Objective To establish siRNA adenovirus vectors targeting rat glucocorticoid-induced leucine zipper ( GILZ) , and infect rat L6 myoblasts cultured in vitro, in order to provide experimental basis for further investigation of regulatory effects of GILZ on skeletal muscle metabolism in sepsis.Methods (1) Three specific siRNA sequences targe-ting rat GILZ mRNA and one control sequence were designed and cloned.Double stranded oligo DNAs were inserted into vector GV119 which had been double-digested by AgeⅠand EcoRⅠto construct the shuttle plasmid.After confirmation by sequencing, the shuttle plasmid and the adenovirus backbone plasmid pBHG lox△E1,3 Cre were co-transfected into HEK293 cells.The recombinant adenoviruses named Ad-siRNA-1-GILZ, Ad-siRNA-2-GILZ and Ad-siRNA-3-GILZ were acquired, propagated and purified, before detection of the recombinant adenovirus titer.(2)0.1, 1.0, 10.0 μL recombinant adenovirus were infected to L6 myoblasts in different media ( complete medium, Enhanced Infection Solution, complete medium with 5μg/mL Polybrene, Enhanced Infection Solution with 5 μg/mL Polybrene) .Expression of green fluorescent protein and morphological changes of L6 myoblasts were observed microscopically to identify the best condition for infection.(3) The L6 myoblasts were infected by negative control adenovirus (negative control group), Ad-siRNA-1-GILZ ( GroupⅠ) , Ad-siRNA-2-GILZ ( GroupⅡ) and Ad-siRNA-3-GILZ ( GroupⅢ) .Ex-pression of GILZ mRNA was determined via real-time PCR.Results (1) DNA sequencing and PCR results demonstra-ted that recombinant adenovirus was successfully constructed, and the titer of recombinant adenovirus was 2.0 ×1010 PFU/mL.(2) The best infection effect was demonstrated in Enhanced Infection Solution with 10μL recombinant adenovirus, in which the infection efficiency of L6 myoblasts was up to 80%.(3) Cell necrosis was not observed in the L6 myoblasts in-fected with the recombinant adenovirus, and there were large amounts of green fluorescent protein expressed in L6 myo-blasts.(4) Real-time PCR showed that there was no difference in GILZ mRNA expression between GroupsⅡ,Ⅲand the negative control group (P>0.05).While expression of GILZ mRNA in GroupⅠwas significantly decreased compared with the negative control group(P<0.01), with a silencing efficiency of 59.8%.Conclusion The siRNA adenovirus vectors targeting rat GILZ are successfully constructed, which markedly down-regulates the expression of GILZ in vitro. The study provides experimental basis for investigating the regulatory effects of GILZ on protein metabolism of skeletal mus-cles in sepsis.%目的：构建靶向大鼠糖皮质激素诱导的亮氨酸拉链蛋白（ GILZ）的siRNA腺病毒载体，并感染体外培养的大鼠L6成肌细胞，为进一步研究GILZ对脓毒症骨骼肌代谢的调控作用提供实验基础。方法（1）设计3条靶向大鼠GILZ mRNA的特异性siRNA序列和1条阴性对照序列，合成各序列的Oligo DNA，退火形成双链后，插入AgeⅠ和EcoRⅠ双酶切的载体GV119，构建腺病毒穿梭质粒，经测序证实构建成功后，与腺病毒骨架质粒pBHG lox△E1，3 Cre共转染HEK293细胞，获得重组腺病毒，分别命名为Ad－siRNA－1－GILZ、Ad－siRNA－2－GILZ和Ad－siRNA－3－GILZ，扩增纯化后测定病毒滴度。（2）0.1、1.0、10.0μL重组腺病毒在不同类型培养液（完全培养液、Enhanced Infection Solution、含5μg／mL Polybrene的完全培养液、含5μg／mL Polybrene的Enhanced Infection Solution）中感染L6细胞，显微镜下观察L6细胞形态改变和绿色荧光蛋白表达情况，确定最佳感染条件。（3）分别以阴性对照病毒（阴性对照组）、Ad－siRNA－1－GILZ（Ⅰ组）、Ad－siRNA－2－GILZ（Ⅱ组）和Ad－siR-NA－3－GILZ（Ⅲ组）感染L6细胞，Real time－PCR检测各组GILZ mRNA的表达量。结果（1）经测序分析和PCR验证，成功构建重组腺病毒载体，经测定滴度为2.0×1010 PFU／mL。（2）10μL重组腺病毒在Enhanced Infec-tion Solution的感染效果最佳，感染效率达80％。（3）重组腺病毒感染L6细胞后，细胞无明显坏死，荧光显微镜下可见大量绿色荧光蛋白表达。（4） Real time－PCR结果显示，Ⅱ、Ⅲ组GILZ mRNA表达量与阴性对照组比较差异无统计学意义（ P＞0.05），Ⅰ组GILZ mRNA表达量较阴性对照组明显降低（ P＜0.01），其沉默效率为59.8％。结论成功构建靶向大鼠GILZ的siRNA腺病毒载体，并可在体外细胞水平明显下调GILZ的表达，为后续研究GILZ对脓毒症骨骼肌蛋白代谢的调控作用提供实验基础。
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