【作者】 蒋代凤；

【导师】 邱宗荫；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2002， 博士

【摘要】 恶性肿瘤细胞的转移是肿瘤发展到晚期的必然结果和导致患者死亡的主要原因，但迄今为止，对肿瘤转移机制的研究尚未获得突破性的进展。通过鉴定在肿瘤转移中起重要作用的关键分子，借以设计抗肿瘤转移的药靶和寻找早期诊断的生物标志，成为当前肿瘤转移的研究热点。迄今为止的研究结果表明，在肿瘤转移的复杂过程中,涉及到了以下几方面的调控因素：1）在转移启动初期，E-cadherin、Ig超家族、选择素、整合素等参与肿瘤细胞间的脱落以及肿瘤细胞与胞外基质的粘附；2）肿瘤细胞或间质细胞分泌的蛋白水解酶及其抑制剂，在降解胞外基质和血管的基底膜过程中发挥重要作用；3）在运动因子，生长因子，归巢因子等的作用下，肿瘤细胞迁移到转移靶器官；4）血管增生因子VEGF、bFGF、IL-8、PDGF等促进新生血管的形成，伴随着肿瘤细胞的增生，形成新的转移灶,完成转移过程。鉴于目前对肿瘤转移机制的研究往往针对单个或少数几个分子以及关注基因水平的改变,缺乏系统性、整合性的分析。因此，在所有这些调控因素中，究竟哪<WP=8>些分子在肿瘤转移中发挥关键作用，仍有待阐明。本研究以肺巨细胞癌高、低转移株为研究对象，采用比较蛋白质组技术鉴定了11个与转移相关的候选蛋白，并探讨了与肿瘤转移关系尚不太明确的新蛋白（IL-18）促进肺癌细胞转移的相关功能，具体研究结果如下：1 肺巨细胞癌高、低转移株模型的体外鉴定本章采用目前公认的transwell-chamber系统进行了细胞的体外迁移和侵袭能力分析，确定了来源于同一母细胞，但在裸鼠体内具有不同转移能力的肺巨细胞癌高、低转移株（分别命名为PLA801D、PLA801C）在体外存在侵袭能力的显著差异，高转移的PLA801D细胞株在体外的侵袭能力是低转移的PLA801C细胞株的4倍，但二者的体外迁移能力无显著差异。进一步通过对一些与肿瘤转移关系比较明确的指标的分析后证实：高转移的PLA801D细胞株分泌的MMP-2活性是低转移的PLA801C细胞株的1 .6倍，且MMP-2活性的增高来源于MMP-2的蛋白和RNA水平增高所致。此外，通过western blot和半定量RT-PCR技术检测到高转移的PLA801D表达P53、PCNA、CK18的蛋白和VEGF mRNA显著上调；而E-cadherin、nm23-H1、P16则在高转移的PLA801D细胞内显著下调；本研究结果还表明：一些据报道与肿瘤转移相关的指标，如MMP-9、TIMP-1、TIMP-2、CD44（V6）等并不是导致PLA801C、PLA801D细胞间出现转移表型差异的原因。这些体外实验的证据一方面解释了<WP=9>PLA801C、PLA801D细胞间出现转移表型差异的原因；另一方面也论证了PLA801C、PLA801D作为肿瘤转移模型的可靠性。2 应用比较蛋白质组技术鉴定了11个与肺癌转移相关的蛋白质本章首先采用宽范围的IPG胶条（pH3-10L）比较了来源于三批样品的共9张肺巨细胞癌高、低转移株的2-DE图谱，经考马斯亮蓝染色分离和鉴定了大约850个蛋白点，其中包括50个仅在PLA801C的2-DE图谱中检测到的蛋白点，69个仅在PLA801D细胞的2-DE图谱中检测到的蛋白点和77个具有2倍以上量变的蛋白点；PLA801C与PLA801D细胞株的2-DE图谱间的相关性为76%，且大部分差异蛋白点主要集中在pH4-7范围内；将其中的9个差异蛋白点进行了MALDI-TOF质谱鉴定。为了提高2-DE的分辨率，获得更多的差异表达蛋白，故进一步选择了窄范围的IPG胶条（pH4-7L）进行IEF，经考马斯亮蓝染色共鉴定了大约900个蛋白点，其中有29个蛋白点仅在PLA801C的2-DE图谱中检测到，66个蛋白点仅在PLA801D的2-DE图谱中检测到，72个具有2倍以上量变的蛋白点；PLA801C与PLA801D细胞株的2-DE图谱间的相关性为82%；将其中的15个差异蛋白点进行了MALDI-TOF鉴定。PLA801C与PLA801D细胞间2-DE图谱大约80%的相关性再次肯定了二者来源于同一母细胞，遗传背景相似的特点；而在高、低转移株细胞株间出现的差异表达蛋白则说明了转移表型的差异。通过对24个送检差异蛋白的MALDI-TOF分析以及数据库查询，成功鉴定了12<WP=10>个转移相关的候选蛋白（有两个蛋白点均鉴定为KCRB）。其中，候选蛋白 CK18, TGLC, GDIR, TPMF, IL-18 和ANX1 在高转移的PLA801D显著高表达，而ER60, CH60, PDX1, CLI1 和KCRB 则在 PLA801D显著低表达。有意思的是：IL-18仅在高转移的PLA801D细胞株的2-DE图谱中检测到。其余的12个蛋白点通过数据库查询后，未能有效鉴定，不能排除是新蛋白的可能。根据已有的文献报道：大部分的候选蛋白可通过调控肿瘤细胞的生长、迁移、粘附、血管生成、凋亡或肿瘤免疫等途径影响肿瘤细胞的侵袭和转移能力。该结果再次证明了比较蛋白质组技术获得多个转移相关蛋白的可靠性。值得一提的是：到目前为止，还未见有关文献报道候选蛋白IL-18和CLI1与肿瘤转移相关的直接证据，提示我们该蛋白有可能是与肿瘤转移相关的新蛋白。3 差异候选蛋白的进一步确证为了保证蛋白质组技术鉴定结果的可靠性，为进一步的功能分析提供保障，故在蛋白和mRNA水平对候选蛋白进行了进一步的验证。采用western blot技术，在蛋白水平的进一步验证结果表明：ANX1和CK18在高转移的PLA801D显著高表达，而CH60在PLA801D显著下调，且IL-18仅在PLA801D检测到，很明显：蛋白水更多还原

【Abstract】 Widespread metastasis is a common phenomenon and the major lethal cause of cancer. So far, however, very little is known about the mechanisms underlying metastasis. Once the key factors in tumor metastasis were identified, the drug targets and diagnosis markers could be obtained accordingly.It has been widely reported that many molecules were involved in the complex processes of tumor invasion and metastasis：1) E-cadherin, immunoglobin superfamily, selectin and integrin have been suggested to take part in the detachment of tumor cells from the primary site and interaction of tumor cells with the surrounding extracellular matrix； 2) Matrix-degrading enzymes and their inhibitors secreted by tumor cells or<WP=14>mesenchymal cells have been indicated to be involved in degradation of extracellular matrix and vascular basement; 3) Some growth factors and movememt factors, such as HGF、EGF、AMF、TGF、IFN-γ、IL-1、3、6 and CD44, have been implicated to play role in migration of tumor cells into secondary sites; 4) Angiogenetic factors, such as VEGF, bFGF, IL-8 and PDGF, have been demonstrated to be important in neoangiogenesis and distant metastasis. At present, most of the research associated with metastasis so far has been focused on the genetic changes of related molecules or single or few proteins without systematic study. But little is known about the key factors to trigger tumorigenic cells to initiate further invasion and metastasis facing with so many regulators up to now.On the basis of these considerations, proteomic strategy, combinedwith two-dimensional electrophoresis (2-DE) separation and mass spectrometry (MS) identification with advantage of high resolution, high reproducibility was used to separate and identify differentially expressed proteins between highly and lowly metastatic subpopulations. In this study we hoped to find out a series of protein cluster deeply involved in metastasis process and addressed the question whether there were new proteins to be associated with metastasis, moreover, to clarify the<WP=15>metastasis-associated function mediated by new candidate proteins. In the present study, 11 metastasis-associated proteins were seperated and identified by comparativeproteome technique using established metastatic model, more importantly, the metastasis-associated function mediated by IL-18 was further characterized.1 Characterization of PLA801C and PLA801D subpopulations with different potency of invasion and metastasis in vitroCell motility and invasion assay in vitro using transwell-chamber system demonstrated the significantly different potential of invasion between PLA801C and PLA801D, i.e. the invasion ability of highly metastatic PLA801D was about 4-fold of PLA801C, in addition, the motility potency was similar between PLA801C and PLA801D. Then, MMP and TIMP important in extracellular matrix degradation, and biomarkers associated cell growth, migration, invasion, angiogenesis and metastasis were all employed to explore the molecules associated with the significant difference of the metastatic potentials between PLA801C and PLA801D. Compared with PLA801C, highly metastatic PLA801D with significantly higher invasion and metastatic ability displayed MMP-2 activity to about 1.6 fold, due to the significant up-regulation of its protein<WP=16>and mRNA. Moreover, the protein level of tumor suppressor protein P53, proliferating cell nuclear antigen PCNA, intermediate filament cytokeratin 18 (CK18) and VEGF mRNA were much higher, while tumor suppressor protein P16, intercellular adhesion molecule E-cadherin and tumor metastasis suppressor protein nm23-H1 were significantly lower in highly metastatic PLA801D compared with lowly metastatic PLA801C, while the expression level of CD44 varient V6, MMP-9, TIMP-1 and TIMP-2 were close between PLA801C and PLA801D, implicating that these factors seemed not associated with metastasis of lung giant cell carcinoma. Obviously, the metastatic phenotype differences between PLA801C and PLA801D were caused by the di更多还原