CCL3促进人骨髓间充质干细胞增殖并抑制其外泌体的分泌

CCL3 promotes h BMSC proliferation and inhibits exosome secretion

目的:探讨趋化因子CCL3对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,h BMSCs)外泌体分泌的调控作用。方法:采用CCK-8法和活细胞计数检测h BMSCs的增殖活性;采用电镜观察、流式细胞术和纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)对外泌体进行定性和定量分析。结果:CCK-8检测显示,CCL3组细胞的存活率大于对照组(P<0.05);活细胞计数结果显示,CCL3组细胞数明显多于对照组(P<0.05),呈浓度依赖性;流式细胞术分析结果显示,h BMSCs表达CCR1、CCR5和CCR9 3种CCL3特异性受体;CCL3作用h BMSCs后,CCR9荧光强度明显增强,CCR5和CCR1荧光强度无显著差异。NTA结果显示,与对照组相比,CCL3组外泌体分泌量明显减小(P<0.05),同时微囊泡(粒径大于100 nm)数明显增多(P<0.05);外泌体流式细胞术分析结果提示,与对照组相比,CCL3组的CD9+外泌体阳性率显著下降(P<0.01)。结论:CCL3促进h BMSCs增殖并抑制其外泌体的分泌,同时影响h BMSCs外泌体的粒径分布,无效的微囊泡增多。

AIM： To explore the regulatory effect of chemokine CCL3 on exosome secretion from human bone marrow mesenchymal stem cells（ h BMSCs）. METHODS： h BMSCs were stimulated with chemokine CCL3 at different concentrations in vitro. The proliferation of h BMSCs was measured by CCK-8 assay and viable cell counting. Exosome secretion from h BMSCs was qualitatively analyzed by transmission electron microscope（ TEM） and flow cytometry,and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis（ NTA）. RESULTS： Compared with control group,the viability of the h BMSCs detected by CCK-8 assay was increased when h BMSCs were treated with CCL3（ P 〈 0. 05）. The results of viable cell counting demonstrated that the number of h BMSCs was raised in CCL3 group in a dose-dependent manner（ P 〈 0. 05）. The results of flow cytometry showed that h BMSCs expressed 3 CCL3-related specific receptors,CCR1,CCR5 and CCR9. Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced. However,no significant difference of fluorescence intensity for CCR5 and CCR1 was observed between the 2 groups. The results of NTA demonstrated that the secretion capacity of CCL3-induced h BMSCs was far less than that in control group（ P 〈 0. 05）. However,the microvesicles larger than 100 nm in CCL3 groups were increased（ P 〈0. 05）. The above results indicated that the higher concentration of CCL3 induced the lower secretion of exosomes. In addition,the results of flow cytometry demonstrated that CCL3-induced h BMSCs showed lower quantity of CD9＋exosomes than those in control group（ P 〈 0. 01）. CONCLUSION： CCL3 promotes the proliferation of h BMSCs but depresses the secretion of exosomes in a dose-dependent manner. CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size. CCL3 induces the expression of CCR9 in h BMSCs.