组织工程与重建外科杂志 ›› 2024, Vol. 20 ›› Issue (6): 672-.

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Yes相关蛋白对脂肪移植存活率的影响及其机制研究

  

  • 出版日期:2024-12-02 发布日期:2025-01-03

Effect of Yes-related protein on the survival rate of fat transplantation and its mechanism

  • Online:2024-12-02 Published:2025-01-03

摘要:

目的 探究Yes相关蛋白(YAP)对脂肪移植存活率的影响及其可能的作用机制。方法 体外培养3T3-L1细胞,分别用YAP siRNA和带有慢病毒载体的YAP过表达质粒进行处理,分为阴性对照siRNA(NC)组、YAP-si1组、YAPsi2组、YAP-si3组,慢病毒载体(Vector)组、YAP过表达(YAP)组。通过EdU检测细胞增殖活性,然后诱导3T3-L1细胞成脂分化,在细胞分化的早期和晚期,油红O染色观察脂肪细胞内脂滴的积聚并使用Image-pro plus6.0软件进行统计,利用甘油三酯检测试剂盒检测细胞的甘油三酯含量,利用PCR和Western blot检测成脂分化相关基因和蛋白的表达。在体内实验中,构建裸鼠皮下脂肪移植模型,8只小鼠随机分为阴性对照 siRNA(NC)组、YAP敲低(si-YAP)组,腺相关病毒(AAV)载体组、YAP过表达(AAV-YAP)组,每组2只。每只小鼠在4个不同部位各注射0.2 mL脂肪,总注射量为0.8 mL。然后在各组的脂肪局部分别注射0.2 mL等体积NC siRNA、YAP siRNA、腺相关病毒载体或腺相关病毒载体介导的YAP过表达质粒。在移植后1、2、4周收集移植脂肪,称量脂肪质量和体积,评估移植后脂肪的存活率,并用HE染色和免疫荧光染色观察脂肪结构、油囊数量、存活脂肪细胞数量。结果 细胞实验结果显示,与对照组相比,YAP敲低组的3T3-L1细胞的增殖活性与凋亡比例无明显变化,油红O染色面积增加(P<0.05),甘油三酯含量增加(P<0.05),成脂分化相关基因和
蛋白表达增加(P<0.05)。反之,与慢病毒载体组相比,过表达YAP组的3T3-L1细胞的增殖活性增加,凋亡比例无明显变化,油红O染色面积减少(P<0.05),甘油三酯含量降低(P<0.05),成脂分化相关基因和蛋白表达降低(P<0.05)。体内实验结果显示,相比NC组,si-YAP组脂肪的质量和体积增加,脂肪结构更加完整,油囊数量减少,存活脂肪细胞增多。而相比AAV载体组,AAV-YAP组脂肪的质量和体积减少,脂肪结构完整性降低,油囊数量增加,存活脂肪细胞减少。结论:YAP对脂肪移植存活率有明显的抑制作用。此外,YAP通过调控PPARγ通路影响3T3-L1成脂分化。

关键词:

Abstract:

Objective To investigate the effect of YAP on fat graft survival and its possible mechanism. Methods 3T3L1 cells were cultured in vitro and treated with YAP siRNA or YAP-overexpression plasmids carried by lentiviral vectors,resulting in the following groups: negative control siRNA (NC), YAP-si1, YAP-si2, and YAP-si3; lentiviral vector(Vector) and YAP-overexpression (YAP). Cell proliferation was assessed using EdU staining. Differentiation into adipocyteswas induced, and early and late stages of differentiation were analyzed by Oil Red O staining, with lipid droplet accumulationquantified using Image-Pro Plus 6.0 software. Triglyceride content was measured using a triglyceride assay kit, and PCR andWestern blot were used to assess the expression of genes and proteins related to adipocyte differentiation. For in vivo experiments, a subcutaneous fat transplantation model in nude mice was established. A total of 8 mice were randomly divided into NC siRNA group, YAP knockdown (si-YAP) group, adeno-associated virus (AAV) vector group, and YAP overexpression (AAV-YAP) groups, with 2 mice per group. Each mouse received 0.2 mL of fat injection at four different sites, totaling 0.8 mL. Subsequent injections of 0.2 mL of NC siRNA, YAP siRNA, AAV vector, or AAV vector-mediated YAP-overexpression plasmid were administered into the transplanted fat. Fat was collected at 1,2, and 4 weeks post transplantation, and fat weight and volume were measured to evaluate survival rates. Hematoxylin-eosin staining and immunofluorescence were used to examine fat structure, number of oil droplets, and viable adipocytes. Results In cell experiments, YAP knockdown did not significantly affect proliferation or apoptosis of 3T3-L1 cells compared to controls, but resulted in increased Oil Red O staining area (P<0.05), higher triglyceride content (P<0.05), and elevated expression of adipogenic genes and proteins (P<0.05). Conversely, YAP overexpression led to increased proliferation of 3T3-L1 cells with unchanged apoptosis rates, decreased Oil Red O staining area (P<0.05), lower triglyceride content (P<0.05), and reduced expression of adipogenic genes and proteins (P<0.05) compared to the lentiviral vector group. In animal experiments, the si-YAP group exhibited increased fat weight and volume, more intact fat structure, reduced oil droplet number, and more viable adipocytes compared to the NC group. In contrast, the AAV-YAP group had decreased fat weight and volume, less intact fat structure, increased oil droplet number, and fewer viable adipocytes compared to the AAV vector group. Conclusion YAP significantly inhibits the survival rate of transplanted fat. Furthermore, YAP affects 3T3-L1 adipogenesis by modulating the PPARγ pathway.

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