【作者】 黄琰菁；

【导师】 于世英；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2010， 博士

【摘要】 第一部分Abil蛋白在脑胶质瘤和乳腺癌组织中的表达目的：研究脑胶质瘤组织中Abil蛋白的表达及其临床意义,以及乳腺癌组织中Abil、c-Abl和WAVE2蛋白的表达及其相互关系。方法：应用免疫组化S-P法检测38例脑胶质瘤组织和7例正常脑组织中Abil蛋白的表达,以及66例乳腺癌组织和24例正常乳腺组织中Abil、c-Abl和WAVE2蛋白的表达。结果：1.脑胶质瘤组织与正常脑组织相比,Abil蛋白表达明显下降。在脑胶质瘤组织中,Abil强阳性率与组织学分级呈负相关。2.乳腺癌组织与正常乳腺组织相比,Abil和WAVE2蛋白表达明显下降,而c-Abl蛋白表达无明显下降,但有蛋白定位的改变。3.Abil强阳性率与乳腺癌组织的肿瘤大小、组织学分级、淋巴结转移及临床分期呈负相关性,与患者年龄无关。c-Abl阳性率及WAVE2强阳性率均与乳腺癌组织的组织学分级、淋巴结转移及临床分期呈负相关性,与患者年龄及肿瘤大小无关。4.乳腺癌中Abil蛋白表达与c-Abl和WAVE2蛋白表达呈正相关。结论：1.Abil在脑胶质瘤和乳腺癌中的低表达均与不良预后因素相关。2.乳腺癌中Abil蛋白表达的变化可影响c-Abl蛋白的定位和WAVE2蛋白的表达。推测Abil在Abl/Abil/WAVE2通路中可能具有至关重要的地位。第二部分稳定转染Abil基因的脑胶质瘤细胞株的建立目的：前期研究表明脑胶质瘤组织中Abil表达明显降低,为探讨Abil与脑胶质瘤发生发展的关系,建立稳定转染Abi1基因的脑胶质瘤细胞株。方法：采用Western blot检测U251 MG、H4、U-87 MG及C6这4株脑胶质瘤细胞Abil蛋白的表达,选取表达最低的细胞株,采用脂质体法进行pEGFP-C3/Abil质粒的稳定转染。用G418筛选转染株2周后,采用单克隆化操作,将细胞接种于96孔板。最终,扩增出的单克隆细胞株用Western blot检测其Abil的表达情况。结果：1.4株脑胶质瘤细胞Abil蛋白表达都降低,其中以U251 MG细胞Abil表达为最低。2.经8周G418筛选以及Western blot鉴定,得到可稳定、较高水平表达Abil的U251 MG细胞株。结论：成功建立了稳定转染Abil基因的人脑胶质瘤U251 MG细胞株。第三部分Abil对脑胶质瘤生物学行为的影响目的：探讨Abil对脑胶质瘤生物学行为的影响,为Abil基因应用于脑胶质瘤基因治疗提供理论依据。方法：研究稳定转染Abil基因的人脑胶质瘤U251 MG细胞株的生物学行为。采用倒置显微镜和吉姆萨染色观察细胞形态。采用流式细胞术检测细胞DNA含量。采用生长实验、软琼脂克隆形成实验及Ki67标记等方法评价细胞增殖能力。采用划痕试验和迁移实验检测细胞迁移能力,采用侵袭实验检测细胞侵袭能力。结果：稳定转染Abil基因的U251 MG与对照组相比,发生了以下变化：1.细胞形态向分化程度高的方向变化。2.多核细胞的比例下降。3.细胞生长曲线下移,克隆形成率和Ki67表达降低。4.细胞迁移能力上调。结论：Abil对脑胶质瘤的恶性转化可能有抑制作用。Abil可以抑制脑胶质瘤细胞增殖,上调细胞迁移能力。更多还原

【Abstract】 Part 1 The expression of Abil in glioma and breast cancer tissuesObjective To investigate the expression and significance of Abil protein in glioma. And to explore the expression of Abil, c-Abl and WAVE2 proteins in breast cancer and their correlation.Methods The expression of Abil was examined in 38 glioma tissues and 7 normal brain tissues by immunohistochemical staining. The expression of Abil, c-Abl and WAVE2 were examined in 66 breast cancer specimens and 24 normal mammary tissues by immunohistochemical staining.Results 1. The strong positive rate of Abil was significantly lower in glioma group than in normal control group. The strong positive rate of Abil was negatively correlated with histological grade of glioma.2. The strong positive rate of Abil and WAVE2 were significantly lower in breast cancer group than in normal control group. The positive rate of c-Abl was not significantly lower in breast cancer group than in normal control group, but its location changed.3. In breast cancer, the strong positive rate of Abil was negatively correlated with tumor size, histological grade, lymph node metastasis and clinical stage, but not with age. The positive rate of c-Abl and the strong positive rate of WAVE2 were negatively correlated with histological grade, lymph node metastasis and clinical stage, but not with age and tumor size.4. In breast cancer, positive correlation was found between the expression of Abil protein and that of c-Abl protein, and between the expression of Abil protein and that of WAVE2 protein.Conclusion 1. The reduction of Abil expression is correlated with poor prognosis of glioma and breast cancer patients.2. The reduction of Abil expression may influence the location of c-Abl protein and the expression of WAVE2 protein. So it is hypothesized that Abil may have a key role in Abl/Abil/WAVE2 passway. Part 2 The construction of glioma cell line stably transfected with Abil geneObjective Our prior studies indicated that the expression of Abil in glioma tissues was relatively low. To explore the relationship between Abil and tumorigenesis and development of glioma, glioma cell line stably transfected with Abil gene was constructed.Methods The expression of Abil in 4 glioma cell lines (U251 MG, H4, U-87 MG and C6) were detected by Western blot. pEGFP-C3/Abil plasmid stably transfected glioma cell line in which Abil expression is lowest among four glioma cell lines by Lipofectamine 2000. Then the cells were cultured in DMEM containing G418. After two weeks, cells were seeded into 96 wells plates. So, different clones were got, which were amplified from a single cell. Abil expression in different monoclones were tested by Western blot.Results 1. Abil expression in U251 MG cells was lowest among four glioma cell lines.2. A glioma cell line which stably expressing Abil at high level was obtained by 8-week G418 selection and Western blot identification.Conclusion Glioma cell line U251 MG stably transfected with Abil gene was successfully constructed.Part 3 The effects of Abil on biological behavior of gliomaObjective To explore the effects of Abil on biological behavior of glioma.Methods To evaluate the effect of Abil on cell morpholoy, U251 MG stably transfected with Abil gene were observed by converted microscope with or without Giemza staining. The cell cycle distribution was analyzed by flow cytometry. Growth assay, soft agar colony formation assay and Ki67 staining were used to analyse cell proliferation. For evaluation of in vitro motility of glioma cells, a monolayer wounding(scratch) assay and transwell migration assay were performed. Matrigel invasion assay was performed to evaluate invasive capability in vitro.Results 1. Upregulation of Abil expression changed morphology of U251 MG cells. 2. Abi1 decreased the proportion of multinucleate cells in U251 MG cells.3. Abi1 downregulated the growth rate of U251 MG cells, decreased cloning efficiency and Ki67 expression.4. Abi1 increased migration of U251 MG cells.Conclusion Abi1 could inhibit malignant transformation of glioma. Abi1 suppressed cell proliferaion of glioma, and increase migration.更多还原