人CD44基因真核表达载体构建及在乳腺癌细胞中的表达_临床医学论文.docVIP

人CD44基因真核表达载体构建及在乳腺癌细胞中的表达_临床医学论文 人CD44基因真核表达载体构建及在乳腺癌细胞中的表达_临床医学论文 作者：房新建 许文林 张徐 钱晖 陈琛 方莉莉 陈巧云 【摘要】 目的： 从人类乳腺癌耐多柔比星细胞株细胞（MCF中克隆CD44基因并构建CD44基因真核表达载体pcDNA3.1将pcNDA3.1稳定转染入人乳腺癌细胞株（MCF）细胞中。方法： 从MCF细胞中提取总RNA进行RT，获得全长的cDNA模板；将含有CD44基因的cDNA模板使用限制性内切酶进行双酶切获得带有酶切位点的CD44基因,再将带有酶切位点的CD44基因经TA克隆后测序验证，然后亚克隆入真核表达载体pcDNA3.1中，双酶切鉴定后再次测序验证。脂质体法将pcDNA3.1质粒转染到MCF细胞中，48小时后RT和流式细胞术检测转染前后CD44基因、蛋白在MCF细胞的表达变化。结果： 成功从人MCF细胞中克隆获得CD44基因并构建真核表达载体，转染后在MCF中检测到CD44基因mRNA和蛋白水平表达上调；将上述表达CD44基因及蛋白的瞬时转染MCF细胞经G418筛选两周后获得阳性克隆。结论： 成功克隆和建立人CD44基因真核表达质粒；pcDNA3.1能在MCF细胞中表达；在MCF中新发现一种CD44基因的变异体(基因库号FJ216964)；这为进一步研究其基因功能打下了基础。 【关键词】 CD44； 多药耐药； 侵袭； 乳腺癌 ［Abstract］ Objective: To clone CD44 gene from multidrug resistant human mammary carcinoma cells(MCFconstruct its eukaryotic expression vector. Which was transfected into MCF7 cells to obtain position clone.Methods： The full length cDNA encoding CD44 was obtained by RTor. The CD44 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotides sequencing, then inserted in the eukaryotic expression vector pcDNA3.1. The recombinant vector pcDNA3.1CD44 was transfected into MCF7 cells, and the expression changes of CD44 gene and protein were detected at 48 hour later, respectively. Results： CD44 gene was cloned from MCFstructed plasmid was confirmed containing the sequence of CD44 gene. After transient transfection, the mRNA and protein level of CD44 gene were obviously upregulated in MCF7 cells. Screened with G418 for two weeks, positive clone was obtained from the above cells. Conclusion： The expression vector pcDNA3.1；Our study may start a new approach to study the function of CD44 gene and provide a method of gene therapy. ［Key words］ CD44； multidrug resistance； invasion； human mammary carcinoma cells CD44分子是一类重要的尚未归类的黏附分子,于1980年由Dalchau等［1］用单克隆抗体技术检测并最初发现,而后经过学者认定属于同一CD44 家族。它是一种跨膜糖蛋白并且是透明质酸(HA)的主要受体。CD44分子的主要功能包括作为导向性受体调节淋巴细胞在血液及淋巴液中运行；促进成纤维与淋巴细胞等与细胞外基质成分HA、硫酸软骨素等的黏附。 最新研究表明CD44分子的异常表达还可显著提高恶性肿瘤细胞的转移能力,与恶性肿瘤的侵袭、转移及耐药密切相