| Multilineage and self-renewal are the basis of adult stem cells for tissue and organregeneration, which can be affected by inflammatory microenvironment. The laryngealmucosa mesenchymal stem cells (LM-MSCs) are isolated from the epiglottis lingualmucosa, and they have the potential for vocal ford tissue injury repair. We cultured andidentified the LM-MSCs, then injected it into the laser damaged vocal folds and observedvocal fords tissue at2w、4w and8w. It showed that LM-MSCs for vocal cord repair caneffectively reduce the formation of the vocal fords edema, scar, and the cells can survivein vivo for up to8w, they even could be transformed into muscle fiber cells. However, theexcision of vocal fords lesion tissue will also cause acute inflammation. Acuteinflammatory on the biological characteristics of LM-MSCs is bound to affect LM-MSCs repairing tissue function.The Wnt signaling pathway is a signaling pathway important for human growth,development and plays an important role in a variety of disease processes.The Wntsignaling pathway were consisted of classical and non-classical Wnt signaling pathway.The classical Wnt signaling pathway is also called Wnt/β-catenin signaling pathway,which determination of cell fate, and the non-classical Wnt signaling pathway is relatedwith tissue polarity and cell movement. Studies have shown that Wnt/β-catenin signalingpathway may be involved in cell proliferation, differentiation in normal and inflammatorymicroenvironment. The osteogenic differentiation of periodontal ligament stem cells(PDLSCs) in the inflammatory microenvironment was decreased in inflammatorymicroenvironment compared with normal microenvironment, while the Wnt/β-cateninsignaling pathway inhibitor DKK1will partly even totally reversed osteogenicdifferentiation of PDLSCs.To sum up, we simulate inflammation microenvironment in vitro to explore the effectsof adipogenic, osteogenic and muscle differentiation ability of LM-MSCs. Considering thespecial struture of the vocal fords tissues, we focued on the effects of inflammatorymicroenvironment on myogenic differentiation ability of LM-MSCs. And with theinhibitor and activator of Wnt/β-catenin signaling pathway, we want to reverse myogenicdifferentiation into LM-MSCs in the inflammatory microenvironment.Objective:The effects of the inflammatory microenvironment on the LM-MSCs multilineagedifferentiation, such as, osteogenic, adipogenic and myogenic differentiation were testedto show the factor of the Wnt/β-catenin signaling pathway. Then by regulating theWnt/β-catenin signaling pathway, we hope to resverse myogenic differentiation ability ofLM-MSCs in the inflammatory microenvironment, finally promote the LM-MSCsrepairing tissue damage.Methods:1. The culturing, identifying and multilineage differentiation of the LM-MSCs: we cultured the laryngeal mucosa and obtained the LM-MSCs, then detected stem cell surfacemarker CD105, CD90, CD44, CD34, CD14and CD31expression by flow cytometry. Thecells were induced into osteogenic and adipogenic cells respectively, then stained with redby alizarin or Oil red O at21d or14d; the cells were induced differentiation into oblastecells up to6w, then detected the myogenic differentiation ability by immunofluorescencestaining and real-time quantitative detection (RT-PCR).2. Effects of inflammatory microenvironment on the differentiation capability ofLM-MSCs: the10ng/ml of TNF-α and5ng/ml of IL-1β in vitro to simulate inflammatorymicroenvironment, and induced the cells in the inflammatory and normalmicroenvironment to osteogenic, adipogenic respectively and myogenic differentiation.The alkaline phosphatase (ALP) and alizarin red staining were used to detect osteogenicdifferentiation, and the mineralized nodules were dissolved for quantitative analysis; theadipogenic differentiation of the LM-MSCs were stained by oil red O at14d, and thedrops were dissolved for quantitative analysis; the proteins and genes, such as Myod1,Myogenin (Myog) and myosion heavy chain (MyHc) of myogenic differentiation of theLM-MSCs were explored at1w,3w and6w by RT-PCR and Western Blot; and theimportant signal molecules in wnt/β-catenin signal pathway, such as GSK3β, P-GSK3βand β-catenin protein and gene expression in inflammatory microenvironment were alsodectected.3. The Wnt/β-catenin signaling pathway on the ability of the LM-MSCs myogenicdifferentiation in inflammatory microenvironment: in vitro, we respectively down-regulated Wnt/β-catenin signaling pathway by DKK1in inflammatory microenvironmentand up-regulated Wnt/β-catenin signaling pathway by Wnt3a in normal microenvironmentof LM-MSCs myogenic differentiation, and detected myogenic differentiation relatedprotein and gene in vitro; prepared cell and collagen mass of muscle differentiation, andtransplanted them into nude mice subcutaneous2W, removed the graft, and observed thehistological behavior by HE staining. We observed cell differentiation by massontrichrome staining, and using immunofluorescence staining to observe the expression ofmyogenic differentiation related protein. Result:1. The culturing, identifying and multilineage differentiation of the LM-MSCs: byisolation and culture of the epiglottis lingual mucosa tissues by enzyme digestion,LM-MSCs can be obtained, which can be adherent growth, long spindle shaped; thepositive expression of mesenchymal stem cell surface molecule were CD105(94.2%),CD90(99.5%) and CD44(99.8%), but not hematopoietic system and endothelial cells ofCD34(0.6%), CD14(0.7%) and CD31(0.5%). Cells with osteogenic differentiation of21d parallel alizarin red staining, showed mineralized nodules; cells with adipogenicdifferentiation of14d oil red staining, showed beaded lipid droplets of red dye; the cellsinto muscle cells induced differentiation of1w,3w and6w, by immunefluorescent stainingand RT-PCR showed the Myod1and Myog expressed in myogenic differentiation after1w, and sustainable expressed in muscle differentiation at6w；MyHc, terminal protein andgene in myogenic differentiation, expressed in myogenic differentiation after3w, andsustainable expressed in myogenic differentiation at6w.2. Effects of inflammatory microenvironment on the differentiation capability ofLM-MSCs2.1Effects of inflammatory microenvironment on osteogenic differentiation ofLM-MSCs:In inflammatory and normal micro environment, cells were induced into osteoblasts,and were subjected to ALP staining, alizarin red staining. And results can be seen, cellswere also blue by ALP in the inflammatory and normal microenvironment osteogenicdifferentiation after7d, but the color was light in inflammatory microenvironment; afterinducing into bone at21d by alizarin red staining, cells in inflammation microenvironmentwith red mineralized nodules were less than them in normal microenvironment; suggesttedthat the inflammatory microenvironment suppressed LM-MSCs osteogenic differentiation.2.2Effects of inflammatory microenvironment on adipogenic differentiation ofLM-MSCs:In inflammatory and normal micro environment, cells were induced into fat cells, andstaining at14d by oil red O, lipid droplet was formed in inflammatory and normal microenvironment of LM-MSCs, and in inflammatory microenvironment, beaded lipiddroplet were less, showed that the inflammatory microenvironment inhibits LM-MSCsdifferentiation.2.3Effects of inflammatory microenvironment on myogenic differentiation ofLM-MSCs:In inflammatory and normal micro environment, cells were induced into muscles, andrespectively induced differentiation at1w,3w and6w, gene and protein extracted fromcells for RT-PCR and Western Blot for the detection of expression; and results showed ininflammation microenvironment and the process of myoblast differentiation, protein andgene related with myoblast were lower expression than those in the control group.3. Regulation of LM-MSCs muscle Wnt/β-catenin signal pathway on theinflammatory microenvironment to the ability of differentiation3.1In vitro experiments: We respectively down-regulated DKK1in inflammatorymicroenvironment and up-regulated Wnt3a in normal microenvironment in LM-MSCsmyoblast differentiation process of Wnt/β-catenin signaling pathway, and detect myogenicdifferentiation related protein and gene expression. The results showed that after DKK1intervention or in inflammatory microenvironment, myogenic differentiation relatedproteins and genes expression was increased. Adding Wnt3a up-regulation in normalmicroenvironment of the LM-MSCs myogenic differentiation, the expression of myogenicdifferentiation related proteins and genes weakened, suggesting that down-regulation ofWnt/β-catenin signaling pathway can reverse the inflammatory microenvironmentinhibitory effect of the LM-MSCs myogenic, and up-regulating can inhibit the LM-MSCmyogenic differentiation in normal microenvironment.3.2In vivo experiments: according to the vitro experiment, we prepared cell and collagenmass, and transplanted them into nude mice subcutaneous for2w, removed the graft, andthen used HE staining to observe the histological behavior; the Masson trichrome stainingwere used to observe myogenic differentiation; and the immunefluorescence staining wereused to observe the expression of myogenic differentiation related protein. The resultswere in accordance with the results in vitro, suggesting that down-regulation of Wnt/β -catenin signaling pathway can reverse the inflammatory microenvironment inhibitoryeffect of myogenic differentiation of the LM-MSCs, and up-regulation of pathway caninhibit the myogenic differentiation of the LM-MSCs in normal microenvironment.Conclusion1The culturing, identifying and multilineage differentiation of the LM-MSCs: theLM-MSCs is belonged to mesenchymal stem cells, and has the ability of osteogenic,adipogenic and myogenic differentiation differentiation.2Effects of inflammatory microenvironment on the multilineage differentiationcapability of LM-MSCs: the inflammatory microenvironment can inhibit the ability ofosteogenic, adipogenic and myogenic differentiation of the LM-MSCs.3The regulation of Wnt/β-catenin signal pathway on the ability of myogenicdifferentiation in inflammatory microenvironment: by down-regulating the expressionof Wnt/β-catenin signaling pathway, we can partially or completely reversed the ability ofthe LM-MSCs myogenic differentiation which was inhibited in inflammatorymicroenvironment. |
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