【作者】 时晓芳；

【导师】 康巧珍；

【作者基本信息】 郑州大学 ， 细胞生物学， 2010， 硕士

【摘要】 研究背景乳腺癌是女性最常见的恶性肿瘤,也是导致女性患者死亡的重要原因之一尽管以手术和放化疗为主的综合治疗方式极大地提高了乳腺癌患者的生存质量,但仍有较高的复发和转移率。未发生转移的乳腺癌患者其治愈率可达98%,当发生转移时,患者预后差,特别是转移至内脏及脑的患者,其5年生存率仅27%。因此寻找新的乳腺癌相关基因和蛋白分子并阐明其与乳腺癌发生转移相关的分子机制,最终提高乳腺癌患者的预防与治疗水平是十分重紧迫的工作。蛋白4.1家族的发现起始于4.1R,它是从人红细胞中分离鉴定得到的一种膜骨架蛋白。随后又在哺乳动物细胞中发现了三种与4.1R有很高同源性的蛋白,分别是4.1G、4.1N、4.1B,它们的典型特征是都具有三个保守结构域：FERM、SABD和CTD结构域,并通过这些结构域与多种蛋白相互作用。对该家族进行的早期研究证实,其在维持细胞形态中发挥重要作用,可在spectrin-actin骨架蛋白与跨膜蛋白之间发挥连接作用。随着蛋白4.1家族与众多跨膜蛋白之间的相互作用不断得到证实,越来越多的研究显示蛋白4.1家族可能参与了细胞的生长与运动调控。同时,许多临床报道表明蛋白4.1在多种肿瘤中表达缺失,如在肺癌、脑膜瘤、乳腺癌、前列腺癌等多种肿瘤中发现了4.1B的缺失,4.1R和4.1G也被证实参与脑部肿瘤的发生。另外,作为细胞膜骨架成分的蛋白4.1在保持细胞-细胞,细胞-基质间的相互作用以及细胞骨架的组织构成方面有重要作用,而在肿瘤复杂的发生和转移过程中均涉及到细胞与细胞、细胞与基质间的相互作用的改变,所以推测蛋白4.1在肿瘤发生和转移过程中可能发挥重要作用。已有研究发现,蛋白4.1B与肉瘤细胞系和前列腺癌的转移有关,但该家族其他成员特别是4.1N在肿瘤转移中的作用还未见报道。本研究首次探究了蛋白4.1与乳腺癌转移的关系,并对蛋白4.1N在乳腺癌细胞增殖和转移过程中的功能进行了初步研究。研究目的1,研究蛋白4.1家族成员4.1R/G/N/B在转移能力不同的三个乳腺癌细胞系中的表达和定位情况,分析蛋白表达量及蛋白定位与乳腺癌细胞转移能力的相关性,筛选出与乳腺癌转移密切相关的蛋白4.1家族成员；2,通过体外稳定转染的方法使筛选到的细胞系重新表达缺失蛋白4.1,建立基因工程细胞系,为研究蛋白4.1功能及分子机制奠定基础；3,通过研究转染前后细胞生物学行为的改变,确定该蛋白在乳腺癌细胞增殖转移过程中的功能。研究方法本实验首先利用蛋白4.1s特异抗体,采用免疫印记(Immunol Bloting,IB)和细胞免疫荧光(Immunoflourence,IF)技术检测4.1R/G/N/B在转移能力不同的乳腺癌细胞系MCF-7(低转移),T-47D(中转移),MDA-MB-231(高转移)中的表达和定位,筛选出与乳腺癌转移密切相关的蛋白4.1分子。继而针对筛选到的蛋白4.1分子,通过构建与绿色荧光蛋白融合表达的真核质粒转染表达缺失的细胞系,经过抗生素筛选获得稳定转染的基因工程细胞系。最后通过细胞增殖实验,细胞粘附实验,细胞迁移实验和侵袭实验研究转染前后细胞生物学行为的改变,以确定该蛋白在乳腺癌增殖、转移过程中的功能。结果1,蛋白4.1R/B/G在三种乳腺癌细胞系中都表达,在MCF-7和T-47D细胞中主要位于细胞-细胞连接处或细胞膜上,在MDA-MB-231细胞中蛋白4.1B位于细胞核,蛋白4.1R/G位于细胞质。蛋白4.1N在MCF-7和T-47D细胞中表达且定位在细胞-细胞连接处,但在MDA-MB-231细胞中不表达,因此推测4.1N与乳腺癌转移密切相关；2,IB和IF检测结果表明成功建立了pEGFP-4.1N稳定转染的MDA-MB-231细胞系,为进一步研究4.1N在乳腺癌发生发展中的作用奠定了基础；3,转染前后细胞生物学行为的比较发现：转染pEGFP-4.1N细胞组与空质粒组和未转染组相比细胞增殖受到抑制(P<0.05),细胞对基质Fn的粘附性降低(P<0.001),细胞的迁移和侵袭能力也受到抑制(P<0.001)。结论1,蛋白4.1N的表达与乳腺癌转移密切相关；2,体外细胞生物学行为研究表明蛋白4.1N是乳腺癌细胞增殖、迁移的抑制因子,可能成为乳腺癌生物治疗与药物研发的新靶点。更多还原

【Abstract】 BackgroundBreast cancer is one of the most common malignancy in women and still represents a great health concern in China. Although the diagnosis and combined treatment, mainly the surgery and the radiotherapy & chemotherapy, of breast cancer have made great progresses, distant metastasis is still the main factor affecting the prognosis of patients. The recovery rate of non-metastatic breast cancer patients has been reported to be 98%, but can dramatically dicrease when the metastasis especially the internal organs and brain metastasis of the patients occur, with the 5-year survival rate being only 27%. So it is important to investegate the genes related to breast cancer genesis and identify the molecular mechanisms of tumor development and metastasis for bettering the clinical breast cancer treatment.Protein 4.1R, the member of the protein 4.1 gene superfamily, was first identified in blood which plays an important role in maintaining mechanical stability of red cell membrane. After that,3 homologous proteins,4.1G,4.1N and 4.1B, were also identified from mammalian cells which share the common homology with 4.1R and all contain three conserved domains:FERM domain (Four.1 protein, Ezrin, Radixin, Moesin), SABD (spectrin-actin-binding domain) and CTD (C-terminal domain). Previous study about protein 4.1 has showed the important function of these proteins in maintaining mechanical stability through co-localization bentween spectin and actin, and also promoting the interaction of spectrin-actin with transmembrane protein. With the identification of protein 4.1 family function in regulating transmembrane protein, more evidence suggest these proteins could have something to do with cell proliferation and motality. It was found that protein 4.1B was low-expressed in lung cancer, meningeoma, breast cancer and prostate cancer, and also found that protein 4.1R and 4.1G are involved in the genesis of brain tunor. Theses clinical evidence suggested the potential role in cancer development.As a cytoskeletal component, it is belived that 4.1s proteins play an important role in maintaining cell-cell and cell-matrix interactions, as well as the organization of the cytoskeleton. The facts that tumor genesis and metastasis process involved in the cell-cell and cell-matrix interaction changes, so 4.1s protein may have some potential roles in tumor metastasis. Previous studies have shown that 4.1B was related to metastasis of sarcoma cell lines and prostate cancer, but the roles of the other 4.1 members in tumor metastasis, especially 4.1N in breast cancer, have not been investigated. Based on these review, the study on relationship between protein 4.1N and breast cancer metastasis, and its functions in tumor development and metastasis were merit further investigated.Objective1. The expression and localization of protein4.1 family members (4.1R/G/N/B) in breast cancer cell lines with different metastatic capability were investigated. The relevance between expression and location of protein 4.1 s and metastatic of breast cancer was analized, followed by screening the 4.1s member which is closely related to breast cancer metastasis.2. The target protien 4.1 that closely related to breast cancer metastasis was expressed in cell line after transfection for the further functional mechanism sduty. 3. The role of screened 4.1 member in breast cancer metastasis was identified by comparision the biological characteristics before and after transfection in vitro.MethodsImmunolbloting and Immunolfluorescence methods were employed to detect the expression and subcellular location of 4.1R/G/N/B in breast cancer cell lines with different metastatic capability, MCF-7 (low metastatic), T-47D (middle metastatic) and MDA-MB-231 (high metastatic), by specific 4.1s antibodies. Protein was screened which is closely related to breast cancer metastasis. Then a plasmid fusion expressing this protein was constructed with EGFP and set up a stable transfected cell line. Finaly, the roles of the protein in breast cancer metastasis were identified by comparing the biological characteristics of breast cancer cells before and after transfection, cell proliferation assay, cell adhesion assay, cell migration and invasion assay.Results 1. Western blot and immunolfluorescence assay showed that 4.1R/B/G were all expressed in MCF-7, T-47D and MDA-MB-231 cells, but the subcellular location was different. In the MCF-7 and T-47D cells 4.1B protein was mainly located in the cell-cell junctions, In the highly metastatic MDA-MB-231 cells it was lacated in the nucleus, but 4.1R/G existed in the cytoplasm. Protein 4.1N was expressed in MCF-7 which was significantly higher than that in T-47D cells (P<005), but no expression was found in MDA-MB-231 cells. This result suggested that 4.1N may be closely related to breast cancer metastasis.2. IB and IF result showed that a stable cell line EGFP-4.1N/MDA-MB-231 was established which will serve as a cell model for the further research on 4.1N in breast cancer metastasis.3. Cell proliferation of MDA-MB-231 transfected with recombinant plasmid pEGFP-4.1N was significantly inhibited (P<0.05) compared to MDA-MB-231 which was transfected with empty vector and non-transfected controls. The adhesion of cells to matrix, cell migration and invasion were also significantly decreased (P<0.001).Conclusion1. Decreased expression of 4.1N protein is closely related to breast cancer metastasis.2. Study on the in vitro biological behavior showed that 4.1N can negatively regulate breast cancer cell proliferation and migration, which could become the potential target for cancer bio-therapy and drug development.更多还原