The Effect Of Integrin β4on The Immune Signal Transmission Of Human Bronchial Epithelial Cells

Airway epithelium is the interface between internal environment and external environment of our body, it acts not only as mechanical barrier but also possess abundant biological function. Dysfunction of homeo stasis in airway epithelium structure or function may cause airway hyperresponssive associated disease such as asthma. Disbalance of Thl/Th2immune reaction is considered as one of the most important mechanism in asthma with essential qualities as chronic inflammation. Bronchila epithelia cells (HBECs) may act as antigen presenting cell, not only express the Toll-like receptors (TLRs) to recognize and catch the extrageneous pathogenic bacteria, but also transfer immune signal to T cell by B7costimulatory factor, impact the T cell and local immune state in airway.Integrin (34located in the basal surface of airway epithelial cells, influence the polarity and3D glandular cavity formation. More importantly, integrin (34is the key component of hemidesmosomes. Integrin (34bands with its specific receptor laminin promote the heterologous adhesion between airway epithelial cells, as well as the homologous adhesion of airway epithelial cells and basilar membrane. Normal expression of integrin β4maintain the anchoring-dependent growth of HBECs. Tracheoscope examine always showed that apoptosis and shedding of HBECs.Our previous study detected the decrease of integrin β4in both peripheral blood leucocyte and airway mucosa in asthma patient. Due to the protective function of integrin β4in HBECs, such as promote the proliferation, inhibit apoptosis, anti-oxidant injuries and reduce the uptake of antigen OVA, we presume that the defect expression of integrin (34may contribute to the dysfunction of homeostasis in airway epithelium structure or function, finally cause asthma. Whether integrin β4act on the immune state of HBECs or regulate the local immunological response still need further study.Here we use the siRNA from chemical synthesis to silence the integrin β4gene in HBECs, dectect the change in B7costimulatory factor, TLR4expression and lymphocyte proliferation stimulated by OVA. The result may offer a new light for the physiological function of integrin β4and elucidate the mechanism of asthma in a different aspect.Part1Synthesis and verification integrin β4siRNA by transient transfectionObjective:Applying the technology of RNA interference to silence the integrin β4in HBECs.Method:Specific integrin β4siRNA fragment was transfected into HBECs by liposomes in suitable condition,48h later cells were collected and the mRNA was detected by real-time PCR, the change in protein lever was examed by western blotting.Result:Both mRNA and protein of integrin β4deceased significantly in HBECs treated by siRNA.Conclution:The specific integrin β4siRNA fragment efficiently caused the integrin β4gene silence in HBECs.Part2Effect of integrin β4gene silence on the exprssion of B7H1and B7DC in HBECsObjective:To observe the effect of integrin (34gene silence on exprssion of costimulatory factor B7H1and B7DC in HBECs.Method:After siRNA treatment cells were collected and analyzed with real-time PCR and flow cytometry to measure whether any change occur in the expression of B7H1and B7DC.Result:B7H1increased in integrin β4silenced HBECs compared to normal control, and B7DC display the similar tendency with B7H1.Conclution:The silence of integrin β4gene promoted the B7H1and B7DC in both mRNA and protein lever.Part3Impact of integrin β4gene silence on the exprssion of TLR4in OVA stimulated HBECsObjective:The part was designed to examine whether integrin β4gene silence act on the exprssion of TLR4in OVA stimulated HBECs.Method:Cells incubated with OVA for1h and immunochemistry was used to deteched the changes of TLR4gene expression in integrin β4gene silenced and normal HBECs.Result:The expression of TLR4was up-regulated in siRNA transfected HBECs stimulated by OVA for1h compared to negtive control.Conclution:The siRNA can effectively down-regulate integrin β4, which also improved the TLR4gene expression in OVA stimulated HBECs.Part4Influence of integrin β4gene silence on the proliferation of lymphocytes co-cultured with HBECsObjective:Detect the influence of integrin (34gene silence on the proliferation of lymphocytes co-cultured with HBECs.Method:Integrin β4gene silenced HBECs or normal HBECs were treated with OVA, then co-cultured with freshly isolated lymphocyte, MTT method was carried out to determine the proliferation activities of lymphocyte.Result:HBECs can act as APC in OVA induced lymphocyte response, increase the proliferation activities of lymphocyte. Integrin β4gene silence in HBECs inhibited the lymphocyte proliferation in a certain extent.Conclution:Integrin (34gene silence in HBECs decreased the lymphocyte proliferation reaction to OVA.It is suggested that integrin β4could act on the expression of B7H1, B7DC, TLR4and lymphocyte proliferation to regulate the immune state of HBECs, which maybe regulate the local immunological response.