p62基因缺失对人脂肪间充质干细胞成脂的促进作用

doi:10.13481/j.1671-587x.20200513

摘要/Abstract

摘要： 目的：探讨p62基因缺失对人脂肪间充质干细胞（hADSCs）自噬活性的影响，阐明脂肪分化的分子机制。方法：应用Ⅰ型胶原酶消化法从人大腿皮下脂肪抽吸液中分离培养hADSCs，使用80 μmol·L^-1油酸（OA）联合地塞米松和胰岛素对hADSCs进行成脂诱导；将hADSCs分为对照组和p62基因缺失组，p62基因缺失组hADSCs转染p62 shRNA慢病毒载体，对照组hADSCs转染hADSCs空载体。采用CCK-8实验绘制2组细胞的生长曲线并观察细胞增殖活性变化；应用尼罗红染色观察脂滴的形成（尼罗红脂滴染色阳性细胞数），Western blotting法检测成脂标志转录因子C/EBPα和微管相关蛋白1轻链3Ⅱ型/微管相关蛋白1轻链3Ⅰ型（LC3Ⅱ/LC3Ⅰ）蛋白表达，磺酰成二胺（MDC）染色检测2组细胞成脂中的自噬活性（MDC染色阳性细胞数），采用LC3与Mitotracker Red荧光共定位检测2组细胞的线粒体自噬活性，使用环孢菌素A（CsA）分别抑制2组细胞线粒体自噬活性并进行成脂诱导，光镜下计数含脂滴细胞数。结果：分离培养的细胞呈多角形或梭形，传代后细胞呈漩涡状排列；OA诱导hADSCs成脂后，细胞内有大量脂滴形成。CCK-8实验，在培养6 d后p62基因缺失组细胞增殖活性较对照组明显降低（P<0.01）。2组细胞诱导成脂后，与对照组比较，p62基因缺失组hADSCs中尼罗红脂滴染色阳性细胞数增多，C/EBPα蛋白表达水平升高（P<0.01），MDC染色阳性细胞数明显升高（P<0.05），LC3Ⅱ/LC3Ⅰ蛋白表达水平升高（P<0.01），细胞中LC3的点状化和颗粒化增多，与线粒体的共定位现象增加。使用CsA抑制线粒体自噬后，光镜下观察，p62基因缺失组含脂滴细胞数降低到与对照组接近的水平。结论：p62基因缺失可通过增强总自噬及线粒体自噬促进hADSCs的成脂。

关键词: 脂肪间充质干细胞, 成脂, 油酸, 细胞自噬

Abstract: Objective: To explore the effect of p62 gene deletion on the autophagy activity of human adipose-derived stromal cells (hADSCs), and to clarify the molecular mechanism of fat differentiation. Methods: The hADSCs were isolated and cultured from human thigh subcutaneous fat tissue by typeⅠcollagenase digestion method. Adipogenic induction of hADSCs was performed with 80 μmol·L^-1 oleic acid(OA) combined with dexamethasone and insulin. The hADSCs were divided into control group and p62 gene deletion group. The hADSCs in p62 gene deletion group were transfected with p62 shRNA lentiviral vectors. The hADSCs in control group were transfected with empty vectors. CCK-8 assay was used to draw the growth curves of cells in two groups,and the changes of cell proliferation activities were observed; Nile red staining was used to observe the lipid droplet formation(number of Nile red staining positive cells); Western blotting method was used to detect the expression levels of adipogenic marker transcription factor C/EBPα and microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ/Ⅰ (LC3Ⅱ/LC3Ⅰ).The autophagy activities (number of MDC staining positive cells) in two groups were detected by MDC staining. The mitochondrial autophagy activities of the cells in two groups were detected by fluorescence colocalization of LC3 and Mitotracker Red. Cyclosporin A(CsA) was used to inhibit the mitochondrial autophagy activities of the cells in two groups to induce adipogenesis, and the number of cells containing lipid droplets was counted under light microscope. Results: The isolated and cultured cells were polygonal or spindle-shaped, and the cells were arranged in a vortex after passage. A large number of lipid droplets were formed in the cells after OA-induced adipogenesis of hADSCs. The CCK-8 assay results showed that the number of cell proliferation activity in p62 gene deletion group 6 d after culture was significantly decreased (P<0.01).After adipogenesis induction,compared with control group, the number of Nile red staining positive cells in p62 gene deletion group was increased, the expression level of C/EBPα protein was increased (P<0.01); the number of MDC staining positive cells was significantly increased (P<0.05), and the expression levels of LC3Ⅱ/LCⅠproteins were increased (P<0.01); the pitting and granulation of LC3 in the cells was increased, and the colocalization with mitochondria was increased. The number of cells containing lipid droplets in p62 gene deletion group was reduced to a level close to that in control group under light microscope after CsA was used to inhibit the mitochondrial autophagy. Conclusion: The deletion of p62 gene can promote the adipogenesis of hADSCs by enhancing the total autophagy and mitochondrial autophagy.

Key words: adipose-derived stromal cells, adipogenesis, oleic acid, autophagy

中图分类号:

R329.4

曾瑞霞, 张轶博. p62基因缺失对人脂肪间充质干细胞成脂的促进作用[J]. 吉林大学学报(医学版), 2020, 46(05): 979-984.

ZENG Ruixia, ZHANG Yibo. Promotion effect of p62 gene deletion on adipogenesis of human adipose-derived stromal cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(05): 979-984.

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