老年小鼠ADSCs与BMSCs外泌体的蛋白质组学分析研究

Proteomic analysis of exosomes ADSCs and BMSCs in aged mice

目的通过无标记定量(Label-free)Label-free蛋白质组学分析老年No.57雄鼠和No.52雌鼠交配获得第六组亚系小鼠(C57BL/6)脂肪间充质干细胞(ADSCs)和骨髓间充质干细胞(BMSCs)外泌体,探讨其细胞治疗的可行性.方法利用梯度密度离心-速差贴壁法分离、培养源于老年C57B/L6小鼠的ADSCs和BMSCs,通过超速离心法提取外泌体,对外泌体进行透射电镜下的形态观察,通过Western blot检测外泌体动力相关蛋白-1(CD9)、溶酶体颗粒膜蛋白(CD63)、四跨膜蛋白(CD81)的表达.并通过Label-free蛋白质组学分析ADSCs和BMSCs外泌体.结果成功获取老年C57B/L6小鼠的ADSCs和BMSCs,透射电镜结果和Western blot结果显示成功获取外泌体;Label-free蛋白质组学分析结果显示ADSCs外泌体较BMSCs外泌体含有更多的金属硫蛋白(MTI)、克拉拉细胞蛋白(CC16)等;经通路分析显示,ADSCs外泌体在抗炎反应等信号通路变化有关,Western blot结果显示MTI在ADSCs外泌体的表达高于BMSCs外泌体.结论老年C57B/L6小鼠的ADSCs较BMSCs外泌体含有更多的MTI,可能为老年供体来源ADSCs外泌体应用于抗炎治疗提供实验依据及奠定理论基础.

Objective To explore the feasibility of cell therapy with adipose tissue-derived stromal cells(ADSCs)and bone marrow mesenchymal stem cells(BMSCs)exosomes from C57 black 6(C57BL/6)mice,the offspring from male mouse No.57 and female mouse No.52,through Label-free proteomic analysis.Methods ADSCs and BMSCs from aged C57BL/6 mice were isolated and cultured by gradient density centrifugation and differential velocity adherent technique.Then the exosomes were extracted by ultracentrifugation and subject to morphology observation under transmission electron microscopy(TEM).The expression of motility related protein-1(CD9),lysosomal membrane glycoprotein(CD63)and transmembrane 4 superfamily(CD81)of exosomes was detected by Western blot.The exosomes of ADSCs and BMSCs were analyzed by label free proteomics.Results ADCs and BMSCs were obtained from aged C57BL/6 mice.The TEM and Western blot results showed that exosomes were obtained and the label-free proteomic analysis showed more metallothionein(MTI)and Clara cell protein(CC16)in ADSC exosomes than in BMSC exosomes.Pathway analysis suggested that the ADSC exosomes were associated with the changes in signal path in anti-inflammatory response.Western blot demonstrated that the expression of MTI in ADSC exosomes was higher than that in BMSC exosomes.Conclusion The exosomes of ADSCs from aged C57BL/6 mice contain more MTI than that of BMSCs,which may provide experimental basis and theoretical basis for the application of ADSC exosomes from aged donors in anti-inflammatory treatment.