The Effect Of Silencing Of Integrin β4on The Expression And Signaling Transduction Of Toll Like Receptor4

At present, the pathogenesis of asthma is till unclear, destruction in airway epithelium structure and integrity,which is often induced by flaws of constitutive adhesion,is the essential link of asthma pathogenicity.Our previous work identified genes that were differentially expressed in the peripheral leukocytes of asthma patients by using cDNA microarray analysis.The results demonstrated that integrin β4was significantly down-regulated in asthma patients,which indicated the passible relationship between integrin β4and asthma.Toll like receptor connect innate immunity and adaptive immunity,and play a role in acute inflammation and signal transduction.TLRs recognize various molecules of microbial origin,called pathogen-associated molecular patterns(PAMPs),and trigger inflammation by inducing the transcription of genes encoding cytokines,including TNF-α and IFN-β.TLR4has universal adaptability.Our previous study have demonstrated the relationship between integrin β4and asthma.It also found that silencing of integrin β4affect on antigen presentation and shut down the Thl pathway.We reason the relationship between silencing of integrin β4and the change of Toll like receptor.Therefore, the aim of the present study is to observe the influence of integrin β4and TLR4. Objective:To study the effect of silencing of integrin α4on the expression of TLR4, and the difference of two pathways.At the same time,we will study the effect of lymphocyte differentiation.Preliminary study on the immunological theory caused by asthma.Method:(1) Integrin β4siRNA were constructed and staly transfected into HBECs.The effect of siRNA were observed by fluorescence microscope.Identify the efficiency of integrin04siRNA by RT-PCR and flow cytometry.(2) MTT was used to detect the best concentration of LPS(3) On effect of LPS, the levels of TLR4in normal HBECs and control siRNA and integrin β4siRNA were detected by flow cytometry(4) On effect of LPS, the levels of MyD88and TRIF in normal HBECs and control siRNA and integrin β4siRNA were detected by Western blot.(5) Normal HBECs and control siRNA and integrin β4siRNA were added LPS,and cultured at37℃for24h,48h and72h.The levels of TNF-a and IFN-β were detected by ELISA.(6) Normal HBECs and control siRNA and integrin β4siRNA were treated with1ng/mL LPS for6h,then cocultured with lymphocyte for72h.The supernatant liquid were harvested respectively, The levels of IFN-y,IL-4and IL-17were detected by ELISA.Result:(1) We successfully constructed integrin β4siRNA and transfected them into HBECs.The transfection efficiency of siRNA was90%by fluorescence microscope.Compared with control siRNA transfected HBECs,the expression of integrin04was significally decreased in integrin β4siRNA transfected HBECs.(2) The best concentration of LPS is1ng/mL(3) On effect of LPS,the expression of TLR4were increased in normal HBECs and control siRNA and integrin β4siRNA. Compared with control siRNA transfected HBECs,the expression of TLR4was signically increased in integrin β4siRNA.(4) On effect of LPS, the expression of MyD88,TRIF were increased in normal HBECs and control siRNA and integrin β4siRNA. Compared with control siRNA transfected HBECs,the expression of MyD88and TRIF was signically increased in integrin β4siRNA.(5) On effect of LPS, the expression of TNF-α and IFN-β were increased in normal HBECs and control siRNA and integrin β4siRNA. Compared with control siRNA transfected HBECs,the expression of TNF-α and IFN-β were signically increased in integrin β4siRNA. TNF-α has not obvious change in24h,48h and72h,but IFB-β has obvious increase in72h.(6) Compared with integrin β4siRNA transfected HBECs, the expression of IL-4,IL-17were signically increased in integrin β4siRNA transfected HBECs,and have no influence on the secretion of IFN-γ. On effect of LPS, the expression of IFN-y were increased in integrin β4siRNA.Conclusion:(1) The silence of integrin α4can significantly increase the expression of TLR4,and two signal pathway on TLR4are affected.The affect of MyD88 dependent signaling pathway happened in24h, but the affect of MyD88independent signaling pathway happened in72h.(2) The silence of integrin (34can significantly decrease the ratio of Thl/Th2and induce the Th17immune.At the same time, LPS (1μg/mL) can induce the Th17immune.