下调Perilipin 1基因表达对3T3-L1细胞脂解的影响  

Effect of Down-regulated Perilipin 1 Gene Expression on Lipolysis of 3T3-L1 Adipocytes

作　　者：赵志武[1] 王君实[1] 马敏[1] 张少华[1] 燕炯[1] 

机构地区：[1]山西医科大学公共卫生学院,太原030001

出　　处：《中国生物工程杂志》2016年第3期17-22,共6页China Biotechnology

摘　　要：目的:研究下调围脂滴蛋白基因(PLIN1)表达对3T3-L1细胞脂解的影响。方法:采用RNA干扰技术,构建3组阳性及1组阴性sh-PLIN1重组载体,并进行菌液PCR和DNA测序鉴定。Western blot测定PLIN1A蛋白表达,评价载体下调效果。细胞转染有效载体2天后,Bodipy 493/503染色脂滴;酶学方法测定细胞中甘油三酯和甘油含量;Western blot检测甘油三酯脂肪酶(ATGL)、激素敏感性脂肪酶(HSL)及其磷酸化蛋白(p-HSL)的表达。酶联免疫吸附法(ELISA)测定细胞中环磷酸腺苷(c AMP)和蛋白激酶A(PKA)的浓度。结果:各sh-PLIN1干扰载体构建成功,且3组阳性载体均能显著下调PLIN1A蛋白的表达(P<0.05)。转染有效载体后,与阴性转染组相比,sh-PLIN1转染组细胞中脂滴减小,甘油三酯含量降低,甘油含量升高,ATGL和HSL相对表达量显著升高(P<0.05),p-HSL相对表达量及c AMP、PKA的浓度无显著性差异(P>0.05)。结论:下调PLIN1基因表达可加快3T3-L1细胞脂解速率,其可能通过上调ATGL和HSL的表达而实现,c AMP/PKA信号通路对其无明显调节作用。Objective： To investigate the effect on lipolysis of 3T3-L1 adipocytes by down-regulating Perilipin 1 gene expression. Methods： Three shRNA recombinant vectors targeting mouse PLIN1 gene and one negative control were designed,synthesized and detected by bacterium liquid PCR reaction and DNA sequencing.Western blot analysis was performed to verify the transfection effects. Bodipy 493 /503 staining was used to observe the lipid droplets size,and the content of cellular triglyceride and glycerol were detected after transfection of adipocytes. The adipose triglyceride lipase（ ATGL）,hormone-sensitive lipase（ HSL） and phosphorylase HSL（ p-HSL） were detected by Western blot,and the cyclic adenosine monophosphate（ c AMP） and protein kinase A（ PKA） were measured by enzyme linked immunosorbent assay（ ELISA）. Results： Every recombinant vector was constructed successfully,and the three positive vectors could significantly inhibit the protein expression of PLIN1A（ P〈0. 05）. Knockdown of PLIN1 gene expression decreased lipiddroplets size and cellular triglyceride level and increased glycerol level. In addition, the level of ATGL and HSL was increased significantly（ P〈0. 05）,and the level of cellular c AMP and PKA had no obvious change（ P〉0. 05）. Conclusion： Downregulating PLIN1 gene could promote lipolysis in 3T3-L1 adipocytes by up-regulating the level of ATGL and HSL,and c AMP-PKA signaling pathway had no significant effect on it.

关 键 词：围脂滴蛋白 RNA干扰 细胞转染 3T3-L1前脂肪细胞 脂解 

分 类 号：Q591.5[生物学—生物化学]