MMP13對肺大細(xì)胞癌血管生成擬態(tài)調(diào)控機制的研究
發(fā)布時間：2019-06-27 11:59
【摘要】：研究目的肺癌是世界上發(fā)病率和死亡率增長最快,對人群健康和生命威脅最大的惡性腫瘤之一。肺大細(xì)胞癌是非小細(xì)胞肺癌中惡性程度最高的一類,早期便易發(fā)生侵襲和轉(zhuǎn)移,預(yù)后差。腫瘤血管生成擬態(tài)(vasculogenic mimicry,VM)是一種存在于高侵襲性腫瘤的微循環(huán)模式,VM的存在與腫瘤的發(fā)展以及不良預(yù)后密切相關(guān)。肺癌中關(guān)于VM的研究較少,本研究擬探討基質(zhì)金屬蛋白酶-13(MMP13)在肺大細(xì)胞癌VM形成中的作用及其相關(guān)分子機制,并初步探討MMP13和MMP2在肺大細(xì)胞癌不同血液供應(yīng)模式中的調(diào)控關(guān)系,為進一步明確腫瘤血管生成提供理論基礎(chǔ)。研究方法1.收集51例人肺大細(xì)胞癌組織標(biāo)本,應(yīng)用免疫組織化學(xué)染色和CD34/PAS雙重染色方法檢測肺大細(xì)胞癌組織中MMP13的表達水平、VM和微血管密度(microvessel density,MVD)。分析MMP13的表達與肺大細(xì)胞癌臨床病理參數(shù)、VM和MVD之間的相關(guān)性及其與肺大細(xì)胞癌患者生存預(yù)后的關(guān)系。2.體外培養(yǎng)肺大細(xì)胞癌H460和H661細(xì)胞系,利用脂質(zhì)體轉(zhuǎn)染方法建立穩(wěn)定轉(zhuǎn)染MMP13過表達和降表達的肺大細(xì)胞癌細(xì)胞系。通過體外細(xì)胞三維培養(yǎng),觀察過表達和降表達MMP13后腫瘤細(xì)胞管道形成能力的變化。在H460和H661細(xì)胞系中加入重組人MMP13和MMP2蛋白及其對層粘連蛋白-5(laminin-5,Ln-5)的降解產(chǎn)物觀察對腫瘤細(xì)胞管道形成能力的影響。3.利用硝酸銀染色技術(shù)檢測MMP13和MMP2對Ln-5的降解片段,利用LC-MS/MS質(zhì)譜技術(shù)檢測MMP13對Ln-5的降解片段以確定其氨基酸序列。利用Western blot和免疫熒光技術(shù)觀察并分析外源性MMP13和MMP2對Ln-5降解片段對肺大細(xì)胞癌細(xì)胞中EGFR,F-actin,α-tublin,vimentin表達的影響。4.應(yīng)用免疫組織化學(xué)染色檢測51例人肺大細(xì)胞癌組織中腫瘤細(xì)胞VE-cadherin的表達。分析MMP13與VE-cadherin表達的相關(guān)性。在肺大細(xì)胞癌細(xì)胞系H460和H661中加入不同濃度外源性MMP13,利用Western blot和免疫熒光染色檢測細(xì)胞VE-cadherin的表達變化。5.體外培養(yǎng)臍靜脈內(nèi)皮細(xì)胞(Human Umbilical Vein Endothelial Cells,HUVEC),觀察外源性MMP13、MMP2和Ln-5中和抗體對其管道形成能力以及遷移能力的影響。免疫組織化學(xué)染色檢測分析肺大細(xì)胞癌組織中MMP13的表達與Ln-5和EGFR的關(guān)系。6.裸鼠皮下接種肺大細(xì)胞癌H460及MMP13過表達H460細(xì)胞建立小鼠移植瘤模型,觀察上調(diào)MMP13對腫瘤生長及血管擬態(tài)形成的影響,免疫組化檢測移植瘤組織中Ln-5和EGFR的表達。不同時間點處死小鼠,獲取瘤組織觀察H460組移植瘤中MMP2和MMP13在腫瘤不同生長時間點的表達變化。研究結(jié)果1.在51例肺大細(xì)胞癌組織中,MMP13呈陽性表達的有20例,陰性表達的有31例。MMP13的表達水平與腫瘤的臨床分期及腫瘤直徑呈正相關(guān)。生存分析結(jié)果顯示,腫瘤細(xì)胞內(nèi)MMP13呈陽性表達的患者總生存時間明顯低于MMP13陰性表達者(P0.05)。統(tǒng)計學(xué)分析顯示MMP13的表達與VM存在呈負(fù)相關(guān)(P0.05);與腫瘤組織中微血管密度呈正相關(guān),差異具有統(tǒng)計學(xué)意義(P0.05)。2.在體外細(xì)胞培養(yǎng)模型中,低表達MMP13的細(xì)胞系H460在體外三維培養(yǎng)中的管道形成能力明顯強于高表達MMP13的細(xì)胞系H661。上調(diào)細(xì)胞系H460中MMP13的表達水平后,其體外三維培養(yǎng)中的管道形成能力減弱;而下調(diào)H661中MMP13的表達水平后,腫瘤細(xì)胞的管道形成能力增強。此外,將外源性MMP13加入H460后其管道形成數(shù)量減少。3.MMP13可以將Ln-5降解成分子量約為20KD大小的片段,后者可以抑制腫瘤細(xì)胞體外管道形成的能力。通過質(zhì)譜分析得出20KD片段為Ln-5蛋白γ2亞基中的肽段,氨基酸序列為Cys540-Arg694。4.Western blot和免疫熒光結(jié)果顯示,MMP2對Ln-5降解產(chǎn)物加入培養(yǎng)的H460和H661細(xì)胞中使促進EGFR和F-actin表達,vimentin和α-tublin表達無明顯變化。而加入MMP13對Ln-5降解產(chǎn)物后細(xì)胞的EGFR及F-actin表達減少,vimentin和α-tublin表達無明顯變化。5.51例肺大細(xì)胞癌組織中,腫瘤細(xì)胞高表達VE-cadherin的有12例,低表達的有39例。在MMP13陽性表達的組織中僅有一例呈VE-cadherin陽性表達,在31例MMP13陰性表達的組織中有11例呈VE-cadherin陽性表達,MMP13與VE-cadherin的表達呈負(fù)相關(guān),差異有統(tǒng)計學(xué)意義(P0.05)。肺大細(xì)胞癌細(xì)胞系H460和H661中加入不同濃度外源性MMP13作用后,Western blot結(jié)果顯示其VE-cadherin蛋白表達隨MMP13濃度增大而減少,免疫熒光顯示受MMP13作用后細(xì)胞表面VE-cadherin蛋白表達減少。6.將外源性MMP13和MMP2用于HUVEC的培養(yǎng),內(nèi)皮細(xì)胞在三維培養(yǎng)基中形成的管道數(shù)目明顯增多。Transwell遷移實驗結(jié)果表明,MMP13和MMP2均可以促進內(nèi)皮細(xì)胞的運動能力,加入Ln-5中和抗體后對內(nèi)皮細(xì)胞的遷移和管道形成能力影響顯著減小,差異具有統(tǒng)計學(xué)意義(P0.05)。7.接種H460 MMP13過表達細(xì)胞組裸鼠移植瘤初期生長速度慢于H460對照組,差異具有統(tǒng)計學(xué)意義(P0.05)。免疫組化染色和endomucin/PAS結(jié)果顯示,H460MMP13過表達組移植瘤VM數(shù)量,Ln-5和EGFR表達水平低于H460對照組,與體外實驗結(jié)果一致。8.MMP13在肺大細(xì)胞癌移植瘤中的表達呈現(xiàn)時間依賴性變化,隨著腫瘤的生長,MMP13在腫瘤細(xì)胞中的表達逐漸升高,且移植瘤中MMP13的表達與血管生成擬態(tài)的數(shù)目呈負(fù)相關(guān),而與MVD呈正相關(guān),差異有統(tǒng)計學(xué)意義(P0.05)。MMP2在大細(xì)胞癌移植瘤生長過程中呈較高水平表達,表達水平未見明顯變化。結(jié)論1.MMP13在肺大細(xì)胞癌內(nèi)的高表達與腫瘤惡性程度和患者不良預(yù)后有關(guān)。2.MMP13可對肺大細(xì)胞癌中VM的形成起到負(fù)向調(diào)控的作用,而對腫瘤內(nèi)皮依賴性血管的生成起到正向調(diào)控作用。3.MMP13可能通過對Ln-5的降解作用而抑制EGFR/F-actin通路影響細(xì)胞骨架變化從而抑制腫瘤血管生成擬態(tài)的形成。4.MMP13可降解細(xì)胞表面VE-cadherin影響細(xì)胞的血管擬態(tài)生成。5.MMP13可增加內(nèi)皮細(xì)胞的遷移運動能力而促進內(nèi)皮依賴性血管生成。6.MMP13可能在腫瘤生長的晚期階段促進內(nèi)皮依賴性血管取代血管生成擬態(tài)成為腫瘤內(nèi)的主要微循環(huán)模式。
[Abstract]:The study of lung cancer is one of the most common malignant tumors in the world, with the fastest rate of morbidity and mortality in the world and the greatest threat to the health and life of the population. The lung-large cell carcinoma is the highest in the non-small-cell lung cancer, and the early-stage is susceptible to invasion and metastasis, and the prognosis is poor. Vasculogenic mimicry (VM) is a kind of microcirculatory pattern in highly invasive tumor, and the existence of VM is closely related to the development of the tumor and the poor prognosis. In this study, the role of matrix metalloproteinase-13 (MMP13) in the formation of pulmonary large cell carcinoma (VM) and its related molecular mechanism were discussed, and the regulation and control of MMP13 and MMP2 in different blood supply modes of large-cell lung cancer were discussed. In ord to further clarify that theoretical basis for tumor angiogenesis. Study Method 1. The expression level, VM and microvessel density (MVD) of the MMP13 in the lung-large cell carcinoma were detected by immunohistochemical staining and CD34/ PAS double staining. The relationship between the expression of MMP13 and the clinicopathological parameters of the large-cell carcinoma of the lung, the correlation between the VM and the MVD and the relationship with the survival and prognosis of the patients with large-cell carcinoma of the lung were analyzed. In vitro, the H460 and H661 cell lines of the lung large cell carcinoma are cultured, and the lung-large cell carcinoma cell lines stably transfected with the MMP13 through-expression and down-expression are established by using the liposome transfection method. The changes of the formation of tumor cells after MMP13 expression and expression of MMP13 were observed by three-dimensional culture in vitro. The effect of the degradation products of the recombinant human MMP13 and MMP2 protein and its p-laminin-5 (Ln-5) on the formation of tumor cells was observed in the H460 and H661 cell lines. The degradation fragment of MMP13 and MMP2 on Ln-5 was detected by silver nitrate staining technique, and the degradation fragment of MMP13 on Ln-5 was detected by LC-MS/ MS mass spectrometry to determine its amino acid sequence. The effects of exogenous MMP13 and MMP2 on the expression of EGFR, F-actin, VEGF-tubulin, vimentin in the lung of large-cell lung cancer cells were observed and analyzed by Western blot and immunofluorescence. The expression of VE-cadherin in 51 human lung cancer tissues was detected by immunohistochemical staining. The relationship between the expression of MMP13 and VE-cadherin was analyzed. Different concentrations of exogenous MMP13 were added to H460 and H661 of large-cell lung cancer cell lines. The expression of VE-cadherin was detected by Western blot and immunofluorescence staining. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro to observe the effects of exogenous MMP13, MMP2 and Ln-5 neutralizing antibodies on their tube formation and migration ability. The relationship between the expression of MMP13 and the expression of Ln-5 and EGFR in the lung-large cell carcinoma was analyzed by immunohistochemical staining. H460 and MMP13 overexpressing H460 cells were inoculated subcutaneously in nude mice to establish a model of mouse transplantation, and the effects of up-regulation of MMP13 on the formation of tumor and the formation of the blood vessel were observed, and the expression of Ln-5 and EGFR in the transplanted tumor tissues was detected by immunohistochemistry. Mice were sacrificed at different time points, and the expression of MMP2 and MMP13 in the transplanted tumor of H460 group was observed at different time points. Study Results 1. In 51 cases of large-cell carcinoma of the lung, there were 20 cases of positive expression of MMP13 and 31 cases of negative expression. The expression level of MMP13 was positively correlated with the clinical stage of the tumor and the tumor diameter. Survival analysis showed that the total survival time of MMP13 in tumor cells was significantly lower than that of MMP13 (P0.05). Statistical analysis showed that the expression of MMP13 was negatively correlated with the presence of the VM (P0.05); the microvessel density was positively correlated with the microvessel density in the tumor tissue (P0.05). In the in vitro cell culture model, the line forming ability of the cell line H460 of the low expression MMP13 in the in vitro three-dimensional culture is significantly stronger than the cell line H661 of the high expression MMP13. After up-regulating the expression level of MMP13 in the cell line H460, the tube forming ability in the three-dimensional culture of the cell line is weakened; and after the expression level of the MMP13 in the H661 is reduced, the pipeline forming ability of the tumor cells is enhanced. 3. MMP13 can degrade Ln-5 into fragments with a molecular weight of about 20KD, which can inhibit the formation of tumor cells in vitro. The results showed that the expression of MMP2 in H460 and H661 cells in cultured H460 and H661 cells showed no significant change in the expression of EGFR and F-actin in H460 and H661 cells. There were no significant changes in the expression of EGFR and F-actin in the cells after the addition of the MMP13 to the Ln-5 degradation products. The positive expression of VE-cadherin in the tissues of the MMP13-positive expression was found in 11 of the 31 MMP13-negative tissues, and there was a negative correlation between the expression of MMP13 and VE-cadherin, and the difference was statistically significant (P0.05). The results of Western blot showed that the expression of VE-cadherin was decreased with the increase of the concentration of MMP13, and the expression of VE-cadherin in the cell surface after MMP13 was decreased by MMP13. Exogenous MMP13 and MMP2 were used for the culture of HUVEC, and the number of ducts formed in the three-dimensional culture medium of the endothelial cells increased significantly. The results of Transwell migration showed that both MMP13 and MMP2 could promote the ability of the endothelial cells to move, and the effects of the addition of Ln-5 and the antibody on the migration of endothelial cells and the formation of the tube were significantly reduced, and the difference was statistically significant (P0.05). The initial growth rate of H460 MMP13 overexpressing cell group was slower than that of H460 control group, and the difference was statistically significant (P0.05). The results of immunohistochemistry and endomucin/ PAS showed that the number of VM, Ln-5 and EGFR in H460MMP13 overexpressing group was lower than that of H460 control group, and the expression of Ln-5 and EGFR was lower than that of H460 control group. The expression of MMP13 in the tumor cells increased gradually, and the expression of MMP13 in the transplanted tumor was negatively correlated with the number of angiogenesis, and the expression of MMP13 was positively correlated with the MVD (P0.05). MMP2 was expressed at a high level in the growth of large cell carcinoma, and no significant change was observed in the expression level. Conclusion 1. The high expression of MMP13 in the large-cell carcinoma of the lung is related to the malignant degree of the tumor and the poor prognosis of the patients. 3. MMP13 can inhibit the changes of the cytoskeleton by the degradation of Ln-5 and inhibit the formation of the tumor angiogenesis. 6. MMP13 may promote the generation of endothelial-dependent blood vessels in the advanced stage of tumor growth.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R734.2
本文編號：2506769
[Abstract]:The study of lung cancer is one of the most common malignant tumors in the world, with the fastest rate of morbidity and mortality in the world and the greatest threat to the health and life of the population. The lung-large cell carcinoma is the highest in the non-small-cell lung cancer, and the early-stage is susceptible to invasion and metastasis, and the prognosis is poor. Vasculogenic mimicry (VM) is a kind of microcirculatory pattern in highly invasive tumor, and the existence of VM is closely related to the development of the tumor and the poor prognosis. In this study, the role of matrix metalloproteinase-13 (MMP13) in the formation of pulmonary large cell carcinoma (VM) and its related molecular mechanism were discussed, and the regulation and control of MMP13 and MMP2 in different blood supply modes of large-cell lung cancer were discussed. In ord to further clarify that theoretical basis for tumor angiogenesis. Study Method 1. The expression level, VM and microvessel density (MVD) of the MMP13 in the lung-large cell carcinoma were detected by immunohistochemical staining and CD34/ PAS double staining. The relationship between the expression of MMP13 and the clinicopathological parameters of the large-cell carcinoma of the lung, the correlation between the VM and the MVD and the relationship with the survival and prognosis of the patients with large-cell carcinoma of the lung were analyzed. In vitro, the H460 and H661 cell lines of the lung large cell carcinoma are cultured, and the lung-large cell carcinoma cell lines stably transfected with the MMP13 through-expression and down-expression are established by using the liposome transfection method. The changes of the formation of tumor cells after MMP13 expression and expression of MMP13 were observed by three-dimensional culture in vitro. The effect of the degradation products of the recombinant human MMP13 and MMP2 protein and its p-laminin-5 (Ln-5) on the formation of tumor cells was observed in the H460 and H661 cell lines. The degradation fragment of MMP13 and MMP2 on Ln-5 was detected by silver nitrate staining technique, and the degradation fragment of MMP13 on Ln-5 was detected by LC-MS/ MS mass spectrometry to determine its amino acid sequence. The effects of exogenous MMP13 and MMP2 on the expression of EGFR, F-actin, VEGF-tubulin, vimentin in the lung of large-cell lung cancer cells were observed and analyzed by Western blot and immunofluorescence. The expression of VE-cadherin in 51 human lung cancer tissues was detected by immunohistochemical staining. The relationship between the expression of MMP13 and VE-cadherin was analyzed. Different concentrations of exogenous MMP13 were added to H460 and H661 of large-cell lung cancer cell lines. The expression of VE-cadherin was detected by Western blot and immunofluorescence staining. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro to observe the effects of exogenous MMP13, MMP2 and Ln-5 neutralizing antibodies on their tube formation and migration ability. The relationship between the expression of MMP13 and the expression of Ln-5 and EGFR in the lung-large cell carcinoma was analyzed by immunohistochemical staining. H460 and MMP13 overexpressing H460 cells were inoculated subcutaneously in nude mice to establish a model of mouse transplantation, and the effects of up-regulation of MMP13 on the formation of tumor and the formation of the blood vessel were observed, and the expression of Ln-5 and EGFR in the transplanted tumor tissues was detected by immunohistochemistry. Mice were sacrificed at different time points, and the expression of MMP2 and MMP13 in the transplanted tumor of H460 group was observed at different time points. Study Results 1. In 51 cases of large-cell carcinoma of the lung, there were 20 cases of positive expression of MMP13 and 31 cases of negative expression. The expression level of MMP13 was positively correlated with the clinical stage of the tumor and the tumor diameter. Survival analysis showed that the total survival time of MMP13 in tumor cells was significantly lower than that of MMP13 (P0.05). Statistical analysis showed that the expression of MMP13 was negatively correlated with the presence of the VM (P0.05); the microvessel density was positively correlated with the microvessel density in the tumor tissue (P0.05). In the in vitro cell culture model, the line forming ability of the cell line H460 of the low expression MMP13 in the in vitro three-dimensional culture is significantly stronger than the cell line H661 of the high expression MMP13. After up-regulating the expression level of MMP13 in the cell line H460, the tube forming ability in the three-dimensional culture of the cell line is weakened; and after the expression level of the MMP13 in the H661 is reduced, the pipeline forming ability of the tumor cells is enhanced. 3. MMP13 can degrade Ln-5 into fragments with a molecular weight of about 20KD, which can inhibit the formation of tumor cells in vitro. The results showed that the expression of MMP2 in H460 and H661 cells in cultured H460 and H661 cells showed no significant change in the expression of EGFR and F-actin in H460 and H661 cells. There were no significant changes in the expression of EGFR and F-actin in the cells after the addition of the MMP13 to the Ln-5 degradation products. The positive expression of VE-cadherin in the tissues of the MMP13-positive expression was found in 11 of the 31 MMP13-negative tissues, and there was a negative correlation between the expression of MMP13 and VE-cadherin, and the difference was statistically significant (P0.05). The results of Western blot showed that the expression of VE-cadherin was decreased with the increase of the concentration of MMP13, and the expression of VE-cadherin in the cell surface after MMP13 was decreased by MMP13. Exogenous MMP13 and MMP2 were used for the culture of HUVEC, and the number of ducts formed in the three-dimensional culture medium of the endothelial cells increased significantly. The results of Transwell migration showed that both MMP13 and MMP2 could promote the ability of the endothelial cells to move, and the effects of the addition of Ln-5 and the antibody on the migration of endothelial cells and the formation of the tube were significantly reduced, and the difference was statistically significant (P0.05). The initial growth rate of H460 MMP13 overexpressing cell group was slower than that of H460 control group, and the difference was statistically significant (P0.05). The results of immunohistochemistry and endomucin/ PAS showed that the number of VM, Ln-5 and EGFR in H460MMP13 overexpressing group was lower than that of H460 control group, and the expression of Ln-5 and EGFR was lower than that of H460 control group. The expression of MMP13 in the tumor cells increased gradually, and the expression of MMP13 in the transplanted tumor was negatively correlated with the number of angiogenesis, and the expression of MMP13 was positively correlated with the MVD (P0.05). MMP2 was expressed at a high level in the growth of large cell carcinoma, and no significant change was observed in the expression level. Conclusion 1. The high expression of MMP13 in the large-cell carcinoma of the lung is related to the malignant degree of the tumor and the poor prognosis of the patients. 3. MMP13 can inhibit the changes of the cytoskeleton by the degradation of Ln-5 and inhibit the formation of the tumor angiogenesis. 6. MMP13 may promote the generation of endothelial-dependent blood vessels in the advanced stage of tumor growth.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R734.2
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