本文選題：線粒體 + 氧化磷酸化　； 參考：《吉林大學》2017年碩士論文

【摘要】：肺癌發(fā)生于支氣管粘膜上皮,是當今世界最常見的惡性腫瘤,其發(fā)病率和死亡率逐年上升,且肺癌的病因復雜,且尚未明晰。由于我國近年來工業(yè)化和城鎮(zhèn)化高速發(fā)展,許多地方空氣污染嚴重,國際癌癥研究機構的研究發(fā)現環(huán)境的PM2.5升高與肺癌發(fā)生呈正相關�，F如今,空氣污染對人們健康的損害已引起中國社會越來越多的關注,與之相關的肺癌發(fā)病和發(fā)展機制的研究也越來越引起人們的關注。肺癌分為鱗狀細胞癌、腺癌、腺鱗癌、小細胞癌、大細胞癌和肉瘤樣癌等六個基本類型,近年來,統(tǒng)計資料表明肺腺癌的發(fā)病率有明顯升高趨勢。目前,肺癌的治療主要采取以手術根治性切除為主,化學療法和放射療法為輔的綜合性治療。隨著醫(yī)療技術的發(fā)展與進步,分子靶向治療、介入治療、免疫治療等技術在肺癌治療上也發(fā)揮著越來越重要的作用。然而,對于肺腺癌來說,其臨床治療效果及預后不如鱗癌,手術切除后五年存活率不到10%,遠處轉移是肺腺癌患者死亡的主要原因。有研究表明,線粒體可能參與腫瘤的發(fā)生、發(fā)展。因此,本研究通過MTT法、免疫組織化學法、劃痕實驗、組織化學法和Mito-tracker Green線粒體特異性標記等方法來探討線粒體在肺腺癌細胞及肺腺癌細胞系A549遷移過程中的作用。方法:本研究分為體內實驗和體外實驗兩部分。體內實驗,于吉林省腫瘤醫(yī)院收集肺腺癌組織,常規(guī)石蠟包埋、切片,石蠟切片厚5μm,進行琥珀酸脫氫酶的免疫組織化學染色。體外實驗,常規(guī)培養(yǎng)A549肺腺癌細胞,加入抗霉素A(線粒體功能抑制劑)、三溴丙酮酸(糖酵解功能抑制劑)及丙酮酸鈉,利用MTT法確定藥物最佳作用濃度,用此藥物濃度分別處理A549細胞。劃痕實驗檢測細胞遷移能力的差異,組織化學法檢測琥珀酸脫氫酶、乳酸脫氫酶的活性差異,Mito-tracker Green線粒體特異性標記法檢測線粒體長度變化情況,免疫組織化學法檢測細胞內線粒體分裂蛋白的表達差異。結果體內實驗:免疫組織化學染色結果為:高分化肺腺癌組織琥珀酸脫氫酶表達低于低分化肺腺癌組織,差異顯著(P0.05);高分化肺腺癌癌巢周邊的癌細胞內琥珀酸脫氫酶表達低于低分化肺腺癌癌巢周邊的癌細胞,差異極顯著(P0.001)。體外實驗:MTT法檢測抗霉素A的最佳藥物濃度為150μM,三溴丙酮酸的最佳藥物濃度為200μM,丙酮酸鈉的最佳藥物濃度為0.02M且與三溴丙酮酸同時加入時作用效果最好。劃痕實驗檢測結果顯示:劃痕48h時,抗霉素A組細胞遷移距離(125.6±30.01μm)與對照組遷移距離(584.1±59.70μm)相比差異極顯著(P0.0001),三溴丙酮酸組細胞遷移距離(322.4±29.16μm)與對照組相比,差異極顯著(P0.001),三溴丙酮酸與丙酮酸鈉聯(lián)合用藥其細胞遷移距離(521.1±32.92μm)與三溴丙酮酸單獨作用組相比,差異顯著(P0.01),抗霉素A組細胞遷移距離與三溴丙酮酸組相比,差異極顯著(P0.001),三溴丙酮酸+丙酮酸鈉組細胞遷移距離與對照組相比,無顯著差異(P0.05)。組織化學法乳酸脫氫酶相對活性檢測結果顯示:當遷移48h時,三溴丙酮酸組乳酸脫氫酶相對活性(0.006±0.003)與對照組(0.008±0.004)相比,無顯著差異(P0.05),三溴丙酮酸+丙酮酸鈉組乳酸脫氫酶相對活性(0.007±0.004)與三溴丙酮酸組相比,無顯著差異(P0.05),抗霉素A組乳酸脫氫酶相對活性(0.020±0.002)高于對照組,差異顯著(P0.01)。組織化學法琥珀酸脫氫酶相對活性檢測結果顯示:當細胞遷移48h時,抗霉素A組琥珀酸脫氫酶相對活性(0.002±0.001)與對照組(0.062±0.008)相比,差異極顯著(P0.0001),三溴丙酮酸組琥珀酸脫氫酶相對活性(0.030±0.007)與對照組相比,差異極顯著(P0.0001),三溴丙酮酸+丙酮酸鈉組細胞的琥珀酸脫氫酶相對活性(0.057±0.005)高于三溴丙酮酸單獨作用組,且差異極顯著(P0.0001),三溴丙酮酸+丙酮酸鈉組琥珀酸脫氫酶相對活性與對照組相比,差異不顯著(P0.05)。Mito-tracker Green線粒體特異性標記法檢測偽足內線粒體長度結果顯示:當遷移48h時,抗霉素A組細胞線粒體長度(1.257±0.257μm)明顯低于對照組細胞線粒體長度(27.88±6.209μm),差異極顯著(P0.0001),三溴丙酮酸組細胞線粒體長度(6.569±2.311μm)與對照組相比,差異極顯著(P0.0001),三溴丙酮酸+丙酮酸鈉組細胞線粒體長度(21.95±6.661μm)與對照組相比,差異不顯著(P0.05)。免疫組織化學染色法檢測線粒體分裂蛋白表達結果顯示:當遷移48h時,抗霉素A組細胞線粒體分裂蛋白表達光密度(0.172±0.011)明顯高于對照組細胞線粒體分裂蛋白表達光密度(0.038±0.008),差異極顯著(P0.0001),三溴丙酮酸組細胞線粒體分裂蛋白表達光密度(0.078±0.006)與對照組相比,差異顯著(P0.01),三溴丙酮酸+丙酮酸鈉組細胞線粒體分裂蛋白表達光密度(0.047±0.004)與對照組相比,差異不顯著(P0.05)。結論:琥珀酸脫氫酶的表達提示,線粒體的功能活動與肺腺癌組織的分化程度及肺腺癌細胞的遷移有關;偽足內線粒體氧化磷酸化和糖酵解均參與肺腺癌細胞的遷移;糖酵解為線粒體氧化磷酸化提供丙酮酸,線粒體通過氧化磷酸化的方式為遷移的肺腺癌細胞提供能量;抑制線粒體氧化磷酸化,線粒體分裂增強,肺腺癌細胞的遷移能力下降。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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