门静脉注射小鼠铁调素基因特异性RNA干扰腺病毒的方法研究  

Methodology of injection from portal vein the hepcidin gene-specific RNA interference adenovirus in mouse

作　　者：朱志鹏[1] 曾从丽[1] 方向明[1] 

机构地区：[1]浙江大学医学院附属第一医院麻醉科,杭州310003

出　　处：《中华实验外科杂志》2013年第5期1035-1037,共3页Chinese Journal of Experimental Surgery

摘　　要：目的门静脉注射小鼠铁调素（Hepcidin）基因特异性小发夹RNA（shRNA）重组腺病毒干扰载体，观察其对肝脏HepcidinmRNA的干扰效果。方法根据Genebank小鼠Hepcidin基因序列及相关文献，设计shRNA并合成l对互补的DNA单链寡核苷酸，退火后克隆至线性化pENTR／u6入门载体，以CMV—EGFP改造载体，利用Invitrogen公司LR重组系统与pAd／BLOCK—IT—DEST进行重组，构建pAD—U6-shRNA—CMV—EGFP腺病毒载体，转染293A细胞进行包装、扩增，获得腺病毒。实验组用BALB／C小鼠麻醉后行病毒门静脉注射，常规饲养10d后取成活小鼠肝组织，Trizol法提取RNA，半定量逆转录聚合酶链反应（SqRT—PCR）法检测mRNA表达。另检测正常小鼠肝脏mRNA表达作对照。结果经测序鉴定证实pAD—U6-shRNA—CMV—EGFP腺病毒载体构建成功，获得病毒的滴度为：2．7×10^9ifu／ml。实验组Hepcidin基因相对表达量为1．30±0．53，明显低于对照组（6．39±1．00）。结论成功构建小鼠Hepcidin基因shRNA重组腺病毒干扰载体，其门静脉注射能够有效抑制目的基因表达。Objective To construct the hepcidin gene-speeific RNA interference （RNAi） adenovirus in mouse and injected from portal vein, and observe its influence of RNA interference to hepeidin. Methods according to gene sequence in Genebank and relative literature, design of short hairpin RNA （shRNA） for hepcidin, synthesied a pair of complementary DNA liogo, annealed and linked with linearized pENTR/U6, modify the vector with cytomegalovirus （CMV）-enhanced green fluorescent protein （EGFP）. The LR recombination reactions were performed between the entry clones and pAd/BLOCK-ITDEST to generate adenovirus vectors pAD-U6-shRNA-CMV-EGFP. Then, the vectors were cotransfeet-ed with viral packaging mix into 293A eells to generate stock, portal vein of halb/c mice was revealled after anesthesiacd, the mice was injected with virus and breed normally for 10d. abstraction was performed in living mice with Trizol method. Expression level of hepcidin mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction （SqRT-PCR）. Results The recombinant adenovirus vectors expressing hepcidin-shRNA was successfully constructed, the titer of adenovial was 2. 7 ± 109 ifu/ml, the expression level in experimental group was 1.30 ± 0. 53, which was lower obviously compared to control group （6. 39 ± 1.00）. Conclusion The recombinant gene-specific RNAi adenovirus vectors expressing hepcidin-shRNA was successfully constructed, which can effective knochdown the expression of targeted gene by portal vein injection.

关 键 词：铁调素 腺病毒载体 门静脉 基因治疗 

分 类 号：R575[医药卫生—消化系统]