【作者】 汪健；

【导师】 王云杰；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 目的与背景肺癌是目前发病率和死亡率最高的恶性肿瘤。转移是其治疗失败和患者死亡的主要原因。已有研究表明趋化因子及其受体与肿瘤侵袭和转移密切相关,可能是通过和炎性细胞浸润相似的机制参与了肿瘤的侵袭和转移。本课题旨在研究趋化因子SDF-1及其受体CXCR4构成的特异性轴在肺癌侵袭和转移中的作用与机制,为肺癌转移的防治提供新的思路和靶点。材料与方法(1)收集临床非小细胞肺癌(NSCLC)组织标本60例,分别取癌组织、癌旁组织及正常肺组织,进行免疫组化和RT-PCR检测其CXCR4的表达,分析肺癌组织与癌旁、正常肺组织间CXCR4表达的差异;按病理类型、分化程度及临床TNM分期分别进行分组,分析CXCR4的表达与病理类型、分化程度及临床TNM分期的相关性。(2)体外构建pEGFP-C1-CXCR4质粒,稳定转染肺癌A549细胞系,建立CXCR4过表达的A549细胞系(CXCR4-A549)。在CXCR4的特异性配体SDF-1的诱导下,分别对CXCR4-A549与A549两组细胞进行体外趋化和侵袭实验,并建立裸鼠皮下接种肺癌细胞的动物模型,分析上调CXCR4的表达后,对肺癌细胞体外迁移能力和体内成瘤及转移的能力的影响。(3)为探讨SDF-1/CXCR4在肺癌侵袭和转移中的作用机制:①利用流式细胞仪检测CXCR4-A549与A549两组细胞的增殖和凋亡,分析上调CXCR4的表达后,对细胞生长周期的影响;②利用流式细胞仪检测两组细胞钙离子内流强度,分析SDF-1/CXCR4对钙离子通道活性的调控;③采用Western blot实验检测两组细胞PI3K/AKT、MAPK/ERK相关信号通路的活性,分析SDF-1/CXCR4可能激活的下游信号分子;④采用Western blot和RT-PCR检测两组细胞肿瘤转移相关基因VEGF-C、MMP-2的表达,探讨SDF-1/CXCR4与VEGF-C、MMP-2的相互作用关系。研究结果(1)免疫组化检测结果显示:肺癌组织中CXCR4的表达主要定位于胞膜和胞浆,呈棕黄色强阳性染色,其表达率为83.3%(50/60),而在正常肺和癌旁组织中则无表达;半定量RT-PCR检测肺癌组织中CXCR4 mRNA表达为83.3% (50/60),与癌旁组织38.3% (23/60)、正常肺组织31.7% (19/60)差异显著;免疫组化检测肺鳞癌和腺癌中CXCR4的表达分别为79.3%(23/29),87.1%(27/31),鳞、腺癌间无显著差异(p>0.05);在高分化癌中CXCR4的表达为76.9%(10/13),中分化癌中为82.1%(23/28),低分化癌为84.2%(16/19),虽然随分化程度降低而呈增高趋势,但三者之间无统计学差异(p>0.05);TNM分期中Ⅰ期患者CXCR4的表达率为72.7%(8/11),Ⅱ期患者为83.9%(26/31),Ⅲ期患者为88.9%(16/18),三期之间表达有显著相关性。实验结果提示CXCR4在非小细胞肺癌中的表达具有:与癌细胞类型无关,随细胞分化程度降低而增强趋势,而与临床TNM分期密切相关,分期越晚、表达越高的特征。表明CXCR4与肺癌侵袭和转移密切相关,检测CXCR4的表达有可能作为判断转移和预后的依据。(2)经脂质体转染A549细胞,建立了稳定转染的CXCR4-A549细胞系,Western blot检测表明,CXCR4-A549较未转染的细胞CXCR4蛋白表达明显增高,说明构建的CXCR4-A549细胞可以上调CXCR4的表达。体外侵袭实验表明,上调CXCR4表达后,CXCR4-A549细胞的侵袭能力平均升高了1.62倍(p<0.05);在SDF-1的诱导作用下,CXCR4-A549细胞趋化作用较A549细胞更为明显(p<0.05)。体内实验中,CXCR4-A549与A549细胞接种的各6只裸鼠致瘤率均为100%,平均瘤重分别为:4.37±0.96g,3.24±1.16g,两组差异显著(p<0.05)。CXCR4-A549组6只鼠肺内均可见明显转移瘤形成,而A549组仅2只肺内有瘤细胞浸润,无转移瘤形成。表明过表达CXCR4的A549细胞在SDF-1的趋化作用下,其侵袭、成瘤及转移能力均有明显增强。(3)流式细胞仪检测显示:与A549细胞相比,CXCR4-A549细胞周期S期和G2期的比例升高,增殖指数(PI)升高9.72%,凋亡细胞比率减少3.60%。提示SDF-1/CXCR4可以促进细胞增殖,但两组无明显差异(p>0.05)。钙离子内流实验中,用100ng/ml SDF-1诱导20s,CXCR4-A549和A5459细胞内荧光显著增强,随时间延后而迅速的下降,两组间钙离子浓度有明显差异(p<0.05),表明SDF-1的作用具有时间依赖性。Western blot检测显示,在SDF-1作用30min时,ERK与AKT磷酸化明显。提示SDF-1/CXCR4可以活化PI3K/ERK和MAPK/AKT,激活下游分子启动肺癌细胞的侵袭和转移。在上调CXCR4表达的CXCR4-A549细胞中,VEGF-C和MMP-2在mRNA和蛋白水平均明显增高,而给予SDF-1后,其增高趋势更为明显(p<0.05)。说明SDF-1/CXCR4与VEGF-C、MMP-2在肺癌侵袭和转移中具有协同作用。结论(1)通过免疫组化法检测,揭示了CXCR4在非小细胞癌中表达的基本特征是:癌组织中高于癌旁和正常肺组织;其表达与病理类型无关,随癌细胞分化程度降低而增高趋势,但与临床TNM分期密切相关,有望成为判断NSCLC转移和预后的指标之一。(2)成功构建CXCR4稳定表达的肺癌A549细胞系,经体外和体内实验证明,可上调CXCR4的表达;在SDF-1作用下,其体外迁移和体内成瘤、转移能力显著增强。说明SDF-1/CXCR4在肺癌侵袭和转移中发挥重要作用。(3)SDF-1/CXCR4轴可通过激活肺癌细胞钙离子内流、MAPK/ERK与PI3K/AKT信号通路,启动肺癌侵袭和转移过程,并与肿瘤转移相关基因MMPs、VEGF在肺癌侵袭和转移中具有协同作用。本研究较系统地分析了SDF-1/CXCR4与肺癌的相关性,证实了SDF-1/CXCR4在肺癌侵袭和转移中的基本作用,初步阐明了SDF-1/CXCR4在肺癌侵袭转移中的作用机制,为进一步研究肺癌转移机制和临床诊断治疗提供了有益的帮助。更多还原

【Abstract】 Backgroud and AimsLung cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related deaths worldwide. This high mortality is probably attributable to early metastasis. The greedy invasiveness of NSCLC cells results in an extremely poor average 5-year survival rate in patients so far. Recent studies indicate that tumor cells express chemokine receptors and may use chemokine-mediated mechanisms during the metastasis process. Among the chemokines and chemokine receptors identified to date, the membranous CXC chemokine receptor 4 (CXCR4) and its ligand, stromal-cell-derived factor 1α(SDF-1αor CXCL12), has been found to play a key role in tumorigenicity, proliferation, metastasis and angiogenesis. Although the mechanisms underlying SDF-1a/CXCR4-mediated tumor invasion have been studied in many cancers, the role of SDF-1a/CXCR4 in the process of lung cancer cells proliferation and migration remains largely unknown.Therefore, the expression of CXCR4 in NSCLC patients was detected by RT-PCR and immunohistochemistry in this study. Based on the results, the role and mechanism of SDF-1/CXCR4 axis in metastasis of lung cancer were further studied by up-regulation expression of CXCR4 in lung cancer cells in vitro and in vivo.Methods(1) Immunohistochemical staining and RT-PCR were used to detect the expressions of CXCR4 in 60 cases of NSCLC tissues. The relationship of CXCR4 experssion in NSCLC was analyzed in the pathologic types, differentiation degrees and TNM stages.(2) CXCR4 stable over expression human lung cancer cell lines (A549) were established by CXCR4 transfection (CXCR4-A549). The migration and invasion assay was performed to detect the migration ability both in CXCR4-A549 and A549 cells. In vivo, CXCR4-A549 and A549 cells were injected subcutaneously into nude mice(each group in 6).Four weeks later the mice were scarified and the tumors in situ and the lungs were taken out to be examined histologically.(3) Th cell cycle distribution was measured by flow cytometry to analyse the effect of SDF-1/CXCR4 in the cell proliferation and atoposis. The changes of the intracellular calcium concentration after SDF-1 activation were aslo measured by flow cytometry. Western blot was used to anlayse the phosphorylation of AKT and ERK, and the expression of MMP-2 and VEGF-C.Results(1) The results of immunohistochemical staining analyzing showed that there were 50 of 60(83.3%) cases with positive staining of CXCR4 protein. Staining of CXCR4 protein was identified in the cell membrane and/or cytoplasm of cancer cells with a rate in 83.3%, but not significantly detected in the normal lung cells taken from non-cancerous regions in 38.3%, adjacent to lung cancer tissue in31.7%. Comparing the expression of CXCR4 protein bewwent adenoid (87.1%) and squamous (79.3%) carcinoma, high, medium and lower differentiation cells, there were no significant differences statistically. However, the expression of CXCR4 was significantly associated with TNM stages, stage I in 72.7%,stage II in 83.9%,stage III in 88.9%(P<0.05). Also the differential expression of CXCR4mRNA was confirmed by semi-quantitative RT-PCR in lung cancer cells and normal lung cells (P<0.05).(2) A stable p-EGFP-C1 expressive plasmid with a CXCR4 sequence was constructed and transfected into A549 cells. With a hogh expression of CXCR4, it demonstrated the CXCR4-A549 cells were able signifcantly to increase expression of the protine by Western blotting analysis. The migration of A549-CXCR4 cells was increased 1.62 times than that of of A549 cells (P<0.05). The chemotactic activity in both of both A549-CXCR4 and A549 cells induced by SDF-1 were improved, but the former much more than the latter in vitro (P<0.05). To assay the tumor formation and metastasis ability induced by over expression of CXCR4 in vivo, twelve nude mice were resepectively inoculated subcutaneously with CXCR4-A549 cells and A549 cells (six mice in each group). The mean weight of the tumors respectively was 4.37±0.96g and 3.24±1.16g in A549-CXCR4 and A549 group (P<0.05). Moreover, the metastatic tumors were affirmatively formed microscopically and pathologically in 6 mice iduced by A549-CXCR4 cells group. In contrast, there were no typical metastatic tumors formed just only 2 mice with invasive tumor cells within the lungs in A549 cells group. It was suggestted the ability of prolifation and invasion of lung cancer cells with CXCR4 over expression.(3) Compared with A549 control cells, CXCR4 over expression increased the proportion of the cells in G2 and S phase in CXCR4-A549 cells and the proliferation index was added 9.72% and proportion of the cells apoptosis was decreased 3.60%. But there was no significant difference between CXCR4-A549 and A549 cells. Subsequently, though intracellular calcium was increased rapidly in a time-dependent mode while both CXCR4-A549 and A549 cells stimulated with SDF-1. However, the mean fluorescence in CXCR4-A549 cells was stronger than that in A549 cells. There was a significant difference in MMP-2 and VEGF-C expression between CXCR4-A549 and A549 cells by Western blot and RT-PCR analysis. These results indicated that regulation of CXCR4 expression was corelated to MMP-2 and VEGF-C activity in lung cancer cells. Phosphorylation of AKT and ERK was strongly increased after stimulation with SDF-1 in 100ng/ml during maximum 30 min. It was suggested that MAPK and PI3K pathway activated by SDF-1 with CXCR4 might be cooperated in metastasis of lung cater cells.Conclusions(1) There were no significant differences in expression of CXCR4 protein between histological types or differentiation degrees of the lung cancer cells. However, it was significantly associated with TNM stages.(2) CXCR4 over expression was functionally actived by SDF-1a. Sequently, migration and invasion of lung cancer cells were promoted the in vivo and in vitro either.(3) The calcium channel might cooperated with MAPK/ERK pathway activated by SDF-1/CXCR4 axis and PI3K/AKT pathway resulting in the activity of downstream mediators, MMP-2 and VEGF-C activity in metastasis of lung cater cells.Based on the results in this study, the role and mechanism of SDF-1/CXCR4 axis in metastasis of lung cancer both in vitro and in vivo were aproached. SDF-1a/CXCR4 axismay be a potential target for NSCLC therapy.更多还原