Experimental study of effect and mechanism of cysteine rich protein 61 on survival of adipose tissues in rats after autologous fat grafting
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摘要:
目的 探索富半胱氨酸蛋白61即CCN家族成员1(CCN1)对自体脂肪移植术后脂肪组织存活的影响及其作用机制。 方法 建立大鼠自体脂肪移植模型1周后,随机分为CCN1组与对照组,每组各20只。比较两组大鼠移植脂肪存活情况; 比较两组大鼠移植脂肪组织形态、活性脂肪细胞比例及新生血管数量; 利用高通量测序技术比较两组大鼠移植脂肪组织差异表达信使核糖核酸(mRNA)并进行聚类分析; 比较两组大鼠移植脂肪组织促炎因子的表达水平。 结果 与对照组比较,CCN1组脂肪组织质量保留率较高,差异有统计学意义(P < 0.05),CCN1组脂肪细胞完整性更高,囊泡化与空泡化程度、炎症细胞聚集程度和纤维化程度更低,差异均有统计学意义(均为P < 0.000 1)。免疫荧光染色结果显示,CCN1组中活性脂肪细胞比例更高且形态均一,对照组存在较少的活性脂肪细胞且大小不一,伴有囊泡化现象。与对照组比较,CCN1组新生血管数量更多,血小板衍生生长因子(PDGF)和成纤维细胞生长因子(FGF)mRNA表达水平均升高,差异均有统计学意义(均为P < 0.05)。高通量测序结果显示两组的转录组水平差异有统计学意义,CCN1组中与M1型巨噬细胞相关的细胞表面标志物、炎症因子及趋化因子基因表达呈下调趋势,实时荧光定量聚合酶链反应(RT-qPCR)结果显示CCN1组白细胞介素(IL)-8、IL-1和Toll样受体(TLR)2 mRNA表达水平较对照组均降低,差异均有统计学意义(P < 0.01~0.05)。 结论 在自体脂肪移植过程中添加外源性CCN1能有效促进脂肪组织新生血管化,提高移植脂肪存活率,其机制可能是通过下调TLR2表达介导巨噬细胞向M2型转化。 Abstract:Objective To evaluate the effect and mechanism of cysteine rich protein 61, namely CCN family member 1(CCN1) on the survival of adipose tissues in rats after autologous fat grafting. Methods At 1 week after the establishment of autologous fat grafting rat models, all animals were randomly divided into the CCN1 group (n=20) and control group (n=20). The survival of fat grafts, the morphology of fat graft tissues, the proportion of active adipocytes and the number of new blood vessels of rats were statistically compared between two groups. The levels of differential expressed messenger ribonucleic acid (mRNA) in the fat graft tissues of rats were compared between two groups by high-throughput sequencing and subsequently subject to cluster analysis. The expression levels of related proinflammatory cytokines of fat graft tissues of rats were statistically compared between two groups. Results The weight retention rate of adipose tissues in the CCN1 group was significantly higher than that in the control group (P < 0.05). In the CCN1 group, the integrity of adipocytes was considerably higher, the degree of vesiculation and vacuolation, the degree of inflammatory cell aggregation and the degree of fibrosis were significantly lower than those in the control group (all P < 0.000 1). Immunofluorescence staining demonstrated that the proportion of active adipocytes with uniform morphology was higher in the CCN1 group, whereas the proportion of active adipocytes was lower and the cells were observed in different sizes accompanied by vesiculation in the control group. Compared with the control group, the quantity of new blood vessels was significantly higher, and the expression levels of platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) mRNA were remarkably higher in the CCN1 group (all P < 0.05). High-throughput sequencing analysis showed that the data at the transcriptome levels significantly differed between two groups. In the CCN1 group, the gene expression levels of cell surface markers, inflammatory cytokines and chemokines related to M1 macrophages tended to decline. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) revealed that the mRNA expression levels of interleukin (IL)-8, IL-1 and Toll-like receptor (TLR) 2 in the CCN1 group were significantly lower than those in the control group (P < 0.01-0.05). Conclusions During autologous fat grafting, supplement of exogenous CCN1 may effectively promote the neovascularization of adipose tissues and improve the survival rate of fat graft probably by mediating the transformation of macrophages into M2 phenotype via down-regulating the TLR2 expression level. -
表 1 高通量测序分析两组mRNA表达水平差异
Table 1. High-throughput sequencing analysis of differenceof mRNA expression levels between two groups
M1型巨噬细胞相关基因 log2(差异倍数) 表达量改变 细胞表面标志物 SOCS3 -1.963 36 下调 TLR2 -1.364 11 下调 TLR7 -1.486 99 下调 TLR9 -1.599 46 下调 IL-18RAP -1.669 3 下调 炎症因子和趋化因子 IL-1β -4.319 61 下调 TNF-αIP3 -1.654 89 下调 IL-23α -1.700 33 下调 IL-6 -2.539 59 下调 IL-18 -1.477 76 下调 CCL3 -4.207 43 下调 CCL4 -1.771 42 下调 CXCL1 -3.184 19 下调 CXCL2 -6.822 16 下调 CXCL6 -3.915 91 下调 -
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