过表达VCAM-1对小鼠间充质干细胞成脂分化的影响与作用机制

Effect of Vascular Cell Adhesion Molecule-1 Overexpression on Adipogenic Differentiation of Murine Mesenchymal Stem Cells and Its Mechanism

目的:探讨基因修饰过表达VCAM-1对小鼠间充质干细胞(MSC)成脂分化能力的影响与作用机制。方法:将稳定过表达VCAM-1的MSC细胞(MIGR1-VCAM-1)和转入空载体的MSC细胞(MIGR1)分别向脂肪细胞诱导分化,以原位油红O染色和real-time PCR检测成脂分化能力与相关关键转录因子C/EBPα和PPARγ的表达;Western blot检测相关信号通路P38、ERK和JNK通路的活化。利用信号通路抑制剂处理MSC,观察其成脂分化能力的变化。结果:无论是自分化组还是诱导分化组,过表达VCAM-1的MIGR1-VCAM-1/MSC与对照组MIGR1/MSC相比,脂滴变大,脂肪细胞数量显著增加(P<0.01);同时在mRNA水平调控成脂分化的关键转录因子C/EBPα和PPARγ表达显著上调;Western blot检测相关信号通路,结果表明JNK通路在VCAM-1调控成脂分化中明显下调,P38及ERK通路则显著上调;加入通路抑制剂后,JNK通路抑制可显著上调MIGR1-VCAM-1/MSC成脂分化能力,脂肪数量明显增加(P<0.01),且相关转录因子C/EBPα和PPARγ的mRNA表达也显著上升;而抑制P38及ERK通路则使MIGR1-VCAM-1/MSC的C/EBPα和PPARγ的mRNA表达水平下调,脂滴及脂肪细胞数则更小、更少。结论:过表达VCAM-1可促进小鼠MSC成脂分化,VCAM-1可能通过抑制JNK信号通路,活化P38及ERK通路促进小鼠MSC成脂分化能力。

Objective： To investigate the effect of vascular cell adhesion molecule-1 （ VCAM-1 ） gene overexpression on adipogenic differentiation of mouse mesenchymal stem cells （MSC） and explore its molecular mechanism. Methods： VCAM-1 overexpression MSC （MIGR1-VCAM-1/MSC） and the empty plasmid transfection MSC （MIGR1/MSC） were induced to adipogenic differentiation, oil-red-O staining and real-time PCR were used to detect the adipogenic differentiation ability and the mRNA expression level of key transcription factors C/EBP α and PPAR γ. The activation of P38, ERK and JNK pathways were analyzed by Western blot. Furthermore, the specific chemical inhibitors of MAPK pathway （SB203580,PD98059 and JNK inhibitor II） were added to the induced culture system and the alteration of the MSC adipogenic differentiation ability were evaluated. Results： no matter in self or induced differentiation groups, the lipid droplets in MIGR1-VCAM-I/MSC became larger, the amount of adipocyte increased than that in MIGR1/MSC（ P 〈 0. 01 ）, the mRNA expression level of C/EBPα and PPARγ/were upregulated, and JNK pathway were down-regulated while the P38 and ERK pathway were significantly up-regulated. The inhibition of JNK pathway of MIGR1-VCAM-1/ MSC could lead to increased mRNA expression level of C/EBP α and PPAR γ, the amount of adipocytes increased （P 〈0.01 ）, however, the inhibition of the P38 and ERK pathway of MIGR1-VCAM-1/MSC could lead to decreased mRNA expression level of C/EBP αand PPAR ,y, and the lipid droplets and the number of adipocytes became smaller and less. Conclusion： The overexpression of VCAM-1 may promote MSC to differentiate into adipocytes through inhibiting JNK signaling pathway, activating P38 and ERK pathways.