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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 genetically modified VCAM-1 on adipogenic differentiation of mouse mesenchymal stem cells (MSCs) and its mechanism. Methods: MSC cells (MIGR1-VCAM-1) overexpressing VCAM-1 and MSC cells transfected with empty vector (MIGR1) were respectively induced to differentiate into adipocytes and were detected by in situ oil red O staining and real-time PCR Adipogenic differentiation and the expression of related key transcription factors C / EBPα and PPARγ. Western blot was used to detect the activation of P38, ERK and JNK pathway. MSCs were treated with signal pathway inhibitor to observe the changes of adipogenic differentiation ability. Results: Compared with control group MIGR1 / MSCs, MIGR1-VCAM-1 / MSC overexpressing VCAM-1 significantly increased lipid droplets and increased number of fat cells (P <0.01), both in autophagy and induced differentiation groups. At the same time, the expression of C / EBPα and PPARγ, the key transcription factors regulating adipogenesis at mRNA level, were significantly up-regulated. Western blot analysis showed that JNK pathway was significantly down-regulated in adipogenic differentiation regulated by VCAM-1, P38 and ERK pathway was significantly (P <0.01), and the mRNA expression of related transcription factors C / EBPα and PPARγ were also significantly increased (P <0.01). The inhibitory effect of JNK pathway on the adipogenic differentiation of MIGR1-VCAM- While the inhibition of P38 and ERK pathway down-regulated the mRNA expression of C / EBPα and PPARγ in MIGR1-VCAM-1 / MSC, with smaller lipid droplets and adipocytes. CONCLUSION: VCAM-1 overexpression can promote adipogenic differentiation of mouse MSC. VCAM-1 may promote adipogenic differentiation of mouse MSC by inhibiting JNK signaling pathway and activating P38 and ERK pathways.