Lck反义RNA重组腺病毒载体的构建及其对肾小管上皮细胞Lck表达的影响  

Construction of Lck Gene Antisense RNA Adenoviral Vector and its Effect on Lck Expression in Renal Epithelial Cells

作　　者：邓英辉[1] 李清刚[2] 任崇余[3] 王质刚[2] 刘文虎[2] 

机构地区：[1]首都医科大学宣武医院肾内科,北京100053 [2]首都医科大学附属北京友谊医院肾内科,北京100053 [3]中国医学科学院,中国协和医科大学基础医学院生理室,北京100730

出　　处：《中国中西医结合肾病杂志》2006年第7期386-390,共5页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：北京市自然科学基金资助项目(No.7022012)

摘　　要：目的:观察淋巴细胞特异性蛋白酪氨酸激酶(Lck)在肾小管上皮细胞中的表达及Lck反义RNA对其表达的影响。方法:采用RT-PCR方法从肾小管上皮细胞中获得Lck目的基因片段,将目的基因片段反向插入pDC316质粒中,构建Lck反义RNA穿梭质粒(pDC316-antisenseLck),经PCR、酶切及测序鉴定正确后,将此穿梭质粒与辅助质粒pBHGloxΔE1,3Cre共转染HEK293细胞,重组产生Lck基因反义RNA腺病毒载体(Ad-antisenseLck)。用此重组腺病毒感染体外培养的肾小管上皮细胞,Westernblotting方法观测此重组腺病毒载体对IL-2诱导的Lck蛋白表达的影响。结果:构建出Lck反义RNA重组腺病毒载体,此重组腺病毒载体感染培养的肾小管上皮细胞后可明显抑制IL-2诱导的Lck蛋白表达(P<0.05),抑制率可达47%。结论:成功构建出Lck反义RNA重组腺病毒载体,此载体可在肾小管上皮细胞内发挥生物学作用,显著阻抑肾小管上皮细胞的Lck蛋白表达。Objective： The objective of this experiment was to construct recombinant adenoviral vector expressing antisense RNA to Lck gene, and investigate its effect on Lck protein expression in renal tubule epithelial ceils （TEC）, Methods： The Lck cDNA was obtained from TEC of BALB/C mice by RT-PCR and partial Lck cDNA was inserted into pDC 316 plasmid reversely to construct the antisense RNA shuttle plasmid（pDC 316-antisenseLck）. The recombinant plasmid was identified through PCR, restriction endonuclease digestion and DNA sequence analysis. This shuttle piasmid and helper plasmid were co-transfected into HEK 293 ceils through lipofectin to produce recombinant adenovirns（Ad- antisenseLck）, then the recombinant adenovirus was used to infect the TEC, The influence of the adenovirus on Lck protein expression induced by IL-2 stimulations was observed by th way of irnmunoblotting, Results：The recombinant adenovirus expressing Lck antisense RNA could inhibit the expression of Lck protein in TEC induced by IL-2 （P 〈 0. 05）, Conclusion： A kind of Recombinant adenovirus expressing Lck antisense RNA is constructed successfully and it could inhibit Lck protein expression induced by IL-2 stimulations in TEC significantly.

关 键 词：淋巴细胞特异性蛋白酪氨酸激酶 肾小管上皮细胞 反义RNA 重组腺病毒 

分 类 号：R692[医药卫生—泌尿科学]