线粒体为肺腺癌细胞迁移提供能量的研究

发布时间：2018-05-28 06:46

本文选题：线粒体 + 氧化磷酸化　；参考：《吉林大学》2017年硕士论文

【摘要】：肺癌发生于支气管粘膜上皮,是当今世界最常见的恶性肿瘤,其发病率和死亡率逐年上升,且肺癌的病因复杂,且尚未明晰。由于我国近年来工业化和城镇化高速发展,许多地方空气污染严重,国际癌症研究机构的研究发现环境的PM2.5升高与肺癌发生呈正相关。现如今,空气污染对人们健康的损害已引起中国社会越来越多的关注,与之相关的肺癌发病和发展机制的研究也越来越引起人们的关注。肺癌分为鳞状细胞癌、腺癌、腺鳞癌、小细胞癌、大细胞癌和肉瘤样癌等六个基本类型,近年来,统计资料表明肺腺癌的发病率有明显升高趋势。目前,肺癌的治疗主要采取以手术根治性切除为主,化学疗法和放射疗法为辅的综合性治疗。随着医疗技术的发展与进步,分子靶向治疗、介入治疗、免疫治疗等技术在肺癌治疗上也发挥着越来越重要的作用。然而,对于肺腺癌来说,其临床治疗效果及预后不如鳞癌,手术切除后五年存活率不到10%,远处转移是肺腺癌患者死亡的主要原因。有研究表明,线粒体可能参与肿瘤的发生、发展。因此,本研究通过MTT法、免疫组织化学法、划痕实验、组织化学法和Mito-tracker Green线粒体特异性标记等方法来探讨线粒体在肺腺癌细胞及肺腺癌细胞系A549迁移过程中的作用。方法:本研究分为体内实验和体外实验两部分。体内实验,于吉林省肿瘤医院收集肺腺癌组织,常规石蜡包埋、切片,石蜡切片厚5μm,进行琥珀酸脱氢酶的免疫组织化学染色。体外实验,常规培养A549肺腺癌细胞,加入抗霉素A(线粒体功能抑制剂)、三溴丙酮酸(糖酵解功能抑制剂)及丙酮酸钠,利用MTT法确定药物最佳作用浓度,用此药物浓度分别处理A549细胞。划痕实验检测细胞迁移能力的差异,组织化学法检测琥珀酸脱氢酶、乳酸脱氢酶的活性差异,Mito-tracker Green线粒体特异性标记法检测线粒体长度变化情况,免疫组织化学法检测细胞内线粒体分裂蛋白的表达差异。结果体内实验:免疫组织化学染色结果为:高分化肺腺癌组织琥珀酸脱氢酶表达低于低分化肺腺癌组织,差异显著(P0.05);高分化肺腺癌癌巢周边的癌细胞内琥珀酸脱氢酶表达低于低分化肺腺癌癌巢周边的癌细胞,差异极显著(P0.001)。体外实验:MTT法检测抗霉素A的最佳药物浓度为150μM,三溴丙酮酸的最佳药物浓度为200μM,丙酮酸钠的最佳药物浓度为0.02M且与三溴丙酮酸同时加入时作用效果最好。划痕实验检测结果显示:划痕48h时,抗霉素A组细胞迁移距离(125.6±30.01μm)与对照组迁移距离(584.1±59.70μm)相比差异极显著(P0.0001),三溴丙酮酸组细胞迁移距离(322.4±29.16μm)与对照组相比,差异极显著(P0.001),三溴丙酮酸与丙酮酸钠联合用药其细胞迁移距离(521.1±32.92μm)与三溴丙酮酸单独作用组相比,差异显著(P0.01),抗霉素A组细胞迁移距离与三溴丙酮酸组相比,差异极显著(P0.001),三溴丙酮酸+丙酮酸钠组细胞迁移距离与对照组相比,无显著差异(P0.05)。组织化学法乳酸脱氢酶相对活性检测结果显示:当迁移48h时,三溴丙酮酸组乳酸脱氢酶相对活性(0.006±0.003)与对照组(0.008±0.004)相比,无显著差异(P0.05),三溴丙酮酸+丙酮酸钠组乳酸脱氢酶相对活性(0.007±0.004)与三溴丙酮酸组相比,无显著差异(P0.05),抗霉素A组乳酸脱氢酶相对活性(0.020±0.002)高于对照组,差异显著(P0.01)。组织化学法琥珀酸脱氢酶相对活性检测结果显示:当细胞迁移48h时,抗霉素A组琥珀酸脱氢酶相对活性(0.002±0.001)与对照组(0.062±0.008)相比,差异极显著(P0.0001),三溴丙酮酸组琥珀酸脱氢酶相对活性(0.030±0.007)与对照组相比,差异极显著(P0.0001),三溴丙酮酸+丙酮酸钠组细胞的琥珀酸脱氢酶相对活性(0.057±0.005)高于三溴丙酮酸单独作用组,且差异极显著(P0.0001),三溴丙酮酸+丙酮酸钠组琥珀酸脱氢酶相对活性与对照组相比,差异不显著(P0.05)。Mito-tracker Green线粒体特异性标记法检测伪足内线粒体长度结果显示:当迁移48h时,抗霉素A组细胞线粒体长度(1.257±0.257μm)明显低于对照组细胞线粒体长度(27.88±6.209μm),差异极显著(P0.0001),三溴丙酮酸组细胞线粒体长度(6.569±2.311μm)与对照组相比,差异极显著(P0.0001),三溴丙酮酸+丙酮酸钠组细胞线粒体长度(21.95±6.661μm)与对照组相比,差异不显著(P0.05)。免疫组织化学染色法检测线粒体分裂蛋白表达结果显示:当迁移48h时,抗霉素A组细胞线粒体分裂蛋白表达光密度(0.172±0.011)明显高于对照组细胞线粒体分裂蛋白表达光密度(0.038±0.008),差异极显著(P0.0001),三溴丙酮酸组细胞线粒体分裂蛋白表达光密度(0.078±0.006)与对照组相比,差异显著(P0.01),三溴丙酮酸+丙酮酸钠组细胞线粒体分裂蛋白表达光密度(0.047±0.004)与对照组相比,差异不显著(P0.05)。结论:琥珀酸脱氢酶的表达提示,线粒体的功能活动与肺腺癌组织的分化程度及肺腺癌细胞的迁移有关;伪足内线粒体氧化磷酸化和糖酵解均参与肺腺癌细胞的迁移;糖酵解为线粒体氧化磷酸化提供丙酮酸,线粒体通过氧化磷酸化的方式为迁移的肺腺癌细胞提供能量;抑制线粒体氧化磷酸化,线粒体分裂增强,肺腺癌细胞的迁移能力下降。
[Abstract]:Lung cancer is the most common malignant tumor in the world, which is the most common malignant tumor in the world. The incidence and mortality of lung cancer are increasing year by year, and the cause of lung cancer is complex and not clear. Because of the rapid development of industrialization and urbanization in China in recent years, the air pollution is serious in many places. The research of international cancer research institutes found the PM2.5 liter of the environment. There is a positive correlation between high and lung cancer. Nowadays, the health damage of air pollution has attracted more and more attention in Chinese society. The research on the pathogenesis and development mechanism of lung cancer is becoming more and more concerned. Lung cancer is divided into six types: squamous cell carcinoma, adenocarcinoma, adenoscale, small cell carcinoma, large cell and sarcomatoid cancer. Basic types, in recent years, statistics show that the incidence of lung adenocarcinoma has an obvious tendency to rise. At present, the treatment of lung cancer mainly adopts radical resection, chemical therapy and radiotherapy combined with comprehensive treatment. With the development and progress of medical technology, molecular targeting therapy, interventional therapy, immunotherapy and other techniques in the lung It is also playing an increasingly important role in cancer treatment. However, for lung adenocarcinoma, its clinical effect and prognosis are not as good as squamous cell carcinoma. The survival rate of five years after resection is less than 10%. Distant metastasis is the main cause of death in lung adenocarcinoma. Method, immunohistochemical method, scratch test, histochemical method and Mito-tracker Green mitochondrial specific markers to explore the role of mitochondria in the migration of lung adenocarcinoma cell and lung adenocarcinoma cell line A549. Methods: This study was divided into two parts, in vivo and in vitro. In vivo, the lung was collected in Jilin tumor hospital. Adenocarcinoma tissue, routine paraffin embedding, section and paraffin section thickness of 5 mu m, immunohistochemical staining of succinic dehydrogenase. In vitro, A549 lung adenocarcinoma cells were routinely cultured, anti mycophenoid A (mitochondrial function inhibitor), three bromide pyruvic acid (glycolytic inhibitor) and sodium pyruvate, and MTT method was used to determine the optimal concentration of drugs. A549 cells were treated with the concentration of the drug. The difference of cell migration ability was detected by scratch test. The activity difference of succinic acid dehydrogenase and lactate dehydrogenase was detected by histochemical method. The mitochondrial specific labeling method was used to detect the change of mitochondrial length. The intracellular mitochondrial mitotic protein was detected by immunohistochemical method. Results the results of immunohistochemical staining in vivo: the expression of succinic dehydrogenase in highly differentiated lung adenocarcinoma tissue was lower than that of low differentiated lung adenocarcinoma (P0.05), and the expression of succinic dehydrogenase in the cancer cells around the highly differentiated lung adenocarcinoma nests was significantly lower than that in the periphery of the poorly differentiated lung adenocarcinoma nests. P0.001) in vitro: the best drug concentration of anti mycin A by MTT method was 150 M, the best drug concentration of three bromo pyruvic acid was 200 mu M, the best concentration of sodium pyruvate was 0.02M and the effect was best when added with three bromo pyruvic acid. The scratch test results showed that the migration distance of the antimycin A group was 125.6 + when the scratch 48h was scratched. 30.01 m) was significantly different from that of the control group (584.1 + 59.70 mu m), and the cell migration distance (322.4 + 29.16 m) in the three bromide pyruvic acid group was significantly different from that of the control group (P0.001). The cell migration distance between three bromo pyruvic acid and sodium pyruvate (521.1 + 32.92 micron m) was compared with the group of three pyruvic acid alone. The difference was significant (P0.01). The cell migration distance in the antimycin A group was significantly different from that of the three bromo pyruvic acid group (P0.001). There was no significant difference between the cell migration distance of the three bromo pyruvate + sodium pyruvate group compared with the control group (P0.05). The relative viability test of the lactate dehydrogenase in the histochemical method showed that the three bromo pyruvic acid group was removed from the lactate group when the 48h was migrated. Compared with the control group (0.008 + 0.004), the relative activity of the hydrogenase (0.006 + 0.003) was not significantly different (P0.05). The relative activity of lactate dehydrogenase (0.007 + 0.004) in the three bromo pyruvate + sodium pyruvate group (0.007 + 0.004) was not significantly different from that of the three bromide pyruvate group (P0.05), and the relative activity of lactate dehydrogenase (0.020 + 0.002) in the antimycin A group was higher than that of the control group, and the difference was significant (P0.01 The relative activity of succinic dehydrogenase in the histochemical method showed that when the cells migrated 48h, the relative activity of succinic dehydrogenase in the antimycin A group (0.002 + 0.001) was significantly different from that of the control group (0.062 + 0.008), and the relative activity of succinic dehydrogenase (0.030 + 0.007) in the three bromide pyruvic acid group (0.030 + 0.007) was significantly different from that of the control group. (P0.0001) the relative activity of succinic dehydrogenase (0.057 + 0.005) in the three bromo pyruvate + sodium pyruvate group (0.057 + 0.005) was higher than that of the three bromide pyruvate alone group, and the difference was very significant (P0.0001). The relative activity of succinic dehydrogenase in the three bromo pyruvate + sodium pyruvate group was compared with the control group, and the difference was not significant (P0.05).Mito-tracker Green mitochondrial specificity The results showed that the length of mitochondria in 48h group (1.257 + 0.257 mu m) was significantly lower than that of the control group (27.88 + 6.209 m), and the length of the cell line (6.569 + 2.311, m) in the three bromo pyruvic acid group (6.569 + 2.311, m) was significantly different (P0.0). 001) the cell mitochondrial length (21.95 + 6.661 mu m) of the three bromo pyruvate + sodium pyruvate group was not significantly different from that of the control group (P0.05). The results of mitochondrial mitotic protein expression by immunohistochemical staining showed that the density of the expression of mitochondrial split protein (0.172 + 0.011) in the anti mycophenolate A group (0.172 + 0.011) was significantly higher than that of the control group when the 48h was migrated. The density of the cell mitochondrial mitotic protein (0.038 + 0.008) was very significant (P0.0001). The light density (0.078 + 0.006) of the mitochondrial mitotic protein expression in the three bromo pyruvic acid group was significantly different from the control group (P0.01), and the density of the cell mitochondrial mitotic protein (0.047 + 0.004) in the three bromo pyruvate + sodium pyruvate group (0.047 + 0.004) was compared with the control group. The difference was not significant (P0.05). Conclusion: the expression of succinic dehydrogenase suggests that the functional activity of mitochondria is related to the degree of differentiation of lung adenocarcinoma tissue and the migration of lung adenocarcinoma cells; the oxidative phosphorylation and glycolysis of the mitochondria in the pseudo foot are involved in the migration of lung adenocarcinoma cells; glycolysis for mitochondrial oxidative phosphorylation provides pyruvate and mitochondria The mode of peroxide acidification can provide energy for the migrated lung adenocarcinoma cells, inhibit mitochondrial oxidative phosphorylation, increase mitochondrial division, and decrease the migration ability of lung adenocarcinoma cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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