| Chronic laryngitis is one of the most common diseases of the larynx,accounting for 10%-20% of all laryngeal diseases.Severe chronic laryngitis directly affects vocalization,swallowing and respiratory function.Besides,long-term chronic inflammation will lead to abnormal proliferation of the laryngeal mucosa,seriously affecting people's health,work and life.At present,there is no specific treatment in clinical treatment,and in principle,general anti-inflammatory and antibacterial treatment is adopted.Clinical studies have shown that conventional treatment can not completely reverse the pathological changes,which may be the reason that it is difficult to cure chronic laryngitis and prevent the recurrence of the disease.Therefore,how to cure chronic laryngitis has become an urgent problem for otolaryngologist.Studies have shown that chronic inflammation can destroy the overall homeostasis of functional cells,interstitial cells and neurovascular in local tissues,leading to abnormal tissue function,and mesenchymal stem cells(MSCs)play an important role in the maintenance of homeostasis.Therefore,it is worth to explore whether functional loss of mesenchymal stem cells causes the development of chronic laryngitis.We isolated laryngeal mucosal mesenchymal stem cells(LM-MSCs)from the lamina propria of the laryngeal mucosa.They highly express the surface markers of mesenchymal stem cells and have the capability of multi-directional differentiation,immune regulation,tissue repair,etc.With the development of molecular biology technology,it has become a new direction to study the development of chronic laryngitis by exploring the molecular regulation mechanism of LM-MSCs dysfunction in chronic inflammatory environment and searching for genes that effectively improve the function of LM-MSCs.In recent years,many studies have found that mesenchymal stem cells play an immunomodulatory role in inflammatory diseases and immune diseases by affecting the apoptosis,proliferation,and migration of T cells.The larynx serves as an intersection of the gastrointestinal system and the respiratory system and exerts an important impact on immune regulation.At present,LM-MSCs have been extensively studied in vocal cord regeneration and repair,but the research of immune regulation function changes in chronic inflammatory environment and relevant mechanisms have not yet been elucidated.Understanding the changes in the immune function of LM-MSCs in a chronic inflammatory environment and the relevant mechanisms will help us to better understand the mechanism of chronic laryngeal injury and provide a theoretical basis for the corresponding prevention and treatment.Previous studies have shown that the functions of MSCs are significantly affected by the inflammatory environment dominated by tumor necrosis factor-alpha(TNF-?).Interestingly,studies have shown that the defects of tissue regeneration function of MSCs persist after being out of the inflammatory microenvironment,and even cannot be reversed after multiple passages in vitro,suggesting that long-term inflammation causes irreversible functional defects of LM-MSCs.It may be one of the reasons that we fail to cure chronic laryngitis.This functional defect that can be stably inherited in the next generation of cells suggests that epigenetics may play an important role.General control non-repressed protein5(GCN5),one of the earliest discovered and most important acetyltransferases,plays a significant role in embryonic development,maintenance of multiple cellular functions and deciding the destiny of MSCs.Therefore,we explored the role of GCN5 in LM-MSCs in chronic inflammatory environment.Objective:1)Isolation of LM-MSCs to investigate the effect of TNF-? on the osteogenic differentiation,adipogenic differentiation and immunomodulatory ability of LM-MSCs2)To explore the changes of GCN5 in LM-MSCs after chronic stimulation of TNF-?,and the relationship between GCN5 and its osteogenic,adipogenic and immunomodulatory abilities.3)To explore the potential molecular mechanism of GCN5 regulating the immune regulation function of LM-MSCs after chronic stimulation of TNF-?.Methods:1.Take normal laryngeal mucosa tissue from specimens of patients with laryngeal cancer,and isolate and culture LM-MSCs by enzymatic digestion.2.A long-term TNF-? stimulation model was established in vitro,and LM-MSCs were stimulated for a long time(3 days and 7 days)with appropriate TNF-? concentration(100 ng/mL).osteogenesis differentiation experiment,adipogenic differentiation experiment and directly co-culture experiment in vitro was performed to detect the differentiation ability and immune regulation ability of LM-MSCs stimulated by TNF-?.3.Western blot and qRT-PCR were used to detect the differential expression of GCN5 in normal LM-MSCs and TNF-? chronically stimulated LM-MSCs.In addition,the effects of GCN5 on osteogenic differentiation,adipogenic differentiation and immunomodulatory ability of LM-MSCs were investigated by GCN5 overexpression and interference vectors.4.Western blot and qRT-PCR were used to explore the molecular mechanism of GCN5-mediated immune regulation of LM-MSCs after chronic stimulation of TNF-?.Results:1 Isolation,culture and identification of LM-MSCsThe flow cytometry showed that LM-MSCs expressed the surface markers of MSCs.LM-MSCs possess the differentiation potential and the ability of immune regulation.2 Comparison of behavioral functions between normal LM-MSCs and chronic TNF-? stimulated LM-MSCsThe results showed that chronic TNF-? stimulation had no significant effect on the proliferation of LM-MSCs,and it inhibited the osteogenic and adipogenic differentiation of LM-MSCs and weakened the immunomodulatory ability of LM-MSCs.3 TNF-? affects the behavioral function of LM-MSCs through GCN5-mediated epigenetic modificationThe result of qRT-PCR and Western blot showed that the expression of GCN5 of LM-MSCs decreased after chronic TNF-? stimulation.LM-MSCs with overexpression or knockdown of GCN5 were used to verify the regulation of GCN5 on osteogenic differentiation,adipogenic differentiation and immunomodulatory capacity of LM-MSCs.The results demonstrated that TNF-? affected the osteogenesis,adipogenesis and immune regulation of LM-MSCs through GCN5-mediated epigenetic modification.4 Mechanism of damaged immune regulation of LM-MSCs caused by long-term TNF-? treatmentIn the long-term TNF-? treatment model in vitro,we found that the expression of FAS and FASL is consistent with GCN5 expression in LM-MSCs.Then,LM-MSCs with overexpression or knockdown of GCN5 were used to testify whether FAS and FASL were regulated by GCN5.The results showed that GCN5 affect the expression of FAS and FASL to regulate the immune regulation of LM-MSCs.Conclusion:In this study,we found that the osteogenic differentiation,adipogenic differentiation ability and immune regulation ability of LM-MSCs were impaired after chronic treatment of TNF-?.The biological function of LM-MSCs in a chronic inflammatory environment is affected via GCN5-mediated epigenetic modification.In terms of immune regulation,GCN5 can affect the immune regulation ability by regulating FAS and FASL expression of LM-MSCs. |
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