摘要
目的:构建针对肿瘤增殖基因Ki67的小干扰RNA(Ki67-siRNA)腺病毒表达载体,体外研究其对人结肠癌细胞株SW620细胞生长影响,为结肠癌的基因治疗提供参考。方法:设计可形成小发夹结构的Ki67-siRNA对应模板DNA序列,克隆至缺陷型腺病毒质粒pCA13,构建Ki67-siRNA表达质粒pCA13-Ki67。鉴定正确后,将pCA13-Ki67与含有腺病毒右臂的质粒pBHGE3共转染至293细胞,包装成复制缺陷型腺病毒Ad-Ki67。大量扩增Ad-Ki67,氯化铯梯度离心纯化,测病毒滴度。Ad-Ki67感染人结肠癌细胞,结晶紫染色法检测细胞病理作用,MTT法检测细胞存活,Western印迹法检测Ki67表达。结果:酶切分析、测序鉴定表明pCA13-Ki67构建成功,PCR分析表明Ad-Ki67含有Ki67-siRNA序列。Ad-Ki67可显著抑制Ki67蛋白表达及结肠癌细胞生长。结论:含有Ki67-siRNA的重组腺病毒构建成功,其能抑制人结肠癌细胞Ki67基因表达和生长。
Objective： To construct a replication-deficient recombinant adenovirus vector that expresses small interfering RNA （siRNA） against the Ki67 gene and to evaluate its potential for suppressing the cell growth of human colon carcinoma SW620 cells in vitro, which will enable development of a gene therapy protocol for the treatment of human colon carcinoma. Methods： A Ki67-siRNA template DNA sequence, capable of forming a small hairpin structure, was designed. After renaturation, it was cloned into the defective adenoviral expression vector pCA13 to construct the Ki67-siRNA expression vector pCA13-Ki67. After verification, the pCA13-Ki67 vector and the plasmid pBHGE3 that contains the right arm of the adenovirus were cotransfected into 293 cells where they were packed as the replication-deficient adenovirus Ad-Ki67. Ad-Ki67 was abundantly amplified and then purified using a cesium chloride density gradient and virus titer was evaluated. The Ad-Ki67 was then used to infect the human colon cancer SW620 cells. The inhibition of SW620 cell proliferation by Ki67 siRNA was assessed using the MTT assay and crystal violet staining. The expression level of the Ki67 gene in adenovirus-infected SW620 cells was determined by Western blot. Results： Restriction digest patterns and sequencing analysis showed that pCA13-Ki67 was constructed successfully. PCR analysis indicated that the Ad-Ki67 contained the Ki67-siRNA sequence. The Ad-Ki67 significantly inhibited expression of Ki67 and growth of the colon cancer cells. Conclusion： Successful construction of the recombinant adenovirus vector containing the Ki67-siRNA gene was achieved and it can be used to significantly inhibit expression of the Ki67 gene and the growth human colon carcinoma cells.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2007年第4期190-193,共4页
Chinese Journal of Clinical Oncology
基金
国家自然科学基金(编号:30570385)
卫生部科研基金资助(编号:WKJ2005-2-026)
作者简介
通讯作者：郑骏年zhangjinnian@sina．com.
