The Role Of CCL3 In Regulating The Secretion Of Exosomes Derived From Bone Marrow Mesenchymal Stem Cells

Background:Exosomes were originally found in sheep reticulocytes[1]and are membranous vesicles of 30-100 nm in size derived from cells originally considered as "cell litter"[2].In recent years,studies have found that exosomes contain RNA and miRNA,and can be transduced into the receptor cell regulatory protein expression.Exosomes is not only a cell signaling molecule,is a genetic material delivery carrier[3]and is the hub of information exchange between cells[4].It has been found that mesenchymal stem cells(MSCs)are the most capable cells producing exosomes and MSC-exosomes have many biological functions similar to MSCs.[5-7]Some studies have found that paracrine regulation of MSCs is mainly achieved by exosomes secreted by cells[8].Therefore,paracrine regulatory of MSCs and exosomes secretion have a direct relationship.Relevant experimental results confirmed that the secretion of MSCs in the inflammatory pathological environment was active.[9]Some scholars use ?-interferon to mimic the inflammatory environment,confirming that excretion of MSCs will increase in this inflammatory environment[10].CCL3 is a cytokine composed of two peptide chains with a relative molecular mass of about 8000 kDa found in macrophage supernatant after LPS induction.A variety of cells have the ability to secrete CCL3,its role without species-specific.It has a strong chemotactic effect and inflammation induction.CCL3 have three receptors:CCR1,CCR5 and CCR9,which are G protein-coupled receptors.CCL3 specific binds with the corresponding receptor cascade-like cell activation.[11]Studies have shown that CCL3 is highly expressed in different inflammatory pathologies.[12-14]At the same time,in vitro experiments have shown that the high concentration of CCL3 can usually be detected in the bone marrow failure model.[15]Objective:To investigate the regulatory effects of CCL3,an inflammatory cytokine,on the exosome secretion of human bone marrow mesenchymal stem cells in vitro.The role of CCL3 in the pathogenesis of aplastic anemias(AA)was also explored.Methods:The research was divided into three parts to complete.Part ?-Isolation and Purification of Human BMSCs:(1)After informed consent from healthy donors and patients with aplastic anemia,10 ml bone marrow fluid was removed from donors' ilium;(2)The fifth generation of human BMSCs were cultured and surface antigen identification and adipogenie,osteogenic induction differentiation identification;(3)Exosones were extracted form supernatants of the third to fifth generation of human BMSCs,transmission electron microscopy was used to identify exosome morphology.The second part-Analysis of exosomes' secretary ability of BMSCsderived from aplastic anemia patients:(1)Microarray analysis campare the secretion difference of CCL3 between MSCs of healthy dornors and patients with aplastic anemia;(2)The secretions of exosomes in normal group were compared by Nanosight(NTA)and flow cytometry(FCM);(3)To identify the expression of CCL3-related receptors(CCR1,CCR5 and CCR9)on the surface of BMSCs derived from patients with aplastic anemia,flow cytometry was used to compare the differences between the normal groupPart three-The regulation of cytokine CCL3 on the exosomes excretion of human BMSCs:(1)CCK-8 and viable cell counting were used to detect the effect of CCL3 on the proliferation of human bone marrow MSCs;(2)Nanosight(NTA)and flow cytometry(FCM)were used to compare the excretion of exosomes in each group.(3)Flow cytometry was used to identify the expression of CCL3-related receptors(CCR1,CCR5 and CCR9)and the expression changes after CCL3 stimulated.Statistical methods:SPSS 20.0 statistical software was used to analyze datas,and measurement datas were expressed in(x ± s),multiple factors analysis using linear regression;More comparison using single factor analysis of variance between groups,when the variance was approximate F inspection Welch method.Inspection level of a= 0.05,double side inspection.Results:The first part:(1)Human BMSCs were isolated by whole bone marrow adherent culture method,and then cultured to the fifth generation,and finally the cells showed fibroblast-like adherent growth.(2)The results of flow cytometry showed strong positive expression of CD73(100%),CD90(99.5%)and CD105(99.7%),weakly positive CD34(0.2%),CD45(0.1%)and CD19(0.1%),in line with the standard BMSCs phenotype.(3)When the cells were induced into adipocytes for 14 days,large and round lipid droplets could be found in the cells,demonstrating that the cells could differentiate into adipocytes.After 21 days of osteoblast induction,a large number of red-stained calcified nodules were found,demonstrating that the cells could differentiate into osteocytes.The second part:(1)CCL3 was highly expressed in BMSCs of AA patients.(2)AA patients did not express CCR1 on the surface of BMSCs,but the expression of CCR9 increased.(3)The exosomes excretion of BMSCs in AA patients was significantly lower than that in healthy donors(P<0.05).The third part:(1)CCK8 test showed that the cell viability in CCL3 group was significantly higher than that in control group(135.9%± 6.4%vs 100%,P<0.05).The number of viable cells in CCL3 group was significantly higher than that in control group(8.08 ± 0.48×105 vs 5.36±0.76×105,P<0.05).(2)Flow cytometry results showed that human BMSCs express CCR1,CCR5 and CCR9 specific CCL3 receptors.After CCL3 treated with human BMSCs,the fluorescence intensity of CCR9 was significantly increased,while the fluorescence intensity of CCR5 and CCR1 was not significantly different.(3)Compared with the control group,the secretion of exosomes in CCL3 group was significantly.decreased(5.00 ± 0.06 ×1010)/mL vs(4.28 ± 0.07×1010)/mL,P<0.05).At the same time,the number of microvesicles(particle size>100nm)increased obviously(4)Exosome flow cytometry results showed that compared with the control group,the positive rate of CD9 + exosome in CCL3 group was significantly decreased(65.60%±2.10%vs 51.33%±5.42%,P<0.01).Conclusion:(1)CCL3 promotes the proliferation of BMSCs but inhibits its secretion of exosomes,showing a certain concentration-dependent;(2)CCL3 increases the average size of exosomes and promotes the formation of nonfunctional vesicles;(3)Exosomes secretory capacity of human bone marrow MSCs derived from AA patients is low but the secretion of CCL3 is extremely high,and the expression of CCR9 cell membrane surface increased,do not express CCR1.