本文選題：肺腫瘤 + 轉移�。� 參考：《北京市結核病胸部腫瘤研究所》2017年博士論文

【摘要】：背景與目的:肺癌因其高發(fā)病率和死亡率,在中國乃至世界范圍內依然是一個重大的健康問題。肺癌普遍發(fā)病隱匿,早期癥狀不明顯,因此在發(fā)病初期易被忽視。加之肺癌的早期診斷及治療進展緩慢,5年生存率僅為18%。大部分肺癌患者在初次就診時,已經發(fā)生了轉移,這也是目前肺癌療效不佳的一個主要原因。作為腫瘤的六大生物學特性之一,“轉移”一直以來都是導致腫瘤復發(fā)以及患者死亡的主要原因。因此,研究腫瘤發(fā)生轉移的機制,闡明參與腫瘤轉移的信號通路,對控制腫瘤的復發(fā)和轉移有著重要的意義。親環(huán)素A作為最早被發(fā)現的親環(huán)素家族成員,在肺癌、肝癌、胰腺癌、乳腺癌等多種腫瘤組織中均呈高表達狀態(tài)。研究發(fā)現,親環(huán)素A在這些腫瘤的發(fā)生和發(fā)展過程中均發(fā)揮著重要的作用。其中,腫瘤的轉移更是與親環(huán)素A的表達水平有著密切的關系。然而,目前針對親環(huán)素A在肺癌的轉移過程中的作用及其相關機制的研究尚比較少。本課題組前期研究發(fā)現,通過RNA干擾和使用抑制劑的方法,抑制非小細胞肺癌細胞中親環(huán)素A的表達水平后,細胞的遷移和侵襲能力均受到了抑制,這提示我們,親環(huán)素A可能能夠促進非小細胞肺癌細胞的遷移和侵襲。隨后我們發(fā)現,在抑制了非小細胞肺癌細胞中親環(huán)素A的表達水平之后,MAPK信號通路中的一個重要分子——P38的磷酸化水平也受到了明顯抑制,這提示我們親環(huán)素A對非小細胞肺癌細胞的遷移和侵襲能力的調控,可能是通過P38 MAPK信號通路來實現的。因此,本課題將分別通過體外和體內實驗來進一步研究親環(huán)素A在非小細胞肺癌轉移過程中的作用,并對其作用機制進行初步探討。方法:首先,在“親環(huán)素A在非小細胞肺癌轉移過程中的作用”的研究中,我們通過慢病毒感染的方法,在兩種人非小細胞肺癌細胞H1299和A549中構建親環(huán)素A低表達細胞株,并在此基礎上構建親環(huán)素A過表達的細胞株,及其對照細胞;然后通過劃痕實驗和Transwell實驗的方法,觀察親環(huán)素A的表達水平對兩種人非小細胞肺癌細胞H1299和A549的遷移和侵襲能力的影響;同時通過Western-blot的方法來觀察親環(huán)素A的表達水平的變化對EMT相關蛋白表達水平的影響。最后通過動物實驗的方法,構建非小細胞肺癌小鼠轉移模型,來進一步探討親環(huán)素A的表達水平對非小細胞肺癌轉移的影響。接著,在“親環(huán)素A在非小細胞肺癌轉移過程中的作用機制的探討”的研究中,我們使用P38抑制劑SB203580,分別對親環(huán)素A低表達和高表達的非小細胞肺癌細胞進行處理,一方面通過細胞遷移和侵襲實驗,觀察兩種抑制劑在親環(huán)素A表達水平不同的情況下,對非小細胞肺癌細胞遷移和侵襲能力的影響;另一方面通過Western-blot的方法來檢測兩種抑制劑在親環(huán)素A表達水平不同的非小細胞肺癌細胞中,對EMT相關蛋白表達水平的影響。結果:1.在A549和H1299兩種非小細胞肺癌細胞中,通過慢病毒感染,成功構建了親環(huán)素A低表達(CypA-KD)和過表達(CypA-KD-OE)細胞株,及其各自的對照細胞;2.劃痕實驗結果顯示,與對照組H1299 NC細胞相比,H1299 Cyp A-KD組細胞的遷移能力明顯降低(P0.01),而H1299 Cyp A-KD-OE組細胞的遷移能力也明顯高于對照組細胞H1299 CypA-KD-Vector(P0.01);Transwell遷移實驗結果同樣表明,與對照組細胞H1299/A549 NC相比,H1299 CypA-KD組(P0.001)和A549 CypA-KD(P0.05)組細胞的遷移能力也顯著降低,同時H1299CypA-KD-OE組細胞(P0.001)和A549 CypA-KD-OE組細胞(P0.05)的遷移能力較對照組細胞H1299/A549 CypA-KD-Vector相比明顯增高;然而,Transwell侵襲實驗的結果則顯示,無論是在A549還是H1299細胞中,親環(huán)素A的表達水平對兩者的侵襲能力都沒有明顯的影響;3.Wester-blot的結果顯示,親環(huán)素A的表達水平降低后,β-catenin,Vimentin和Snail蛋白的表達水平在H1299和A549細胞中都受到了明顯的抑制,而隨后在親環(huán)素A過表達之后,三者的表達水平較對照組又明顯升高。4.非小細胞肺癌小鼠轉移模型的結果顯示,14周后,A549組和A549CypA-KD-OE組的各自有5/5和5/5只小鼠肺部出現了轉移病灶,而A549CypA-KD組肺部出現轉移病灶的小鼠數量為0/5只;此外,在A549和A549CypA-KD-OE組中,各有一只小鼠出現了骨轉移。5.在H1299和A549細胞中,親環(huán)素A表達水平降低后,P38的磷酸化水平都出現了明顯的降低,而當親環(huán)素A的表達水平再次上調時,P38的磷酸化水平也隨之上升,差異均具有統計學意義(P0.05);6.使用P38抑制劑SB203580處理細胞后,H1299 CypA-KD-OE細胞的遷移能力較對照組(DMSO)明顯降低(劃痕實驗P0.01,Transwell遷移實驗P0.01),而H1299 CypA-KD細胞的遷移能力與對照組(DMSO)相比,沒有顯著的統計學差異;7.經P38抑制劑SB203580處理后,H1299 CypA-KD-OE組細胞中,β-catenin,Vimentin和Snail蛋白的表達水平于對照組(DMSO)相比均受到明顯的抑制,并且差異具有統計學意義(P0.05);而在H1299 Cyp A-KD組的細胞中,與對照組相比,三者的表達水平并未明顯受到SB203580的影響。結論:1.體外實驗的結果表明,親環(huán)素A能夠明顯促進非小細胞肺癌細胞的遷移能力,同時能夠促進細胞發(fā)生EMT;2.動物實驗的結果表明,親環(huán)素A能夠促進非小細胞肺癌在體內發(fā)生轉移;3.P38是親環(huán)素A介導的非小細胞肺癌轉移過程中的重要因素,因此親環(huán)素A在非小細胞肺癌轉移過程中的作用能夠通過P38 MAPK信號通路來實現的。
[Abstract]:Background and objective: lung cancer is still a major health problem in China and in the world because of its high incidence and mortality. Lung cancer is generally insidious and early symptoms are not obvious, so it is easily ignored in the early stage of the disease. In addition, the early diagnosis and treatment of lung cancer are slow, and the 5 year survival rate is only 18%. most of the lung cancer patients. As one of the six major biological characteristics of cancer, "metastasis" has been the main cause of tumor recurrence and the death of the patients. Therefore, the mechanism of tumor metastasis and the signal pathways involved in tumor metastasis have been studied. As a member of the most early discovered cyclophilin family, cyclophilin A is highly expressed in various tumor tissues, such as lung cancer, liver cancer, pancreatic cancer, and breast cancer. It is found that cyclophilin A plays an important role in the development and development of these tumors. The metastasis is closely related to the level of the expression of cyclophilin A. However, there are few studies on the role of cyclophilin A in the metastasis of lung cancer and its related mechanisms. In our previous study, we found that the expression of cyclophilin A in non-small cell lung cancer cells was suppressed by RNA interference and the use of inhibitors. After leveling, cell migration and invasion were inhibited, which suggests that cyclophilin A may be able to promote the migration and invasion of non small cell lung cancer cells. Subsequently, we found that after inhibiting the expression of cyclophilin A in non-small cell lung cancer cells, a key molecule in the MAPK signaling pathway, phosphorylated water of the P38 It is also significantly inhibited, which suggests that the regulation of the migration and invasion of non small cell lung cancer cells by cyclophilin A may be achieved through the P38 MAPK signaling pathway. Therefore, this topic will further study the role of cyclophilin A in the metastasis of non-small cell lung cancer through in vitro and in vivo experiments. Methods: first, in the study of the role of cyclophilin A in the metastasis of non-small cell lung cancer, we constructed a cyclophilin A low expression cell line in two human non-small cell lung cancer cells, H1299 and A549 by the method of lentivirus infection, and on this basis, we construct a cyclophilin A overexpressed cell. The effects of the expression level of cyclophilin A on the migration and invasion of two human non-small cell lung cancer cells H1299 and A549 were observed by scratch test and Transwell test, and the expression level of the expression level of cyclophilin A on the expression level of EMT related proteins was observed by Western-blot method. Finally, the effect of the expression level of cyclophilin A on the metastasis of non-small cell lung cancer was further explored by the method of animal experiment to further explore the effect of the expression level of cyclophilin on non-small cell lung cancer. Then, in the study of the mechanism of the role of cyclophilin A in the metastasis of non-small cell lung cancer, we use the P38 inhibitor SB203580. Do not treat the low expression and high expression of non small cell lung cancer cells with cyclophilin A. On the one hand, the effects of two inhibitors on the migration and invasion ability of non small cell lung cancer cells in different levels of cyclophilin A expression are observed by cell migration and invasion experiments. On the other hand, two kinds of cells are detected by Western-blot method. The effect of inhibitors on the expression level of EMT related proteins in non small cell lung cancer cells with different levels of cyclophilin A. Results: 1. in two non small cell lung cancer cells of A549 and H1299, the low expression of cyclophilin A (CypA-KD) and overexpression (CypA-KD-OE) cell lines and their respective control cells were successfully constructed, and their respective control cells were constructed, 2. The results of the scratch test showed that compared with the control group H1299 NC cells, the migration ability of the cells in the H1299 Cyp A-KD group was significantly lower (P0.01), while the migration ability of the H1299 Cyp A-KD-OE group was significantly higher than the H1299 CypA-KD-Vector (P0.01) in the control group, and the results of the migration experiment also showed that the cells were compared with the control group. The cell migration ability of 99 CypA-KD group (P0.001) and A549 CypA-KD (P0.05) group also decreased significantly, while the migration ability of H1299CypA-KD-OE group (P0.001) and A549 CypA-KD-OE group cell (P0.05) was significantly higher than that of the control group cell H1299/A549 CypA-KD-Vector. In H1299 cells, the expression level of cyclophilin A has no significant influence on the invasiveness of the two. 3.Wester-blot results showed that the expression level of cyclophilin A decreased, and the expression level of beta -catenin, Vimentin and Snail protein was significantly inhibited in H1299 and A549 cells, and then after the expression of cyclophilin A, three The expression level of the.4. non small cell lung cancer mice was significantly higher than that in the control group. After 14 weeks, the A549 and A549CypA-KD-OE groups had 5/5 and 5/5 mice with metastatic lesions, while the number of mice with metastatic lesions in the A549CypA-KD group was 0/5 only; in addition, in the A549 and A549CypA-KD-OE groups, Each mouse had bone metastasis.5. in H1299 and A549 cells. After the level of cyclophilin A expression decreased, the phosphorylation level of P38 decreased significantly. When the expression level of cyclophilin A was up, the phosphorylation level of P38 increased, the difference was statistically significant (P0.05); 6. the P38 inhibitor SB203580 was used. After the cells, the migration ability of H1299 CypA-KD-OE cells was significantly lower than that of the control group (DMSO) (scratch test P0.01, Transwell migration test P0.01), while the migration ability of H1299 CypA-KD cells was no significant difference compared with the control group (DMSO); 7. The expression level of ntin and Snail protein was significantly inhibited compared with the control group (DMSO), and the difference was statistically significant (P0.05); in the cells of the H1299 Cyp A-KD group, the expression level of the three was not significantly affected by SB203580. Conclusion: the results of 1. in vitro showed that the cyclophilin A could be significantly promoted. The migration of non small cell lung cancer cells can also promote the cell formation of EMT. 2. animal experiments showed that procyclin A could promote the metastasis of non-small cell lung cancer in the body; 3.P38 was an important factor in the metastasis of non small cell lung cancer mediated by cyclophilin A. Therefore, cyclophilin A is in the process of metastasis of non-small cell lung cancer. Its role can be achieved through the P38 MAPK signaling pathway.
【學位授予單位】：北京市結核病胸部腫瘤研究所
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R734.2

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