摘要
目的构建针对血管内皮生长因子受体Ⅱ(VEGFR2),又称激酶功能区受体(KDR)的pSilencerTM3.1siRNA(smallinterferingRNA)表达载体,并在前列腺癌细胞PC3中观察其干扰效果.方法设计针对KDR基因编码区的寡核苷酸链,体外退火后克隆入经BamHⅠ,HindⅢ双酶切线性化的干扰载体pSilencerTM3.1-H1neo中,对重组质粒进行酶切分析和DNA系列测定.以脂质体法将pSilencerTM3.1-H1neo空载体和3个(pSilencer3.1-KDR1,pSilencer3.1-KDR2和pSilencer3.1-KDR3)重组质粒分别导人PC3前列腺癌细胞系.72h后用RT-PCR和Westernblotting技术检测各实验组前列腺癌细胞内KDRmRNA及蛋白水平的表达情况.结果经酶切鉴定及DNA测序,重组质粒中已插入了目的基因片断.转染KDR-siRNA的前列腺癌细胞,72h后,而以pSilencer3.1-KDR3的抑制KDRmRNA及蛋白表达效果最为有效,其抑制率约为55%.结论成功构建了针对KDR的RNA干扰表达载体pSilencer3.1-KDR1,pSilencer3.1-KDR2和pSilencer3.1-KDR3,pSilencer3.1-KDR3能有效抑制KDR在前列腺癌细胞中表达,为将其进一步应用于前列腺癌的治疗研究奠定了基础.
AIM： To construct pSilencer^TM 3. 1-H1 small inferfefing RNA （siRNA） expression vector targeting human KDR gene and to observe its silencing effect in the PC3 prostate cancer cell line. METHODS： The designed oligos targeting KDR were cloned into the pSilencer^TM3.1-H1 neo vector, which was lineated after Bam HⅠ and Hind Ⅲ digestion. The recombimant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The reeombimant vectors of pSilencer 3.1-KDR1, pSilencer 3.1-KDR2 and pSilencer 3. 1-KDR3 were transfected by liposome-mediated tranafection into the PC3 prostate cancer cell line with high metastasis potential. At 72 h after transfection, the expression of KDR at the levels of mRNA and protein was detected by RT-PCR and Western blotting. RESULTS： The pSilencerTM 3.1-H1 siRNA expression vectors were constructed and confirmed after the enzyme digestion analysis and the DNA sequencing. The siRNA expression vectors of pSilencer 3.1-KDR1, pSilencer 3.1- KDR2 and pSilencer 3. 1-KDR3, were successfully constructed. pSllencer 3.1-KDR3 was most effective in the 3 which down-regulated 55% mRNA and protein of KDR at 72 h after transfection. CONCLUSION： pSilencer^TM 3. 1-H1 siRNA expression vectors targeting KDR were successfully constructed. The expression of KDR gene was inhibited effectively in PC3 cells transfacted by pSilencer 3.1-KDR3 ,which laid a basis for its application in the treatment of prostate cancer.
出处
《第四军医大学学报》
北大核心
2006年第10期898-901,共4页
Journal of the Fourth Military Medical University
基金
国家863计划资助项目(2001AA215321)
