本細(xì)胞株耐受阿霉素的IC50更高,并且生長速度、葡萄糖攝取量顯著高于親本細(xì)胞株,GLUT1的表達量也顯著高于親本細(xì)胞株。2、通過藥物WZB117的低毒性濃度來抑制人乳腺癌耐阿霉素細(xì)胞株MCF-7/ADR的GLUT1的功能,可以顯著提升阿霉素對耐藥細(xì)胞株的細(xì)胞毒性、顯著降低其IC50,達到部分逆轉(zhuǎn)耐藥性的目的。3、WZB117可以抑制人乳腺癌耐阿霉素細(xì)胞株MCF-7/ADR遭受阿霉素作用時的有氧糖酵解效應(yīng),其提升阿霉素對耐藥細(xì)胞株的細(xì)胞毒性的分子機制可能與AMPK/mTOR信號通路激活、BAX蛋白向線粒體移位有關(guān)。
[Abstract]:Objective: To observe the human breast cancer cell line resistant to doxorubicin and its parent cell line relative to the glucose uptake rate, the change of GLUT1, to explore the use of drugs that inhibit human breast cancer cell line GLUT1 after the change on adriamycin sensitivity of cell lines, and to explore its mechanism. Background: breast cancer has become the new disease incidence rate in today's world and the mortality of female malignant tumor. Including the highest breast cancer patients, and some should be attributed to the failure of chemotherapy in treatment of cancer patients and 90% cancer cells. Aerobic glycolysis effect is the most important difference between the putative cancer cell characteristics in normal tissue derived cells of the energy produced relatively normal cells need to consume more glucose. Many studies revealed a high expression of high glucose consumption and GLUTs with malignant tumors was associated with histological grade, and Closely associated with the clinical prognosis is relatively poor; directly showed that over expression of resistance to chemotherapy of malignant tumor and aerobic glycolysis and GLUTs studies, the correlation between radiation tolerance. Methods: using concentration gradient method to construct induced resistance to adriamycin in human breast cancer cell line MCF-7/ADR respectively; evaluation of adriamycin concentration gradient, WWZB117 concentration gradient 72h drug resistant strains and parental strain IC50, glucose detection two cells grown under natural environment 72h consumption, analysis of two cells, glucose consumption rate, growth rate, difference between resistant strains and parental strain GLUT1 expression. According to the 72h WZB117 single drug IC50, WZB117 concentration of adriamycin concentration gradient of low toxicity. Treatment of adriamycin resistant cell line 72h, IC50 changes the combination determination of doxorubicin conditions. The resistant cell lines respectively in 0.5 g/ml doxorubicin monotherapy, 10 MWZB117 Single drug, 0.5 g/ml and 10 MWZB117 of adriamycin in combination with 72h treatment alone, no drug treatment group compared the apoptosis of negative cell line rate, migration ability, growth ability, glucose uptake, lactate production difference; changes in 48h processing time when the AMPK/mTOR signaling pathway and BCL-2 protein family and analysis of the above four groups of cells. Results: 1, increasing the concentration gradient induced by construction of human breast cancer cell line MCF-7/ADR resistant to adriamycin adriamycin IC50 (21.88 + 1) g/ml, MCF-7 (0.29 + 0.1) g/ml, RI is 75.44; the 2,3d resistant cell line growth rate, glucose uptake rate was higher than the parent cell line (P0.05); the expression of multidrug resistant cell line GLUT1 was significantly higher than that of IC50 WZB117.2,72h in MCF-7/ADR cells. Under the condition of 257.04 + 3.4 M; 5 M WZB117 72h 8.07 IC50 combined with adriamycin + 0.9 g/ml, 10 M WZB117 72h IC50 combined with adriamycin was 1.18 + 0.8 g/ml, RF were 2.71 and 18.54, the drug resistance reversed significantly (P0.01). The combination of two drugs can significantly increase the apoptosis rate (P0.01); doxorubicin monotherapy group, cell proliferation velocity the treatment group were significantly lower than the negative control group (P0.01), but the growth rate of doxorubicin monotherapy group 3D improved significantly, higher than the combination group (P0.01); WZB117 group, combination group long action time (2 weeks) visible cell colony number was significantly lower than the negative control group adriamycin, single drug group (P0.01); inhibitory effect of adriamycin on the migration of resistant strains was much higher than WZB117 (P0.05), but the two kinds of combination of drugs when cell migration inhibition did not show enhanced WZB117 monotherapy or combination therapy under.3,72h, MCF-7/AD The consumption of R glucose was significantly lower than the negative control group (P0.05); glucose doxorubicin monotherapy on cancer cells did not show significant effects of consumption; but doxorubicin monotherapy in MCF-7/ADR would significantly increase cell production of lactic acid (P0.01), adriamycin and WZB117 will produce significant inhibition of lactic acid (P0.01). The treatment of 48h, WZB117 group, combination group AMPK phosphorylation activation was increased, and its downstream signal molecule mTOR phosphorylation state inactivation increased; single adriamycin treatment did not show a similar phenomenon; adriamycin, WZB117 monotherapy group and combination group were resulted in the translocation of BAX to mitochondria however, WZB117 monotherapy, the combination group BAX to promote the transfer to mitochondria is stronger than doxorubicin monotherapy group, intracellular BCL-2 protein expression level was not due to overall treatment of two kinds of drugs and the emergence of significant Change. Conclusion: 1. Increasing the concentration gradient induced by the construction of human breast cancer cell line MCF-7/ADR resistant to adriamycin than the parental cell tolerance of adriamycin IC50 is higher, and the growth rate, glucose uptake was significantly higher than that of the parental cell line, the expression of GLUT1 was significantly higher than that of the parent cell line.2 by WZB117, drug toxicity the concentration of inhibition of human breast cancer cell line MCF-7/ADR in adriamycin resistant GLUT1 function, can significantly enhance the cytotoxicity of adriamycin resistant cell lines, reduce the IC50, achieved a partial reversal of drug resistance to.3, WZB117 can inhibit human breast cancer cell line MCF-7/ADR by adriamycin adriamycin resistant effect of aerobic glycolysis effect the molecular mechanism of cell toxicity, enhance adriamycin on multidrug resistance cell line might activate AMPK/mTOR signaling pathway, BAX protein to mitochondria translocation.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9
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本文編號：1487861