本文選題：線粒體 + 氧化磷酸化　； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：肺癌發(fā)生于支氣管粘膜上皮,是當(dāng)今世界最常見(jiàn)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(fù)雜,且尚未明晰。由于我國(guó)近年來(lái)工業(yè)化和城鎮(zhèn)化高速發(fā)展,許多地方空氣污染嚴(yán)重,國(guó)際癌癥研究機(jī)構(gòu)的研究發(fā)現(xiàn)環(huán)境的PM2.5升高與肺癌發(fā)生呈正相關(guān)。現(xiàn)如今,空氣污染對(duì)人們健康的損害已引起中國(guó)社會(huì)越來(lái)越多的關(guān)注,與之相關(guān)的肺癌發(fā)病和發(fā)展機(jī)制的研究也越來(lái)越引起人們的關(guān)注。肺癌分為鱗狀細(xì)胞癌、腺癌、腺鱗癌、小細(xì)胞癌、大細(xì)胞癌和肉瘤樣癌等六個(gè)基本類(lèi)型,近年來(lái),統(tǒng)計(jì)資料表明肺腺癌的發(fā)病率有明顯升高趨勢(shì)。目前,肺癌的治療主要采取以手術(shù)根治性切除為主,化學(xué)療法和放射療法為輔的綜合性治療。隨著醫(yī)療技術(shù)的發(fā)展與進(jìn)步,分子靶向治療、介入治療、免疫治療等技術(shù)在肺癌治療上也發(fā)揮著越來(lái)越重要的作用。然而,對(duì)于肺腺癌來(lái)說(shuō),其臨床治療效果及預(yù)后不如鱗癌,手術(shù)切除后五年存活率不到10%,遠(yuǎn)處轉(zhuǎn)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(guò)MTT法、免疫組織化學(xué)法、劃痕實(shí)驗(yàn)、組織化學(xué)法和Mito-tracker Green線粒體特異性標(biāo)記等方法來(lái)探討線粒體在肺腺癌細(xì)胞及肺腺癌細(xì)胞系A(chǔ)549遷移過(guò)程中的作用。方法:本研究分為體內(nèi)實(shí)驗(yàn)和體外實(shí)驗(yàn)兩部分。體內(nèi)實(shí)驗(yàn),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(jìn)行琥珀酸脫氫酶的免疫組織化學(xué)染色。體外實(shí)驗(yàn),常規(guī)培養(yǎng)A549肺腺癌細(xì)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(xì)胞。劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力的差異,組織化學(xué)法檢測(cè)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(biāo)記法檢測(cè)線粒體長(zhǎng)度變化情況,免疫組織化學(xué)法檢測(cè)細(xì)胞內(nèi)線粒體分裂蛋白的表達(dá)差異。結(jié)果體內(nèi)實(shí)驗(yàn):免疫組織化學(xué)染色結(jié)果為:高分化肺腺癌組織琥珀酸脫氫酶表達(dá)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(xì)胞內(nèi)琥珀酸脫氫酶表達(dá)低于低分化肺腺癌癌巢周邊的癌細(xì)胞,差異極顯著(P0.001)。體外實(shí)驗(yàn):MTT法檢測(cè)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(shí)加入時(shí)作用效果最好。劃痕實(shí)驗(yàn)檢測(cè)結(jié)果顯示:劃痕48h時(shí),抗霉素A組細(xì)胞遷移距離(125.6±30.01μm)與對(duì)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(xì)胞遷移距離(322.4±29.16μm)與對(duì)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(xì)胞遷移距離(521.1±32.92μm)與三溴丙酮酸單獨(dú)作用組相比,差異顯著(P0.01),抗霉素A組細(xì)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(xì)胞遷移距離與對(duì)照組相比,無(wú)顯著差異(P0.05)。組織化學(xué)法乳酸脫氫酶相對(duì)活性檢測(cè)結(jié)果顯示:當(dāng)遷移48h時(shí),三溴丙酮酸組乳酸脫氫酶相對(duì)活性(0.006±0.003)與對(duì)照組(0.008±0.004)相比,無(wú)顯著差異(P0.05),三溴丙酮酸+丙酮酸鈉組乳酸脫氫酶相對(duì)活性(0.007±0.004)與三溴丙酮酸組相比,無(wú)顯著差異(P0.05),抗霉素A組乳酸脫氫酶相對(duì)活性(0.020±0.002)高于對(duì)照組,差異顯著(P0.01)。組織化學(xué)法琥珀酸脫氫酶相對(duì)活性檢測(cè)結(jié)果顯示:當(dāng)細(xì)胞遷移48h時(shí),抗霉素A組琥珀酸脫氫酶相對(duì)活性(0.002±0.001)與對(duì)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(duì)活性(0.030±0.007)與對(duì)照組相比,差異極顯著(P0.0001),三溴丙酮酸+丙酮酸鈉組細(xì)胞的琥珀酸脫氫酶相對(duì)活性(0.057±0.005)高于三溴丙酮酸單獨(dú)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(duì)活性與對(duì)照組相比,差異不顯著(P0.05)。Mito-tracker Green線粒體特異性標(biāo)記法檢測(cè)偽足內(nèi)線粒體長(zhǎng)度結(jié)果顯示:當(dāng)遷移48h時(shí),抗霉素A組細(xì)胞線粒體長(zhǎng)度(1.257±0.257μm)明顯低于對(duì)照組細(xì)胞線粒體長(zhǎng)度(27.88±6.209μm),差異極顯著(P0.0001),三溴丙酮酸組細(xì)胞線粒體長(zhǎng)度(6.569±2.311μm)與對(duì)照組相比,差異極顯著(P0.0001),三溴丙酮酸+丙酮酸鈉組細(xì)胞線粒體長(zhǎng)度(21.95±6.661μm)與對(duì)照組相比,差異不顯著(P0.05)。免疫組織化學(xué)染色法檢測(cè)線粒體分裂蛋白表達(dá)結(jié)果顯示:當(dāng)遷移48h時(shí),抗霉素A組細(xì)胞線粒體分裂蛋白表達(dá)光密度(0.172±0.011)明顯高于對(duì)照組細(xì)胞線粒體分裂蛋白表達(dá)光密度(0.038±0.008),差異極顯著(P0.0001),三溴丙酮酸組細(xì)胞線粒體分裂蛋白表達(dá)光密度(0.078±0.006)與對(duì)照組相比,差異顯著(P0.01),三溴丙酮酸+丙酮酸鈉組細(xì)胞線粒體分裂蛋白表達(dá)光密度(0.047±0.004)與對(duì)照組相比,差異不顯著(P0.05)。結(jié)論:琥珀酸脫氫酶的表達(dá)提示,線粒體的功能活動(dòng)與肺腺癌組織的分化程度及肺腺癌細(xì)胞的遷移有關(guān);偽足內(nèi)線粒體氧化磷酸化和糖酵解均參與肺腺癌細(xì)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(guò)氧化磷酸化的方式為遷移的肺腺癌細(xì)胞提供能量;抑制線粒體氧化磷酸化,線粒體分裂增強(qiáng),肺腺癌細(xì)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R734.2

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