人骨唾液蛋白非融合荧光蛋白载体的构建及在乳腺癌细胞中的表达(英文)

Construction of pIRES2-HBSP-EGFP vector and its expression in breast cancer cells

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： Bone sialoprotein （BSP） gene is expressed in human breast caneer cells, in which bone metastasis occurs easily outside the mineralized tissue. Clinical observation shows that the expression level of BSP of breast cancer cells at bone metastasis is higher that at the primary site; therefore, BSP may be closely related to tumor specific bone metastasis. The study on breast cancer bone metastasis can provide new drug target for clinical prevention and treatment. OBJECTIVE： To establish breast cancer cell strains of BSP with stable expression and observe the effect of BSP in the whole process of breast cancer bone metastasis. DESIGN： Controlled experiment. SETTING： College of Biological Sciences and Engineering, South China University of Science and Technology; Medical Experiment Center, Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA. MATERIALS： This experiment was conducted in the Medical Experimental Center, Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA,betweer November 2003 and March 2004. pIRES2-EGFP vector （5.3 kb） was purchased from BD Biosciences Clontech Inc.; E.Coli.Top10, pB-hBSP plasmid containing the coding region of hbsp, and human breast carcinoma cells, MDA-MB-231BR that was specifically transferred to brain and MDA-MB-231-BO that was specifically transferred to bone. METHODS： hbsp gene was subcloned from pB-hBSP vector by PCR. Bgl Ⅱ and Pst Ⅰ restriction enzyme sites were inserted at 5＇ and 3＇ ends, orientation cloned to eukaryon expression vector pIRES2-EGFP, and constructed recombinant vector pIRES2-EGFP. The constructed recombinant vector was transfected into MDA-MB-231BR that was specifically transfe ned to brain and MDA-MB-231BO that was specifically transferred to bone. MAIN OUTCOME MEASURES： Construction of pIRES2-hBSP-EGFP recombinant expression vector, recombinant expression vector pIRES2-hB- SP-EGFP transfecting breast cancer ceils. RESULTS： ① pIRES2-hBSP-EGFP was successfully constructed. ②Breast cancer strains specific in bone metastasis and brain metastasis were successfully transfected. The fluorescence labeling could be observed under the fluorescence microscope, and BSP had corresponding expression. CONCLUSION： The successful construction and transfection of pIRES2-hBSP-EGFP of eukaryon expression vector would lay foundation for further study on the role of BSP in breast cancer metastasizing to bone in vivo or in vitro.