论文部分内容阅读
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 study the effect of down-regulating PLIN1 expression on lipolysis in 3T3-L1 cells. Methods: RNA interference technology was used to construct three groups of positive and one negative sh-PLIN1 recombinant vector, and the bacterial liquid PCR and DNA sequencing identification. The expression of PLIN1A protein was detected by Western blot to evaluate the down-regulation effect of vector. Two days after transfection of the vector, the lipid droplets were stained with Bodipy 493/503, the contents of triglyceride and glycerol in the cells were determined by enzymology method. The levels of triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and Its phosphorylated protein (p-HSL) expression. The concentration of cAMP and protein kinase A (PKA) in the cells was determined by enzyme-linked immunosorbent assay (ELISA). Results: The sh-PLIN1 interference vector was constructed successfully, and all the three positive vectors could significantly down-regulate the expression of PLIN1A protein (P <0.05). Compared with the negative transfected group, the lipid droplets in the sh-PLIN1 transfected group decreased, the triglyceride content decreased, the glycerol content increased, and the relative expression levels of ATGL and HSL increased significantly (P < 0.05). The relative expression of p-HSL and the concentration of cAMP and PKA had no significant difference (P> 0.05). CONCLUSION: Down-regulation of PLIN1 gene expression can accelerate the lipolysis rate of 3T3-L1 cells, which may be achieved by up-regulating the expression of ATGL and HSL. However, cAMP / PKA signaling pathway has no significant effect on it.