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背景:人骨唾液蛋白基因在矿化组织以外的易发生骨转移的人乳腺癌中表达。临床观察显示骨转移处的乳腺癌细胞骨唾液蛋白的表达量要高于原发部位的乳腺癌细胞,因此骨唾液蛋白有可能与肿瘤特异性骨转移的关系密切。研究乳腺癌骨转移可为将来临床的预防和治疗提供新的药物靶点。目的:建立骨唾液蛋白的稳定表达乳腺癌细胞系,观察骨唾液蛋白在乳腺癌骨转移的整个过程中的作用。设计:对照实验。单位:华南理工大学生物科学与工程学院,解放军广州军区广州总医院医学实验中心。材料:实验于2003-11/2004-03在解放军广州军区广州总医院医学实验室完成。质粒、菌种和细胞:pIRES2-EGFP载体质粒,E.Coli.Top10、含有人骨唾液蛋白基因全长的克隆载体pB-hBSP和发生特异性骨转移以及脑转移的人乳腺癌细胞株MDA-MB-231BO和MDA-MB-231BR。方法:将人骨唾液蛋白基因通过聚合酶链式反应的方法从构建好的pB-hBSP载体中亚克隆出来,在其5’和3’端分别引入BglⅡ和PstⅠ限制性酶切位点,定向克隆至真核表达载体pIRES2-EGFP,构建重组载体pIRES2-hBSP-EGFP。利用脂质体转染的方法将构建好的重组质粒转入特异性脑转移和骨转移的乳腺癌细胞株MDA-MB-231BR和MDA-MB-231BO中。主要观察指标:pIRES2-hBSP-EGFP重组表达载体的构建。重组表达载体pIRES2-hBSP-EGFP转染乳腺癌细胞。结果:①成功构建人骨唾液蛋白和绿色荧光蛋白非融合表达的真核表达载体pIRES2-hBSP-EGFP。②成功转染特异骨转移和脑转移的乳腺癌细胞株,可在荧光显微镜下观察到荧光蛋白标记,人骨唾液蛋白得到相应表达。结论:真核表达载体pIRES2-hBSP-EGFP的构建及转染可为骨唾液蛋白在乳腺癌骨转移中的作用的体内、外研究奠定一定的基础。
BACKGROUND: Human bone sialoprotein gene is expressed in human breast cancer prone to bone metastases other than mineralized tissue. Clinical observation shows that the expression of bone sialoprotein in breast cancer cells at the bone metastasis is higher than that of the primary breast cancer cells, so bone sialoprotein may be closely related to tumor-specific bone metastases. Studying bone metastasis of breast cancer can provide new drug targets for future clinical prevention and treatment. OBJECTIVE: To establish a stable expression of bone sialoprotein in breast cancer cell lines and to observe the role of bone sialoprotein in the process of bone metastasis of breast cancer. Design: Control experiment. Unit: College of Biological Sciences and Engineering, South China University of Technology, PLA General Hospital of Guangzhou Military Region Medical Experiment Center. Materials: The experiment was performed at the Medical Laboratory of Guangzhou General Hospital of the Guangzhou Military Region of PLA from November 2003 to March 2004. Plasmids, strains and cells: pIRES2-EGFP vector plasmid, E. coli. Top10, cloning vector pB-hBSP containing the full length of human bone salivary protein gene and human breast cancer cell line MDA-MB with specific bone metastases and brain metastases -231BO and MDA-MB-231BR. METHODS: The human bone salivary protein gene was subcloned from the constructed pB-hBSP vector by polymerase chain reaction. The BglII and PstI restriction sites were introduced into the 5 ’and 3’ To eukaryotic expression vector pIRES2-EGFP to construct recombinant vector pIRES2-hBSP-EGFP. The constructed recombinant plasmids were transfected into breast cancer cell lines MDA-MB-231BR and MDA-MB-231BO with specific brain metastases and bone metastases by liposome transfection method. MAIN OUTCOME MEASURES: Construction of pIRES2-hBSP-EGFP recombinant expression vector. The recombinant expression vector pIRES2-hBSP-EGFP was transfected into breast cancer cells. Results: ①The eukaryotic expression vector pIRES2-hBSP-EGFP was successfully constructed, in which human bone salivary protein and green fluorescent protein were not fused. ② Breast cancer cell lines successfully transfected with specific bone metastases and brain metastases can be observed under fluorescence microscope fluorescent protein markers, human bone saliva protein expression. CONCLUSION: The construction of eukaryotic expression vector pIRES2-hBSP-EGFP and its transfection may provide a basis for in vitro and in vivo studies on the role of bone sialoprotein in bone metastasis of breast cancer.