The Study Of The Biological Characteristics Of Laryngeal Mucosa Mesenchymal Stem Cell From Chronic Inflammation Of The Laryngeal Tissues

The sound is produced by the lamina propria of vocal fold, and it could bedamaged by the trauma or infection of lamina propria. How to prevent and reduce vocalcord atrophy and scar formation of the vocal cords during the repair process has becomean urgent p issueneeded to be improved and resolved.Homeostasis and differentiation of adult stem cells in human tissues is thebiological basis of tissue and organ regeneration. There is a kind of pluripotent stemcells which have a good capacity of self-renewal and multi-directional induction ofdifferentiation potential exist in the lamina propria of laryngeal mucosa, and we namedas laryngeal mucosa mesenchymal stem cells (LM-MSCs). We envision that it would be a new hope for vocal cord injury patients if we transplanted the LM-MSCs in thedamaged vocal cords situ. In our previous study we found that under fluorescencemicroscope the implanted LM-MSCs in the injury area of vocal cord could observed8weeks after post-surgery, but as time went by, the number reduced gradually. A few ofimplanted LM-MSCs could transforme into fibroblasts and myofibroblastsin thedamaged vocal fold lamina propria, and this suggested that the LM-MSCs directlyinvolved in the injury repair of vocal cord. As we all known that it is difficult fororganism to complete the diseased tissue regeneration and self-repair under theinflammatory microenvironment. Could the inflammatory microenvironment effects theregenerative capacity of extracellular matrix component and LM-MSCs?In this study, we use the normal (samples were obtained from patients withlaryngeal cancer with total laryngectomy, normal side of vocal cord specimens) andinflammatory mucous membrane of the larynx (samples were obtained from patientswith laryngeal cancer with total laryngectomy, lesion side of vocal cord specimens) toobserve the expression and distribution of LM-MSCs in the normal and inflammatorytissue, and detect the proliferation activity, amplification effect, colony-forming ability,multi-differentiation capacityof LM-MSCs obtained by limiting dilution method fromnormal and inflammatory tissues. Then we compared biological characteristics ofLM-MSCs from the normal and inflamed tissue to clear the effect of inflammatorymicroenvironment in vitro. This research is useful to further reveal the impaction on theLM-MSCs under the inflammatory microenvironment and the molecular mechanisms ofinflammation.We provide a theoretical basis for using LM-MSCs to solve the vocal cordtissue regeneration and defects repair.Objective:Analysis the differences of extracellular matrix components between normal andinflammation of the laryngeal mucosa lamina propria, then observe the expression anddistribution of the LM-MSCs in normal and inflammatory laryngeal mucosa. Utilizethe identification and comparison of the proliferation and differentiation capacity oflaryngeal mucosa mesenchymal stem cell from inflammatory tissue (I-LM-MSCs) andhealthycontrol (H-LM-MSCs) to study the biological characteristics of I-LM-MSCs,andto explore the effect of inflammatory microenvironment on LM-MSCs biologicalbehavior.Methods:1. Use of the methods of HE staining, Masson trichrome staining and Elastin VGstaining to compare the histological differences of the healthy and inflammationvocal folds tissue.2. Use immunofluorescence staining to detect the expression and distribution ofSTRO-1in the lamina propria of normal and inflammation vocal folds.3. We isolated stem cells from the inflammation vocal folds tissue (I-LM-MSCs) andhealthy vocal folds tissue (H-LM-MSCs). We used the method of limiting dilutiontechnique to obtain clonal purification and analyzed the cell phenotype byimmunocytochemistry and flow cytometry(FCM).4. Study the proliferative ability by using the methods of clone-forming ability, MTTviability assay, cell cycle and apoptosis analysis.5. Osteogenic and adipogenic induction were operated. Alizarin Red staining, Ca2+quantitative, ALP staining and ALP activity were used to investigate the osteogenicability. Using Oil red O staining to detect the adipogenic ability.Results:1. The vocal cord tissue from inflammation environment showed the features ofthicken epithelial, local visibility inflammatory cell infiltration, higher collagenfiber content, lower elastic fiber content. Stem cell phenotype is existed both innormal and chronic inflammation of the vocal folds, and there is no difference inexpression of the marker protein. 2. Immunofluorescence staining showed that there are STRO-1expression in bothnormal and inflammation vocal folds, scattered in the organization of the laminapropria and muscularis, and there is no significant difference.3. We isolated LM-MSCs from laryngeal cancer patients and healthy controlssuccessfully.7–10days after the cells were plated at low density, we can see singlecolonies of LM-MSCs were formed. Substantially, both H-LM-MSCs andI-LM-MSCs presented long and thin fibroblastic spindle morphology. Two types ofLM-MSCs both expressed stem cell markers of MSCs and the patterns of overallexpression were similar.4. We got a conclusion that the proliferative ability of I-LM-MSCs was strikinglyhigher than that of H-LM-MSCs by the methods of clone-forming ability, MTTviability assay and cell cycle analysis. We did not see significant differencesbetween H-and I-LM-MSCs in the apoptosis analysis. However, the apoptosis ofI-LM-MSCs was significantly increased after osteogenic or adiposis differentiation.5. The data of ALP staining, ALP activity, Alizarin red staining and quantitativeindicated that the ability of osteogenic differentiation of I-LM-MSCs was lowerthan H-LM-MSCs. Oil red O staining was used to confirm the adipogenicdifferentiation. The experimental results showed that the amount of formation lipiddroplets of I-LM-MSCs was significantly more than the H-LM-MSCs. However,we could not explain why the adipogenic differentiation ability of I-LM-MSCs washigher than H-LM-MSCs.Conclusion:1. The epithelial hyperplasia, thicken lamina propria, increased collagen fiber content,decreased elastic fibers of inflammatory vocal cord tissue, resulting in the lowerelasticity in chronic inflammation of the vocal cord tissue than normal control.2. Both H-LM-MSCs and I-LM-MSCs have shown the biological characteristics ofstem cells. 3. The proliferation ability of I-LM-MSCs is higher than H-LM-MSCs.4. The osteogenic potential of H-LM-MSCs is more powerful than I-LM-MSCs, whilethe adipogenic capacity is lower.