葡萄糖转运蛋白1与乳腺癌耐药性相关性研究

发布时间：2018-02-03 16:41

  本文关键词： 乳腺癌 化疗耐药 阿霉素 葡萄糖转运蛋白1 耐药逆转　出处：《南京大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:观察人乳腺癌耐阿霉素细胞株相对于其亲本细胞株的葡萄糖摄取率、GLUT1的变化,探究利用药物抑制人乳腺癌耐药细胞株GLUT1后对细胞株对阿霉素敏感性的变化,并尝试探索其作用机制。背景:乳腺癌已经成为当今全世界女性恶性肿瘤中新发病率和死亡率最高的疾病。包括乳腺癌患者在内,约有90%的癌症病人治疗失败应当归因于化疗耐药。癌细胞有氧糖酵解效应是公认的癌细胞的最重要区别于正常组织来源细胞的特征之一,其能量的产生相对正常细胞需要消耗更大量的葡萄糖。许多研究揭示了高葡萄糖消耗及GLUTs的表达与恶性肿瘤的高组织学级别之间具有相关性,并且与临床预后相对较差密切关联;众多研究直接表明有氧糖酵解和GLUTs的过表达与恶性肿瘤的化疗耐药、放疗耐受存在相关性。方法:运用浓度梯度递增诱导法构建人乳腺癌耐阿霉素细胞株MCF-7/ADR;分别评价阿霉素浓度梯度、WWZB117浓度梯度作用72h下耐药株与亲本株的IC50,检测两细胞株自然生长状态下72h的葡萄糖消耗,分析两细胞株的葡萄糖消耗率、生长速度,比较耐药株和亲本株的GLUT1表达差异。根据72h的WZB117单药IC50,选择低毒性的WZB117药物浓度联合阿霉素浓度梯度,处理耐阿霉素细胞株72h,测定联合用药情况下的阿霉素IC50变化情况。将耐药细胞株分别于0.5 μg/ml阿霉素单药、10μMWZB117单药、0.5μg/ml阿霉素和10μMWZB117联合用药单独处理72h,同阴性无药物处理组比较细胞株的凋亡率、迁移能力、生长能力、葡萄糖摄取率、乳酸产生量的差异;并分析以上四组细胞于48h处理时长时的AMPK/mTOR信号通路和BCL-2蛋白家族的变化情况。结果:1、浓度梯度递增诱导法构建人乳腺癌耐阿霉素细胞株MCF-7/ADR阿霉素的IC50 为(21.88 ± 1.0)μ g/ml,MCF-7 为(0.29 ± 0.1)μ g/ml,RI 为 75.44;耐药细胞株的第2、3d生长速度、葡萄糖摄取率高于亲本细胞株(p0.05);耐药细胞株的GLUT1表达水平也显著高于亲本细胞株。2、72h 条件下 WZB117 作用于 MCF-7/ADR 的 IC50 为 257.04 ± 3.4μ M;5μM 的 WZB117 联合阿霉素 72h 的 IC50 为 8.07 ± 0.9μg/ml,10μM 的 WZB117联合阿霉素72h的IC50为1.18 ± 0.8 μ g/ml,RF分别为2.71和18.54,使耐药性显著逆转(P0.01)。联合应用两种药物能够显著增加癌细胞的凋亡率(P0.01);阿霉素单药组、联合用药组的细胞增殖速度均显著低于阴性对照组(P0.01),但阿霉素单药组于3d时的生长速度明显提升、高于联合用药组(P0.01);WZB117单药组、联合用药组较长作用时间下(2周)的肉眼可见细胞集落数目均显著小于阴性对照组、阿霉素单药组(P0.01);阿霉素的对耐药株迁移能力的抑制作用要显著强于WZB117(P0.05),但两种药物联合应用时细胞迁移抑制能力并未表现出进一步增强。3、72h的WZB117单药或联合用药作用下,MCF-7/ADR的葡萄糖消耗量均显著低于阴性对照组(P0.05);阿霉素单药对癌细胞的葡萄糖消耗并未显示出显著影响;但是阿霉素单药作用于MCF-7/ADR则会显著增加细胞的乳酸产生量(P0.01),联合阿霉素和WZB117则会显著抑制乳酸的产生(P0.01)。处理48h时,WZB117单药组、联合用药组的AMPK磷酸化活化状态含量升高,而其下游信号分子mTOR的磷酸化失活状态随之升高;单独阿霉素处理并未表现出类似现象;阿霉素、WZB117单药组和联合用药组均导致BAX转位至线粒体上,但是WZB117单药、联合用药组的促进BAX转移至线粒体的能力要强于阿霉素单药组,细胞内BCL-2蛋白总体表达水平并未因两种药物的处理而出现显著改变。结论:1、浓度梯度递增诱导法构建的人乳腺癌耐阿霉素细胞株MCF-7/ADR较其亲本细胞株耐受阿霉素的IC50更高,并且生长速度、葡萄糖摄取量显著高于亲本细胞株,GLUT1的表达量也显著高于亲本细胞株。2、通过药物WZB117的低毒性浓度来抑制人乳腺癌耐阿霉素细胞株MCF-7/ADR的GLUT1的功能,可以显著提升阿霉素对耐药细胞株的细胞毒性、显著降低其IC50,达到部分逆转耐药性的目的。3、WZB117可以抑制人乳腺癌耐阿霉素细胞株MCF-7/ADR遭受阿霉素作用时的有氧糖酵解效应,其提升阿霉素对耐药细胞株的细胞毒性的分子机制可能与AMPK/mTOR信号通路激活、BAX蛋白向线粒体移位有关。
[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.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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本文编号：1487861