深度烧伤痂脂肪干细胞源性外泌体蛋白组学差异的初步研究

Preliminary proteomic analysis of adipose-derived stem cell exosomes in severe burn patients

目的:分析不同脂肪组织来源干细胞源性外泌体(ADSC-Exos)蛋白质组学差异,为深度创面修复提供理论依据。方法:取烧伤痂组织与正常皮下脂肪组织,分离脂肪干细胞(ADSC),通过光镜观察、Western blot(WB)、成骨分化、成脂分化鉴定ADSC。差速离心方法收集脂肪干细胞外泌体(ADSC-Exos),通过电镜观察、WB法、Nanosight分析仪鉴定ADSC-Exos;通过非标记定量蛋白质谱(LFQ)技术分析两组患者ADSC-Exos蛋白质表达水平差异。结果:成功分离ADSC,光镜下呈纤维状,WB显示细胞稳定表达CD29及CD105,成骨诱导分化细胞内见红色密集钙结节、成脂诱导分化细胞内存在折光性好的透亮脂滴。成功分离ADSC-Exos,电镜下呈双膜性结构,WB显示外泌体可稳定表达表面标志物CD63及CD81,Nanosight显示外泌体均匀对称分布,直径30~150 nm。两组表达水平差异蛋白质184种,差异倍数显著上调前10种蛋白:丝裂原活化蛋白激酶3、丝裂原活化蛋白激酶1、转化生长因子β-1前蛋白、细胞凋亡调节因子BAX、热休克蛋白HSP 90α、微管蛋白α-1B链、腺病毒E1B19kDa相互作用蛋白2、钙调素-3、热休克蛋白HSP 90β、蛋白激酶Cβ型;差异倍数显著下调前10种蛋白:激肽源B1、激肽源1、补体家族(C3、C1q C链、C1q B链、C5、C1q A、C4B、C4A、C9)。结论:烧伤痂下组织来源ADSC-Exos蛋白与正常组织的表达存在明显差异,可能与烧伤创面修复、抑制瘢痕过度增生及抗炎与机体免疫等密切相关。

Objective:To provid theoretical basis for deep wound repair by analyzing the proteomic differences of adipose-derived stem cell-derived exosomes(ADSC-Exos)from different adipose tissue sources.Methods:The adipose tissue derived under the eschar of burn patients and normal subcutaneous adipose tissue were used for isolating adipose-derived stem cells(ADSC).ADSC were identified through light microscopy,Western blot(WB),osteogenic differentiation and adipogenic differentiation.ADSC-Exos were collected using differential centrifugation method.ADSC-Exos were identified by electron microscopy,Western Blot,and Nanosight analyzer.The proteomics differential expression of ADSC-Exos was analyzed by using label-free quantitative proteomics(LFQ)technique between the two groups.Results:ADSC was successfully separated,presenting a fibrous shape under the light microscope,the stable expression of CD29 and CD105 were showed by Western Blot analysis.Red dense calcium nodules were observed in osteogenic induced differentiation cells,and there were transparent lipid droplets with good refractive properties in adipogenic induced differentiation cells.ADSC-Exos were successfully isolated,exhibiting a bilayered structure under electron microscopy,the stable expression of surface markers CD63 and CD81 were revealed by Western Blot analysis,the uniform and symmetrical distribution of ADSC-Exos were showed by Nanosight analysis,with a diameter ranging 30~150 nm.There were 184 differentially expressed proteins,top 10 proteins exhibiting a significant upregulation in fold change were selected,including Mitogen-activated protein kinase 3,Mitogen-activated protein kinase 1,Transforming growth factor beta-1,Apoptosis regulator BAX,Heat shock protein HSP 90-alpha,Tubulin alpha-1B chain,adenovirus E1B 19kDa interacting protein 2,Calmodulin-3,Heat shock protein HSP 90-beta,Protein kinase C beta type.As well as the top 10 proteins showing a significant downregulation in fold change,including kallikrein B1,kininogen 1,and complement family members(C3,C1q C chain,C1q B chain,C5,C1q A,C4B,C4A,C9).Conclusion:Significant differences are observed in the protein expression of ADSC-Exos from different sources,and these differential proteins may play important roles in burn wound healing,reducing excessive scar formation,exerting anti-inflammatory and immune-modulating effects on the body.