MMP13对肺大细胞癌血管生成拟态调控机制的研究
发布时间：2019-06-27 11:59
【摘要】：研究目的肺癌是世界上发病率和死亡率增长最快,对人群健康和生命威胁最大的恶性肿瘤之一。肺大细胞癌是非小细胞肺癌中恶性程度最高的一类,早期便易发生侵袭和转移,预后差。肿瘤血管生成拟态(vasculogenic mimicry,VM)是一种存在于高侵袭性肿瘤的微循环模式,VM的存在与肿瘤的发展以及不良预后密切相关。肺癌中关于VM的研究较少,本研究拟探讨基质金属蛋白酶-13(MMP13)在肺大细胞癌VM形成中的作用及其相关分子机制,并初步探讨MMP13和MMP2在肺大细胞癌不同血液供应模式中的调控关系,为进一步明确肿瘤血管生成提供理论基础。研究方法1.收集51例人肺大细胞癌组织标本,应用免疫组织化学染色和CD34/PAS双重染色方法检测肺大细胞癌组织中MMP13的表达水平、VM和微血管密度(microvessel density,MVD)。分析MMP13的表达与肺大细胞癌临床病理参数、VM和MVD之间的相关性及其与肺大细胞癌患者生存预后的关系。2.体外培养肺大细胞癌H460和H661细胞系,利用脂质体转染方法建立稳定转染MMP13过表达和降表达的肺大细胞癌细胞系。通过体外细胞三维培养,观察过表达和降表达MMP13后肿瘤细胞管道形成能力的变化。在H460和H661细胞系中加入重组人MMP13和MMP2蛋白及其对层粘连蛋白-5(laminin-5,Ln-5)的降解产物观察对肿瘤细胞管道形成能力的影响。3.利用硝酸银染色技术检测MMP13和MMP2对Ln-5的降解片段,利用LC-MS/MS质谱技术检测MMP13对Ln-5的降解片段以确定其氨基酸序列。利用Western blot和免疫荧光技术观察并分析外源性MMP13和MMP2对Ln-5降解片段对肺大细胞癌细胞中EGFR,F-actin,α-tublin,vimentin表达的影响。4.应用免疫组织化学染色检测51例人肺大细胞癌组织中肿瘤细胞VE-cadherin的表达。分析MMP13与VE-cadherin表达的相关性。在肺大细胞癌细胞系H460和H661中加入不同浓度外源性MMP13,利用Western blot和免疫荧光染色检测细胞VE-cadherin的表达变化。5.体外培养脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVEC),观察外源性MMP13、MMP2和Ln-5中和抗体对其管道形成能力以及迁移能力的影响。免疫组织化学染色检测分析肺大细胞癌组织中MMP13的表达与Ln-5和EGFR的关系。6.裸鼠皮下接种肺大细胞癌H460及MMP13过表达H460细胞建立小鼠移植瘤模型,观察上调MMP13对肿瘤生长及血管拟态形成的影响,免疫组化检测移植瘤组织中Ln-5和EGFR的表达。不同时间点处死小鼠,获取瘤组织观察H460组移植瘤中MMP2和MMP13在肿瘤不同生长时间点的表达变化。研究结果1.在51例肺大细胞癌组织中,MMP13呈阳性表达的有20例,阴性表达的有31例。MMP13的表达水平与肿瘤的临床分期及肿瘤直径呈正相关。生存分析结果显示,肿瘤细胞内MMP13呈阳性表达的患者总生存时间明显低于MMP13阴性表达者(P0.05)。统计学分析显示MMP13的表达与VM存在呈负相关(P0.05);与肿瘤组织中微血管密度呈正相关,差异具有统计学意义(P0.05)。2.在体外细胞培养模型中,低表达MMP13的细胞系H460在体外三维培养中的管道形成能力明显强于高表达MMP13的细胞系H661。上调细胞系H460中MMP13的表达水平后,其体外三维培养中的管道形成能力减弱;而下调H661中MMP13的表达水平后,肿瘤细胞的管道形成能力增强。此外,将外源性MMP13加入H460后其管道形成数量减少。3.MMP13可以将Ln-5降解成分子量约为20KD大小的片段,后者可以抑制肿瘤细胞体外管道形成的能力。通过质谱分析得出20KD片段为Ln-5蛋白γ2亚基中的肽段,氨基酸序列为Cys540-Arg694。4.Western blot和免疫荧光结果显示,MMP2对Ln-5降解产物加入培养的H460和H661细胞中使促进EGFR和F-actin表达,vimentin和α-tublin表达无明显变化。而加入MMP13对Ln-5降解产物后细胞的EGFR及F-actin表达减少,vimentin和α-tublin表达无明显变化。5.51例肺大细胞癌组织中,肿瘤细胞高表达VE-cadherin的有12例,低表达的有39例。在MMP13阳性表达的组织中仅有一例呈VE-cadherin阳性表达,在31例MMP13阴性表达的组织中有11例呈VE-cadherin阳性表达,MMP13与VE-cadherin的表达呈负相关,差异有统计学意义(P0.05)。肺大细胞癌细胞系H460和H661中加入不同浓度外源性MMP13作用后,Western blot结果显示其VE-cadherin蛋白表达随MMP13浓度增大而减少,免疫荧光显示受MMP13作用后细胞表面VE-cadherin蛋白表达减少。6.将外源性MMP13和MMP2用于HUVEC的培养,内皮细胞在三维培养基中形成的管道数目明显增多。Transwell迁移实验结果表明,MMP13和MMP2均可以促进内皮细胞的运动能力,加入Ln-5中和抗体后对内皮细胞的迁移和管道形成能力影响显著减小,差异具有统计学意义(P0.05)。7.接种H460 MMP13过表达细胞组裸鼠移植瘤初期生长速度慢于H460对照组,差异具有统计学意义(P0.05)。免疫组化染色和endomucin/PAS结果显示,H460MMP13过表达组移植瘤VM数量,Ln-5和EGFR表达水平低于H460对照组,与体外实验结果一致。8.MMP13在肺大细胞癌移植瘤中的表达呈现时间依赖性变化,随着肿瘤的生长,MMP13在肿瘤细胞中的表达逐渐升高,且移植瘤中MMP13的表达与血管生成拟态的数目呈负相关,而与MVD呈正相关,差异有统计学意义(P0.05)。MMP2在大细胞癌移植瘤生长过程中呈较高水平表达,表达水平未见明显变化。结论1.MMP13在肺大细胞癌内的高表达与肿瘤恶性程度和患者不良预后有关。2.MMP13可对肺大细胞癌中VM的形成起到负向调控的作用,而对肿瘤内皮依赖性血管的生成起到正向调控作用。3.MMP13可能通过对Ln-5的降解作用而抑制EGFR/F-actin通路影响细胞骨架变化从而抑制肿瘤血管生成拟态的形成。4.MMP13可降解细胞表面VE-cadherin影响细胞的血管拟态生成。5.MMP13可增加内皮细胞的迁移运动能力而促进内皮依赖性血管生成。6.MMP13可能在肿瘤生长的晚期阶段促进内皮依赖性血管取代血管生成拟态成为肿瘤内的主要微循环模式。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2
本文编号：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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2
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