摘要
目的构建表达端粒酶逆转录酶（hTERT）基因小干扰RNA（siRNA）腺病毒载体，观察其对肾癌细胞系Ketr-3生长抑制作用。方法将病毒Ad—hTERT体外感染人肾癌细胞系Ketr-3，分别用结晶紫染色、噻唑蓝（MTT）法检测其对细胞生长的影响，逆转录-聚合酶链反应（RT-PCR）测定hTERT基因mRNA的表达水平，免疫组织化学检测hTERT抗原的表达。结果结晶紫、MTT结果表明：在MOI=50时，Ad-hTERT感染的Ketr-3细胞发生明显病变；MOI=50的Ad—hTERT感染Ketr-3细胞后，在第3、5、7天细胞存活率分别为（92．3±1．2）％、（82．0±1．0）％和（72．0±1．0）％，与对照组比较差异有统计学意义（P〈0．05）；RT—PCR结果表明：病毒Ad-hTERT感染Ketr-3细胞3d后，hTERTmRNA表达是对照组的（50．5±1．7）％，差异有统计学意义（P〈0．01）；免疫组织化学结果表明：Ad—hTERT处理组的hTERT抗原表达较对照组显著减少。结论转录hTERT基因siRNA的重组腺病毒载体为肾癌基因治疗提供了新的工具。
Objective To construct recombinant adenoviruses vector expressing the small interfering RNA （silLNA） against hTERT gene and evaluate its potential for inhibiting the cell growth of a human renal carcinoma cells （Ketr-3） in vitro. Methods To construct the recombinant adenoviruse, the plasmid pCA13-hTERT was transfected in 293 cells together with pBHGE3. The recombinant adenovimses were verified by PCR. Viruses were purified ,propagated in HEK293 cells and purified according to sthe tandard techniques ,then functional PFU titers were determined. The inhibition of Ketr-3 cell proliferation by hTERT siR- NA was determined by MTT assay and crystal violet dye method. The expression of hTERT gene in adenovims-infected Ketr-3 cells was detected by RT-PCR and immunocytochemical staining. Results The successful cloning of hTERT-siRNA template DNA sequences into pCA13 was confirmed by the analysis of restriction enzyme digestion and nucleotides sequencing technique. The analysis of PCR indicated the recombinant adenovirus containing hTERT-siRNA gene. MTT assay and crystal violet dye method showed obvious cytopathic effect was observed in group treated with Ad-hTERT at 50 MOI. At 3rd,5th,7th day,Kerr-3 cell via- bihty was （92.3 ± 1.2） %, （82.0 ±1.0） % and （72.0 ± 1.0） % in group infected with Ad-hTERT at 50 MOI when compared with the control group （P 〈 0.05）. RT-PCR analysis revealed the expression of hTERT mRNA was （50.5 ± 1.7）% on day 3 postinfection with Ad-hTERT,much less than that of the control group （P 〈0.01 ）. Immunocytochemical staining showed the expression of hTERT-siRNA gene delivered by recombinant adenovirus Ad-hTERT could effectively inhibit hTERT gene expression in protein levels in vitro. Conlusion Recombinant adenovirus vector expressing siRNA against hTERT gene offers a new method and tool for human renal carcinoma gene therapy.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第1期77-79,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30570385）
卫生部科研基金资助项目（WKJ2005-026）
