癌症作为世界上仅次于心脑血管疾病的人类第二大杀手，无论是对于其发病机理还是治疗对策，长期以来都占据着研究的热点。本论文通过免疫蛋白印迹 (Western-blot)、免疫组织化学染色 (IHC) 和酶联免疫吸附 (ELISA) 的方法在几种肿瘤组织或肿瘤患者血浆中鉴定了一种过表达的蛋白——人多种凝血因子缺失综合症蛋白2 (hMCFD2)，该蛋白在过去的研究中很少有涉及肿瘤领域的报导。我们在研究中发现，hMCFD2不仅与肿瘤的发生具有正相关性，并且在MTS细胞增殖实验中，可以显著促进多种肿瘤细胞的生长。利用transwell方法还发现hMCFD2可以显著的促进肿瘤细胞EJ的侵袭能力。但是在Matrigel胶成管实验中，我们没有发现hMCFD2对于血管内皮细胞HUVEC和肿瘤细胞T24的成管情况有影响。接着在对hMCFD2发挥生物学功能的作用机制的研究中，利用细胞增殖实验和western-blot方法对其增殖信号通路进行研究，发现二者对FGFR及其下游Akt、Erk的磷酸化影响一致，猜测二者可能都是通过FGFR信号通路来发挥功能的。FGFR受体的激活涉及到其配体的结合以及发生二聚化的程度。因此，我们先确定了MCFD2与FGF2的相互作用情况。通过表面等离子共振 (SPR) 和活性电泳方法检测到MCFD2与FGF2没有结合作用，用BS3交联方法也没有看到MCFD2对FGF2的二聚化产生影响，于是设计实验来对hMCFD2对FGFR的影响是否存在。首先用SPR的方法确认了二者可以发生结合，并且结合位点与FGF2-FGFR结合位点有部分重叠；其次用DSS交联方法发现hMCFD2可以显著促进FGFR的二聚化；最后通过细胞免疫荧光 (ICC) 手段得出hMCFD2还可以通过增强FGFR胞吞转运至细胞核表面的过程来促进细胞增殖。对于hMCFD2是否完全依赖于FGFR信号通路来完成促增殖作用，我们利用FGFR特异性抑制剂SU5402和BGJ398做了验证，证明hMCFD2确实完全依赖于FGFR信号来发挥功能。在论文的最后，我们用体外结合实验，即：ELISA、体外细胞黏附实验等确认了hMCFD2结合在FGFR胞外区的位置在D2区段，体内免疫共沉淀CoIP实.验也验证了体外结合的实验结果。我们在细胞增殖实验中发现D2的结合可以抑制hMCFD2的生物学功能。通过我们的实验，不仅证明了hMCFD2可作为一种出色的肿瘤分子标记物用于肿瘤的诊断，并且结合多种生物学方法研究了它在肿瘤细胞增殖中的功能以及发挥这种促增殖功能的途径，同时也在分子层面上研究清楚了hMCFD2的作用靶点以及结合位置，对于hMCFD2可能是一种癌基因的猜测以及相应的治疗手段提供了很大的参考价值; As world No.2 killer following cardiovascular disease, cancer always draws the attention of scientists to the mechanisms of tumorigenesis and cancer therapy.In the project, a protein, namely human multiple coagulation factor deficiency protein 2 (hMCFD2), was found to be overexpressed in several tumor tissues or the plasma of cancer patients, which was confirmed by western-blot analysis, immunohistochemistry staining together with enzyme-linked immunosorbent assay (ELISA). The protein was rarely been reported to be involved in cancer before. In our study, hMCFD2 proved to be directly related to tumorigenesis and was found to promote proliferation of several tumor cell lines in MTS experiment. Besides, hMCFD2 was notably found to enhance invasion capacity of EJ cell line using transwell method. But in tube formation assay based on Matrigel, hMCFD2 was found to have no effect on such abilities of human umbilical vein endothelial cells (HUVEC) and T24 cell lines.Further studies were carried out to reveal the molecular mechanism of it. Results from tumor cell proliferation assay and western-blot analysis indicated that hMCFD2 could enhance phosphorylation level of FGFR and its downstream, Akt and Erk, accordingly, it may perform this fuction via the same pathway with FGF2. Activation of FGFR involved binding to its ligands and dimerization of both receptors and ligands. Thus, we first tested whether MCFD2-FGF2 interaction existed. With the negative result of SPR and active electrophoresis, hMCFD2 had also no impact on FGF2 dimerization using BS3 cross-linking method. Afterward, the study was focused on hMCFD2-FGFR. Firstly, still using SPR method, hMCFD2 was found to be capable of binding to FGFR, and the binding sites partly overlapped with those between FGF2 and FGFR. Secondly, hMCFD2 was found to promote the level of FGFR dimerization. Lastly, hMCFD2 was observed to promote cell proliferation by strengthening endocytosis of FGFR to the surface of nucleus in cell immunofluorescence. FGFR specific inhibitors, SU5402 and BGJ398, were used to confirm that hMCFD2 was functionally dependent completely on FGFR signaling. Finally, in vitro assays such as ELISA and cell adhesion assay confirmed that hMCFD2 bound to D2 that belonged to the extracellular domains of FGFR. In vivo, Co-immunoprecipitation assay also verified this result. D2 segment was found to sufficiently inhibit hMCFD2 function in MTS assay.Our study demonstrated that hMCFD2 would be an excellent tumor marker in tumor diagnosis. Its role in tumor cell proliferation and the pathway involved in it were studied thoroughly using many biological methods. The study also obtained a doubtless result of hMCFD2 binding target on the cell surface and binding regions on it. All these conclusions provided valuable clues to hypothesize that hMCFD2 could be an oncogene and possible therapeutic target to this kind of cases.
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