【作者】 王辉；

【导师】 崔泽实；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 前言腺癌是全球发病率较高的恶性肿瘤,是对人类健康与生命危害最大的恶性肿瘤之一。在我国,近二十年来乳腺癌发病率呈迅速上升趋势。已成为城市女性人群恶性肿瘤死亡的首要因素。因此,对乳腺癌相关基因的研究及探讨及发病机制及诊断标准、治疗方法具有极其重要的意义。BAMBI（BMP and activin membrane-bound inhibitor）基因是由Onichtchouk等于1999年发现的,定位于10号染色体p12.3-11.2区域,编码产物为一个由260个氨基酸组成的跨膜糖蛋白,分子量29108道尔顿,又称作伪受体（pseudoreceptor）、NMA（non-metastatic gene A protein）,属于BAMBI家族。该家族成员与转化生长因子-βⅠ型受体胞外区都具有相似的结构。因此BAMBI能够整合到配体-受体复合物中,并与TGF-βⅡ型受体形成聚合物,但由于它不具有TGF-βⅠ型受体所特有的丝氨酸/苏氨酸激酶结构域,所以不能磷酸化胞浆内的Smad蛋白,从而在受体水平上阻滞TGF-β信号传导。有研究表明,对于乳腺癌等上皮组织来源的肿瘤而言,TGF-β可以抑制肿瘤细胞的增殖,而BAMBI在TGF-β信号通路中的负调节机制对肺癌的作用目前尚不清楚。另外,据报道BAMBI在人肺癌、结肠癌、肝癌中均存在表达上调,但在乳腺癌中的表达尚未报道。目的检测BAMBI在乳腺癌（breast cancer）细胞系中的表达与分布,探讨其与乳腺癌临床病理因素之间的关系。实验材料与方法一、乳腺癌细胞系三株细胞系MDA-MB-435s、MDA-MB-231、MCF-7分别为乳腺癌高度转移、中度转移和低度转移细胞系,用于免疫荧光、RT-PCR以及Western blot检测。二、半定量RT-PCR用RNAout提取RNA,合成BAMBI、GAPDH引物,应用两步法RT-PCR试剂盒合成cDNA链,然后进行PCR扩增。产物经2%琼脂糖凝胶电泳,用自动电泳凝胶成像分析系统采集图像,测定条带积分光密度值。三、细胞培养三株乳腺癌细胞系MDA-MB-435s、MDA-MB-231、MCF-7培养于含10%胎牛血清的DMEM培养基,在37℃、5%CO₂条件下孵育。四、Western blot检测离心收集细胞。加入适量裂解液,离心取上清。蛋白定量后电泳、转膜,封闭加入一抗（1∶1000）4℃过夜。次日加入二抗（1∶10000）常温2h,加入ECL试剂暗室曝光,用自动电泳凝胶成像分析系统采集图像,测定条带积分光密度值,计算BAMBI蛋白的相对含量。五、免疫细胞荧光染色收集细胞,细胞爬片,用多聚甲醛固定,封闭后加入一抗（1∶200）4℃过夜,次日加入FITC标记的二抗（1∶100）室温2h,PI（1∶1000）核复染,封片。应用激光共聚焦扫描显微镜系统观察采集图像。结果BAMBI蛋白主要表达在胞膜和靠近胞膜的胞浆,BAMBImRNA在MDA-MB-435s中的表达高于MDA-MB-231且其表达在MDA-MB-231中高于MCF-7。分别比较,差异具有显著意义（P＜0.05）。MDA-MB-435s、MDA-MB-231、MCF-7的蛋白相对含量分别为0.963±0.06 1,0.957±0.048,0.769±0.103,差异有显著性意义（P＜0.05）。结论实验结果揭示:BAMBI的异常高表达与乳腺癌的发生有关,且其表达高低与乳腺癌细胞系的转移能力有关。更多还原

【Abstract】 IntroductionBreast cancer is the most common human cancer and is the primary factor in cancer death worldwide.Therefore,as the strengthening of breast cancer in molecular biology and other fields as the basis for the etiology of breast cancer,exploring its pathogenesis and treatment is extremely important.BAMBI（BMP and activin membrane-bound inhibitor）gene in chromosome 10p12.3-p11.2 region,coding for a product by the 260 amino acid composition of the transmembrane glycoprotein,molecular weight of 29 108 Dalton,which also known as fake receptor（pseudoreceptor）,NMA（non-metastatic A protein gene）,belonging to the BAMBI family.The family members have similar extracellular domain structure with type I receptor ligand,such as TGF-β（the transforming growth factor-β,TGF-β）, Bone morphogenetic protein（bone morphogenetic protein,BMP）,activin.Therefore BAMBI can integration of ligand-receptor complexes,and form TGF-forming polymer with TGF-βⅡreceptor.But since it does not have TGF-typeⅠreceptor-specific serine/threonine kinase domain.It can not phosphorylated Smad located in cytoplasm of protein.,thereby blocking receptor of TGF-signal transduction.It is reported BAMBI were up-regulated expression of existence in lung colon carcinoma,liver cancer and whether express in breast cancinoma yet not clearly.ObjectiveTo study the expression and its significance of BAMBI in breast cancer and explore the relation beween BAMBI and clinical and pathological factors of breast cancer. Materials and Methods1.Breast Cancer Cell LinesThree Breast cancer cell lines including MDA-MB-435s、MDA-MB-231、MCF-7 were used for immunofluorescence technology,RT-PCR and Western blot.2.Semi-quantitative RT-PCRRNAout used to extract the total RNA,synthesis of BAMBI and primer of GAPDH.To synthesis Cdna chain by two-step RT-PCR kit,then amplification.The gel electrophoresis imaging analysis system were used to image acquisition of production by 2%agarose gel electrophores,then we calculated the integral optical density value of the band.3.Cell CultureMDA-MB-435s、MDA-MB-231、MCF-7 cells were cultured in the presence of 10%fetal calf serum DMEM.The conditions of incubation are 37℃,5%CO₂.4.Western blot ProtocolCentrifugal collection of cells adding some lysate,take the supernatant.After SDS-PAGE electrophoresis,transfer the membrane,incubation in primary antibody （1:1000）-4℃overnight after blockage.Incubation in HRP-conjugated secondary antibody（1:10000）for 2h at room temperature next day,using ECL reagent in darkroom and exposure,Gel electrophoresis imaging analysis system for capturing the image and calculating integral optical density value of the bands.5.Immunofluorescence ProtocolThe coverglass-cultured cell were fixed by formalin,incubation in primary antibody（1:200）,-4℃overnight after blockage.Incubation in FITC-conjugated secondary antibody（1:100）for 2h at room temperature next day,nuclear reataining with PI（1:1000）,mounting.The immunofluorescence were observed and imaged by the laser scanning confocal microscope image acquisition system. ResultsBAMBI protein expressed mainly in the membrane and the cytoplasm close to the membrane.BAMBI mRNA expression in the MDA-MB-435s was higher in MDA-MB-231 and its expression in the MDA-MB-231 was higher than MCF-7. Respectively,the difference was significant（P＜0.05）.The relative amount of BAMBI protein in MDA-MB-435s,MDA-MB-231 and MCF-7 were 0.963±0.061, 0.957±0.048,0.769±0.103,and the difference was significant（P＜0.05）.ConclusionThe result of experiments has revealed that the abnormally high expression of BAMBI has relationship to breast cancinogenesis.The expression of BAMBI has relationship with the metastasis of breast cancer.更多还原