腺病毒载体介导的siRNA对3T3-L1细胞脂联素表达的影响  

Effects of recombinant adenovirus-mediated siRNA on adiponectin expression in 3T3-L1 cells

作　　者：孟凡萍[1] 杨刚毅[2] 李钶[2] 李伶[1] 齐晓亚[2] 刘建雷[1] 李荣[2] 

机构地区：[1]重庆医科大学：医学检验系临床生物化学教研室,重庆400010 [2]重庆医科大学：附属第二医院内分泌科,重庆400010

出　　处：《第三军医大学学报》2008年第14期1312-1315,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30771037,30270631,30370671);国家教育部“春晖计划”(2003-56);美国NIH基金(R01HL073267,DK066003)~~

摘　　要：目的构建携带小鼠脂联素(Acrp30)siRNA腺病毒载体,并检测其对小鼠脂肪细胞Acrp30表达的影响。方法首先用KpnⅠ+NotⅠ双酶切本课题组前期构建的质粒pSilencer1.0-U6-Acrp30-1和pSilencer1.0-U6-Acrp30-3,得到目的片段U6-Acrp30-1和U6-Acrp30-3,并将目的片段亚克隆入穿梭质粒pShuttle,用PmeⅠ线性化穿梭质粒pShuttle-U6-Acrp30-1和pShuttle-U6-Acrp30-3,并与pAdeasy-1在BJ5183内同源重组,筛选、鉴定、测序后,在XL10-Gold中扩增重组腺病毒质粒,最后在293细胞内包装扩增为重组腺病毒Ad-Acrp30-1和Ad-Acrp30-3。用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达。结果设计并构建了小鼠Acrp30基因特异性siRNA腺病毒载体,该载体感染脂肪细胞后,能显著抑制其Acrp30 mRNA和蛋白表达。结论构建的Acrp30基因特异性siRNA腺病毒载体能有效的抑制脂联素基因在3T3-L1脂肪细胞中的表达。Objective To construct the RNA interfered adenovirus expression vector specific for Adiponectin （Acrp30） gene and observe its effect on the Acrp30 expression in 3T3-L1 cells. Methods Firstly, fragments U6-Acrp30-1 and U6-Acrp30-3 were obtained by using Kpnl ＋ Notl to digest the plasmids pSilencerl. 0- U6-Acrp30-1 and pSilencerl. 0- U6-Acrp30-3. Two adenovirus vector plasmids, Ad-Acrp30-1 and Ad-Acrp30-3 were constructed according to a two-step transformation protocol. Two newly constructed plasmids were transfected into 293 packaging cells to produce adenovirus, which were further multiplied and purified. Then 3T3-L1 cells were infected with the 2 recombinant adenoviruses respectively. The mRNA expression and protein levels of Acrp30 in these cells were evaluated by semi-quantitative RT-PCR and ELISA. Results Two recombinant adenoviral plasmids Ad-Acrp30-1 and Ad-Acrp30-3 were successfully constructed. They -remarkably downregulated the expression of Acrp30 at both the mRNA and protein levels in transfected 3T3-L1 cells. Conclusion The siRNA eukaryotic expression vectors against Acrp30 mRNA effectively inhibit the expression of Acrp30 in 3T3-L1 adipocytes.

关 键 词：RNA干扰 腺病毒载体 脂联素 3T3-L1细胞 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]