| Objective Mesenchymal stem cells(MSCs)are multipotent stem cells,which can be isolated and extracted from a variety of tissues and organs.MSCs has a high degree of self-renewal and differentiation potential.Bone marrow mesenchymal stem cells(BM-MSCs)are the most abundant source of MSCs,which is easy to operate and obtain.Toll like receptor(TLRs)is an important protein molecule involved in innate immunity and acquired immunity.Micro RNA(mi RNA)is a class of non coding small molecule RNA,which has the function of regulation and control,and the size is about 19-23 nucleotides.It has been recently reported that mi RNAs play critical roles in important physiological and pathological processes of MSCs,such as proliferation,differentiation,migration,survival,cytokine secretion and the hematopoietic support.Presently,in various cell types,it has been demonstrated that TLR signaling regulates mi RNA expression,while mi RNA is also an important regulatory molecule to control TLR signaling.Therefore,there is reason to think that TLR activation associated mi RNAs are more likely to be involved in regulating the function of MSCs.In our previous work,we confirmed that the expression of TLR2 and TLR4 in BM-MSCs,which can regulate the migration,differentiation and other biological functions.Furthermore,we adopted Illumina high-throughput sequencing analysis to examine changes in the mi RNAs of BM-MSCs after stimulation by Pam3CSK4(TLR2 agonist)and LPS(TLR4 agonist).According to the mi RNA profile,we selected 4 kinds of mi RNAs as the research objects:up-regulated mi R-146a、mi R-214,and down-regulated mi R-323b、mi R-425,which were significantly related to the TLR activation.And then evaluated the effect of them on the migration ability of BM-MSCs,addtionally,we detected the effects of these mi RNAs on the migration and differentiation of hematopoietic stem cells induced by BM-MSCs in vitro.Methods BM-MSCs were extracted and cultured from healthy volunteers.After transfected with mimic or inhibitor of mi R-214,mi R-146 a,mi R-323 b and mi R-425 by liposome respectively,quantitative real-time polymerase chain reaction(q RT-PCR)were used to measured expression of mi RNA in different groups.Both the migration ability of adherent BM-MSCs and effect of the supernatant on migration ability of human umbilical cord blood CD34~+ was observed by chemotaxis assay.Alterations of chemokine SDF-1 secreted by BM-MSCs in the supernatant was assayed by enzyme-linked immunosorbent assay(ELISA)and flow cytometry was used to detect the expression of chemokine receptor 4(CXCR4)on BM-MSCs surface.In addition,we established a co-culture system of BM-MSCs and CD34~+ cells for 2 weeks,and observed the differentiation of hematopoietic stem cells by flow cytometry.Hematopoietic growth factor IL-1,IL-8 and G-CSF levels in culture supernatant was quantitatively measured by ELISA.Results 1.Compared with the control group,expression of mi R-214,mi R-146 a,mi R-323 b and mi R-425 were significantly up-regulated after transfection with their mimics and down-regulated after transfection with their inhibitors.2.The migration ability of BM-MSCs transfected with mi R-323 b mimic was inhibited,while cell migration was enhanced after transfection with mi R-323 b inhibitor.Flow cytometry was used to measure the CXCR4 expression on the treated cells surface,and the level of CXCR4 in mi R-323 b mimic group was decreased.3.In the CD34~+ cell migration experiment,compared with the blank group,supernatant of BM-MSCs significantly promote CD34~+ cell migration;and compared with the group without transfection or negative control group,mi R-214 mimic group remarkably promote CD34~+ cell migration and mi R-214 inhibitor group showed the migration surpression of CD34~+cells.There was no obvious difference in SDF-1 concentration between the two groups assayed by ELISA.4.After the cells were co-cultured with CD34~+ cells for a total of two weeks,the differentiation of CD34~+ cells presented differences and the CD34~+ cells in mi R-425 mimic group were significantly differentiated into myeloid cells.5.The changes of IL-1β,IL-8 and G-CSF levels in the culture supernatant of BM-MSCs: the mi R-425 mimic group has the largest concentration of G-CSF compared with the control group.Conclusion 1.The high expression of mi R-323 b can inhibit the migration of BM-MSCs,which may be related to the inhibition of CXCR4 expression on the cell surface.2.mi R-214 signalings may indirectly increase the migration of CD34~+hematopoietic stem/progenitor cells by modulating BM-MSCs functions,which is not significantly correlated with chemokine SDF-1 produced by BM-MSCs.3.mi R-425 upregulated BM-MSCs can promote umbilical cord blood CD34~+ cells to differentiate into myeloid lineage,which may be related to the secretion of G-CSF by BM-MSCs.4.TLR2/TLR4 activation related mi RNA has a certain role in the regulation of hematopoietic function of BM-MSCs. |
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