Mechanism Of BMMSCs Alleviating Asthma By Upregulating The Expression Of Tyro3 In Lung

Part I:Expression of Tyro3 in acute bronchial asthmaObjective: To clarify the changes of Tyro3 expression in acute bronchial asthma.Methods:1.The expression level of Tyro3 in peripheral blood serum of patients with acute asthma exacerbation and healthy volunteers was analyzed by automated latex immunoassay in strict accordance with the inclusion and exclusion criteria.2.The acute asthma model of Balb/c mice was established by the method of inhalation and inhalation after sensitization by intraperitoneal injection of OVA,and a blank control group was established.The pathological changes of lung tissue were confirmed by HE,Masson,and AB-PAS staining and IF were detected.Tyro3 expression in lung tissue,serum Ig E and Tyro3,BALF supernatant Tyro3 content were determined by ELISA.Result:1.The serum Tyro3 level in patients with acute asthma attack was significantly lower than that in healthy volunteers(P<0.001);2.Compared with the blank control group,the serum Ig E content of the acute asthma mice was significantly increased(P<0.001),the lung tissue inflammatory cell infiltration,goblet metaplasia,mucin secretion,airway smooth muscle fiber hyperplasia and peribronchial collagen deposition,Model build successfully.The levels of Tyro3 in lung tissue,serum(P=0.002)and BALF supernatant(P=0.003)were significantly lower than those in the blank control group.Conclusion: Compared with the control group,Tyro3 expression was decreased in acute bronchial asthma.Part II: Effects of BMMSCs intervention on the expression of Tyro3 in acute asthmatic miceObjective: To explore the effect of BMMSCs intervention on the expression of Tyro3 in acute asthma.Methods:1.Isolation,culture and identification of primary BMMSCs: The mouse bone marrow was extracted and cultured,and the morphology,surface markers,and adipogenic and osteogenic differentiation potential were identified.2.To clarify that BMMSCs relieve acute asthma: Balb/c mice were randomly divided into blank control group,asthma model group,MSCs intervention asthma group,and MSCs control group.After intraperitoneal sensitization with OVA,aerosol challenge,BMMSCs suspension was injected into tail vein for intervention,clinical symptoms were scored,serum Ig E,Th2-type inflammatory factor and anti-inflammatory factor IL-10 content in BALF supernatant were detected by ELISA,HE,AB-PAS and Masson staining were used to observe the pathological changes of lung tissue.3.To detect the effect of BMMSCs on the expression level of Tyro3 in asthmatic mice:RT-PCR was used to detect the content of Tyro3 m RNA in lung tissue,and immunohistochemistry,immunofluorescence and Western Blot were used to detect the expression level of Tyro3 protein in lung tissue.Tyro3 protein content in serum and BALF supernatant was analyzed by ELISA.Result:1.BMMSCs were successfully isolated and cultured.2.BMMSCs significantly improved the condition of acute asthmatic mice:2.1 Compared with the asthma model group,the clinical symptom scores of the mice in the MSCs intervention asthma group were decreased,and the levels of Th2-related cytokines in serum Ig E and BALF were decreased(IL-4:P<0.001;IL-5:P=0.002;IL-13 :P=0.005),IL-10 increased.2.2 After BMMSCs intervention,lung histopathology was improved,inflammatory cell infiltration,collagen fiber and smooth muscle hyperplasia were reduced,and goblet metaplasia and mucus secretion were reduced.2.3 Compared with asthma model group,after BMMSCs intervention,Tyro3 m RNA content in lung tissue increased(P=0.035),Tyro3 protein in lung tissue(P=0.032),BALF supernatant(P=0.043),and serum(P=0.009)expression level increased.Conclusion: BMMSCs significantly inhibited the down-regulation of Tyro3 expression in the lungs of asthmatic mice.Part III: The role and mechanism of Tyro3 in the intervention ofBMMSCs in asthmaObjective: To explore the important role of Tyro3 in the intervention of BMMSCs in asthma.Methods:1.Constructing adenovirus Ad-sh Tyro3 interference vector,knocking down the expression level of Tyro3 in lung tissue in vivo,taking the empty adenovirus Ad-NC as control,and according to the OVA/BMMSCs intervention method,Balb/c mice were randomly divided into 8 groups:Control(Ad-NC),Control(Ad-sh Tyro3),Asthma(Ad-NC),Asthma(Ad-sh Tyro3),Asthma+MSCs(Ad-NC),Asthma+MSCs(Ad-sh Tyro3),MSCs(AdNC),MSCs(Ad-sh Tyro3).Lung adenovirus transfection was observed by small animal imaging and frozen section immunofluorescence.2.To analyze the changes of Tyro3 expression in lung tissue of mice after adenovirus transfection: RT-PCR was used to detect the content of Tyro3 m RNA in lung tissue,and immunohistochemistry,immunofluorescence and Western Blot were used to detect the expression of Tyro3 protein in lung tissue.The Tyro3 protein content in serum and BALF supernatant was analyzed by ELISA,and the effect of Ad-sh Tyro3 transfection on the expression level of Tyro3 in mice was comprehensively analyzed.3.To evaluate the role of Tyro3 in the improvement of acute asthma by BMMSCs:ELISA was used to measure the changes of asthma-related inflammatory factors and pathological assessment of asthma severity after Tyro3 was knocked down in vivo.4.Preliminary study on the mechanism of Tyro3 involved in the intervention of BMMSCs in asthmaFlow cytometry was used to analyze the proportion of eosinophils in immune cells in lung tissue,the type and expression of Tyro3-expressing immune cells,the proportion and activation of DCs,and the activation of CD4+ T cells in mediastinal lymph nodes.Result:1.The adenovirus vector was successfully constructed and infected lung tissue,but the heart,liver,spleen and kidney were not infected.2.Transfection of Ad-sh Tyro3 successfully reduced the expression level of Tyro3 in mouse lung tissue,serum and BALF supernatant: In the asthma model group and the MSCs intervention asthma group,the Ad-sh Tyro3 injection between the same groups was more than that of the Ad-NC injected mouse lung.The content of Tyro3 m RNA in tissue was significantly decreased(Ad-NC vs Ad-sh Tyro3 in asthma model group: P=0.008;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P=0.040),lung tissue(Ad-NC vs Adsh Tyro3 in asthma model group: P=0.056;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group:P<0.001),BALF supernatant(Ad-NC vs Ad-sh Tyro3 in asthma model group:P<0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P=0.043),serum(Ad-NC vs Ad-sh Tyro3 in asthma model group: P=0.043;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P=0.035)significantly decreased Tyro3 protein expression levels.Immunohistochemistry and immunofluorescence also showed that the content of Tyro3 in the lung tissue of mice injected with Ad-sh Tyro3 decreased compared with that of mice injected with Ad-NC in the same group.3.After knocking down the expression level of Tyro3 in the lung tissue,the asthma model mice became more ill,and the effect of BMMSCs on asthma was weakened: after knocking down Tyro3,compared with the mice without Tyro3 knockdown in the same group,the asthma model group and MSCs intervention asthma group The levels of serum Ig E in the two groups of mice(Ad-NC vs Ad-sh Tyro3 in asthma model group: P=0.03;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P<0.001),IL-4 in BALF supernatant(AdNC vs Ad-sh Tyro3 in asthma model group: P<0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P<0.001),IL-5(Ad-NC vs Ad-sh Tyro3 in Asthma model group:P<0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P<0.001),IL-13(AdNC vs Ad-sh Tyro3 in asthma model group: P<0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P=0.140)The content of IL-10 was significantly decreased(AdNC vs Ad-sh Tyro3 in asthma model group: P=0.014;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group: P=0.003).In the asthma model group and the MSCs-intervention asthma group,the Ad-sh Tyro3 injection between the same groups increased the inflammatory cell infiltration,collagen fiber and smooth muscle hyperplasia,goblet metaplasia and mucus secretion in the lung tissue of the mice injected with Ad-NC.4.After knocking down of Tyro3,compared with the mice without knockdown of Tyro3,the proportion of CD45+CD3+CD4+ T cells expressing CD69 in the mediastinal lymph nodes of the mice in the asthma model group and the MSCs intervention asthma group(Ad-NC vs Ad-sh Tyro3 in the asthma model group:P=0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group:P<0.001),CD45+CD3+CD4+T cell CD69 MFI level in mediastinal lymph node(Ad-NC vs Ad-sh Tyro3 in asthma model group:P<0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group:P<0.001),the proportion of eosinophils in lung tissue immune cells(Ad-NC vs Ad-sh Tyro3 in Asthma model group:P=0.009;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group:P=0.023),the proportion of DCs expressing Tyro3 in lung tissue(Ad-NC vs Ad-sh Tyro3 in asthma model group:P<0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group:P<0.001),the proportion of DCs in total CD45+ cells in lung tissue(Ad-NC vs Ad-sh Tyro3 in Asthma model group:P<0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group:P<0.001),CD80 MFI level of DCs in lung tissue(Ad-NC vs Ad-sh Tyro3 in asthma model group:P<0.001;Ad-NC vs Adsh Tyro3 in MSCs intervention asthma group:P=0.001)and MHCII MFI level(Ad-NC vs Ad-sh Tyro3 in asthma model group:P<0.001;Ad-NC vs Ad-sh Tyro3 in the MSCs intervention group:P<0.001)were significantly increased,and the Tyro3 MFI level of the DCs was significantly decreased(Ad-NC vs Ad-sh Tyro3 in the asthma model group:P=0.001;Ad-NC vs Ad-sh Tyro3 in MSCs intervention asthma group:P<0.001).Only DCs in the immune cells of mouse lung tissue can express Tyro3,and compared with the blank control group,the proportion of DCs expressing Tyro3 in the lung tissue cells of the mice in the asthma model group to the total number of DCs decreased(blank control group vs asthma model group in Ad-NC:P<0.001;blank control group vs asthma model group in Ad-sh Tyro3:P<0.001),increased after BMMSCs intervention(blank control group vs asthma model group in Ad-NC:P<0.001;blank control group vs asthma Model group in Adsh Tyro3:P<0.001),while other immune cells did not express Tyro3.Although non-immune cells also expressed Tyro3,there was no significant difference in its level among different treatment groups(P>0.05).Conclusion: The increased expression of Tyro3 in DCs is involved in the process of BMMSCs intervention in acute asthma.