| BackgroundBronchiliotis is an inflammation of the small bronchioles and surrounding tissues caused by viral infections.The age varies from 6 months to 24 months according to different guidelines.Bronchiolitis is characterized by dyspnea,cough,hyperinflation,trigeminal signs,wheezing and gasp in clinial.However,bronchiolitis is a disease with undefined and highly heterogeneous diseases with clinical manifestations,age of infection,predisposing factors,and potential genetic mechanisms.RSV and RV are the main pathogenic factors of bronchiolitis,which can affect the airway for a long time and can cause airway hyperresponsiveness and wheezing later.Early childhood bronchiolitis can increase the incidence of asthma in the future.Some cohort studies have shown that the ratio can be as high as 30%-40%.If the causative factor of bronchiolitis is RSV,the risk of asthma in children is 2-3 times higher.Bronchiolitis caused by RSV infection is the main independent risk factor for subsequent asthma,but its pathogenesis remains unclear.Asthma is characterized by airway chronic inflammation,airway remodeling,and airway hyperresponsiveness,which are caused by the interaction of the host variety and environmental factors.Currently,the main drugs for treating asthma are glucocorticoids.Due to the long-term use of hormones and long-term side effects,not only the treatment compliance decreases,but also the psychological and economic burden on children with asthma and their families increases.Bronchiolitis also causes airway inflammation and airway hyperresponsiveness,and both of them have imbalance of Thl/Th2 and Treg/Th17,which is similar to asthma in imuune dysfunction.RSV is currently considered to be an early marker that triggers later wheezing in children.Therefore better understanding of the individual and environmental factors of viral infecton maybe a new strategy for preventing asthmaattacks in the future and may reduce the risk of asthma attacks in the future.Th clinical treatment of bronchiolitis is still controversial,and there is no effective or specific treatment at present.The international consensus and domestic guidelines are relatively consistent that corticosteroids or antibiotics are not recommended.Palizumab is a monoclonal antibody against RSV.Early vaccination can reduce severe RSV infection and reduce recurrent wheezing in premature infants during 6 years of follow-up,but does not affect the incidence of asthma in later years.Therefore,it is imperative to find a drug that can effectively regulate the immune function and have few side effects to treat bronchiolitis.Studies have shown that mesenchymal stem cells(MSCs)have the functions of repair and immunoregulatory properties,and have become potential therapeutics for cell therapy.MSCs have been used in some pulmonary diseases and nervous system diseases.Human placental mesenchymal stem cells(hPMSCs)are more likely to be obtained,more easily to induce immune tolerance,and closer to the clinical environment.It has not been reported whether hPMSCs can be used in the clinical treatment of children with bronchiolitis,how to regulate the immunology,and reduce the incidence of asthma in the future are unknown..Notch signaling pathway is a highly conserved signaling pathway,which plays an important role in the differentiation of CD4 T cells.Receptors of Notch signal can interact with ligands to regulate the generation and polarization of Th cytokines.Studies have shown that hPMSCs play an important role in immune regulation of Notch signal expression.Based on this,we believe that hPMSCs may regulate the pathogenesis of bronchiolitis in rats by regulating the expression of Notch signal,but the mechanism and specific effect of hPMSCs in the pathogenesis of bronchiolitis need to be further studied.This study evaluated the pathogenesis of bronchiolitis and asthma immunological pathogenesis through the clinical and basic research.To evaluate hPMSCs after transplantation of bronchiolitis rats model Thl/Th2 cells imbalance,the influence of the Notch signal pathway,explore hPMSCs adjust the immune function of children with bronchiolitis molecular mechanism,to provides the theory basis for clinical treatment of bronchiolitis.Part One Immunological Imbalance in Children with Different Severity of RSV Bronchiolitis and its Effect on Future AsthmaObjectiveTo compared the percentage of CD4 T lymphocyte subsets Thl,Th2,Treg,Th17 cells in peripheral blood,and related cytokines levels,which in healthy children and RSV bronchiolitis children of acute phase with different severity.To follow up the incidence of asthma and the change of Th1,Th2 cytokines and IgE levels in peripheral blood of children with RSV bronchiolitis at the age of 4,in order to understand the immune imbalance in RSV bronchiolitis of different severity and its impact on asthma in the future.MethodsSixty-eight bronchiolitis children with RSV-positive were enrolled,including 32 in the mild group,36 in the moderate-to-severe group,and 30 in the normal control group.Flow cytometry(FCM)was used to detect the proportion of CD4 T cells in Thl,Th2,Treg and Th17 cells in peripheral blood mononuclear cells(PBMC),and cytokine IFN-y,IL-4,IL-10 and IL-17 levels were detected by enzyme-linked immunosorbent assay(ELISA)method.Subsequently,the incidence of asthma in bronchiolitis children aged 4 years old was followed up by a combination of asthma questionnaire,telephone and hospital review.The levels of IFN-y and IL-4 in peripheral blood serum were detected by ELISA,and the level of IgE in serum of peripheral blood was determined by chemiluminescence method.Results1.Compared with the control group,the percentage of Thl subgroup cells and the relative expression of IFN-y were significantly reduced in the mild bronchiolitis group(P<0.05),while the percentage of Th2 subgroup cells and the expression of IL-4 were significantly increased(P<0.05).Compared with the mild bronchiolitis group,the percentage of Th1 subgroup cells and the relative expression of IFN-y were significantly reduced in the moderate and severe bronchiolitis group(P<0.05),while the percentage of Th2 subgroup cells and the expression of IL-4 were significantly increased(P<0.05).2.Compared with the control group,the percentage of Treg cell subsets and IL-10 in the mild bronchiolitis group were significantly decreased(P<0.05),while the percentage of Thl 7 cell subsets and IL-17 were increased(P<0.05).Compared with the mild bronchiolitis group,the percentage of Treg cell subsets and IL-10 in PBMC of the moderate and severe bronchiolitis group were significantly reduced(P<0.05),while the percentage of Th17 cell subsets and IL-17 were increased(P<0.05).3.50 children with bronchiolitis were followed up successfully,including 2 children with asthma after mild bronchiolitis,22 children with non-asthma(24 cases in mild follow up group);8 children with asthma after moderate to severe bronchiolitis,18 children with asthma(26 cases in moderate to severe follow up group).The incidence of asthma in mild follow up group was moderate to severe.There was significant difference in the degree of follow up group(P<0.05).4.Compared with the normal control group,the expression of IFN-y in mild follow-up group and moderate and severe follow-up group tended to decrease,but there was no statistical significance(P>0.05).Compared with the normal control group,the expression of IL-4 in mild follow-up group tended to increase,but there was no significant difference(P>0.05),while the expression of IL-4 in serum of moderate and severe follow-up group increased significantly(P<0.05).5.The expression of IgE in serum of mild follow up group tended to increase,but its expression value was within the normal range.The expression of IgE in moderate and severe follow up group was significantly higher than that in normal group,and the expression of IgE in serum was significantly higher than that in mild follow up group(P<0.05).Conclusions1.There is a Thl/Th2 cell imbalance and related cytokine changes in bronchiolitis children.2.There is a Treg/Th 17 cell imbalance and related cytokine changes in bronchiolitis children.3.The more severe of the disease,the more obvious Thl/Th2,Treg/Th17 cell imbalance and related cytokine changes.4.RSV infection can affect children with long-term effects,resulting in increased expression of IL-4 and IgE.5.The severity of RSV bronchiolitis at the initial diagnosis was associated with the higher incidence of asthma in the future.Part Two The Changes of Notch Signaling Pathway inBronchiolitis Rats and the Effect of hPMSCs TransplantationObjectiveTo investigate the effect and mechanism of human placental mesenchymal stem cells(hPMSCs)on respiratory syncytial virus(RSV)in bronchiolitis rats.MethodsThirty SD rats were randomly divided into RSV bronchiolitis group,hPMSCs transplantation group and normal control group.RSV bronchiolitis model was established by RSV nasal drop induction,and hPMSCs intervention model was established by tail vein injection of hPMSCs,in order to observe the changes of airway inflammation,AHR,and BLAF cells.To detect Thl and Th2 related proteins in each group,and expression levels of receptors and ligands associated with Notch signaling in each group of rats.ResultsThe normal control group showed normal symptoms.The rats in the RSV bronchiolitis group and the hPMSCs transplantation group showed different degrees of bronchiolitis-like seizures after modeling,and no rats died during the whole modeling and drug intervention.The bronchial and alveolar structures were generally normal in the normal control group;the bronchial and perivascular inflammatory cells were extensively infiltrated in the RSV bronchiolitis group;the degree of inflammatory response was reduced in the hPMSCs transplantation group compared with the RSV group.Compared with the normal control group,the relative content of T-bet in the lung homogenate of RSV bronchiolitis group decreased,and the relative content of GATA-3 increased(P<0.05).Compared with the RSV bronchiolitis group,the relative content of T-bet was increased and the relative content of GATA-3 was decreased in the hPMSCs transplantation group(P<0.05).Compared with hPMSCs and the control group,the expressions of Notch1,Notch2 and Jaddgedl in the lung tissues of RSV bronchiolitis group were significantly increased(P<0.05),and the expressions of Notch3,Delta4 were significantly decreased(P<0.05).Compared with RSV bronchiolitis group,the mRNA expressions of Notch 1,Notch2 and Jaddgedl in blood,lung tissue and lymph,were significantly down-regulated in hPMSCs transplantation group(P<0.05),while the mRNA expressions of Notch3 and delta4 were significantly down-regulated(P<0.05).Conclusions1.The rat model of bronchiolitis and the hPMSCs intervention model are successfully established.2.Notch signaling is involved in the pathogenesis of bronchiolitis.3.hPMSCs can down-regulate the expression of Notch signaling and reduce the severity of bronchiolitis. |
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