| 摘要: |
| 【目的】探讨猪脂肪甘油三酯水解酶(adipose triglyceride lipase, ATGL)基因在脂肪细胞脂解中的作用。【方法】构建针对猪ATGL基因CDS区113和1 358位点的慢病毒干扰载体，包装后用其感染猪成熟脂肪细胞，检测ATGL和激素敏感脂酶(hormone-sensitive lipase,HSL)基因mRNA及其蛋白的表达情况；用甘油试剂盒检测细胞甘油释放量。【结果】成功构建了ATGL基因的慢病毒干扰载体，用其感染猪成熟脂肪细胞后，RT-PCR分析和Western印迹检测结果表明，试验组脂肪细胞ATGL的表达显著下调，HSL的表达没有明显变化；甘油释放量显著降低，其中针对ATGL基因CDS区113位点的siRNA1对ATGL mRNA的干扰效率达55%，甘油释放量降低了49%。【结论】成功构建了猪ATGL基因慢病毒干扰载体，其能显著降低ATGL基因mRNA及其蛋白在猪成熟脂肪细胞中的表达，降低脂肪细胞的甘油释放量，表明ATGL在猪脂肪细胞脂解中有着重要作用，且113位点是ATGL基因CDS区的最佳干扰靶位点。 |
| 关键词: ATGL 慢病毒 猪成熟脂肪细胞 脂肪分解 |
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| 基金项目:国家转基因生物育种培育重点专项“肉用家畜肌肉发育和脂肪沉积关键基因的克隆及功能验证”（2009ZX08009-157B）；西北农林科技大学创新团队建设计划项目 |
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| siRNA interference of ATGL gene expression suppress porcine adipocyte lipolysis |
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| Abstract: |
| 【Objective】The aim of this study was to determine the role of ATGL for lipolysis in porcine adipocytes.【Method】In order to construct lentivirus vector for RNA interference targeting 113 and 1 358 points of ATGL gene CDS,lentivirus were produced by packing plasmids into 293T cells and transfecting LentiH1-ATGL siRNA vector.The culture supernatant was harvested after 48 h and concentrated,then the lentivirus infected porcine mature adipocytes.Semi-quantitative RT-PCR and Western-blotting were used to detect the expression levels of ATGL and hormone-sensitive lipase(HSL) genes mRNA and protein;commercial kits were used to measure the glycerol release.【Result】ATGL mRNA and protein expression in group infected with lentivirus interfering vector targeting 113 points of ATGL gene CDS were significantly decreased by 55% compared with the control group(P＜0.05) and the glycerol releases were also reduced by 49%.ATGL silencing did not affect the expression of HSL.【Conclusion】This study successfully constructed the recombinant lentivirus interfering vector target on porcine ATGL gene,the lentivirus effectively inhibited ATGL mRNA and protein expression and glycerol release in porcine adipocytes.The results suggest that ATGL plays a key role in porcine fat cell lipolysis,and 113 point is the best interference target site of ATGL gene CDS. |
| Key words: ATGL lentivirus porcine mature adiopocyte lipolysis |
