CRISPR/Cas9敲除PLIN1基因增强3T3-L1脂肪细胞的脂解作用

CRISPR/Cas9 knockout plin1 enhances lipolysis in 3T3-L1 adipocytes

摘要:

应用CRISPR/Cas9技术敲除3T3-L1前脂肪细胞plin1，观察PLIN1缺失对脂肪细胞中脂肪水解的影响并探究可能机制。常规培养3T3-L1前脂肪细胞，电穿孔法转染plin1敲除载体，嘌呤霉素培养基挑选plin1敲除细胞，观察转染及筛选后的细胞存活率。“鸡尾酒”法诱导3T3-L1前脂肪细胞分化，酶法测定甘油和TG含量，油红O染色观察脂滴形态及数目的变化。Western blotting检测PLIN1、PPARγ、Fsp27和脂肪酶的蛋白表达；RT-PCR检测PLIN1和脂肪酶的mRNA表达。对照组细胞诱导分化后，微小脂滴数目较少，单房脂滴数目较多并围绕细胞核呈环型排列。相较于对照组，敲除组细胞诱导分化后微小脂滴数目增加，单房脂滴体积缩小，数目减少；细胞中PLIN1 mRNA及蛋白表达被显著抑制 (P<0.05)；甘油水平显著上升 (0.098 4±0.007 6)，TG含量显著下降 (0.031 0±0.005 3)；HSL和ATGL两种脂肪酶的mRNA及蛋白表达均升高 (P<0.05)；PPARγ和Fsp27的表达未有明显变化。上述结果表明plin1敲除后通过暴露脂滴中脂质以及上调脂肪酶等效应增强了3T3-L1脂肪细胞的脂解作用。

Abstract:

We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized “cocktail” method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4± 0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.