| Hearing loss is a global question. Today, exceed 10% aulduts have sensorineural hearing loss in defferent degree and the ratio must be increased as the elder increase,especially in China. Many kinds of sensorineural hearing loss are related to immunity,but this is always ignored in clinical work. There is damnification of immunity in some disease,such as : some of sudden deafness ,Meniere disease, otosclerosis, chronic otitis media, the trauma of temporal bone and some systemic immunologic disease in inner ears, and"auotoimmune inner ear disease(AIED)"is the tentative name of all those immunologic disease in inner ears. Study had detected different non-specific inner ear tissue antigens and one of them was hsp70. because of etogy's unclear, the pathogenesis's complicated,not determined the specific inner ear antigen attacked by immune system and no speficic experimental targets, the therapy of AIED are controversy. There havn't prooves demonstrated that wether the produce of hsp70 is a major element or an affiliated element in AIED. The aim of this research is to establish an AIED animal model which causes high positive hearing impairment and probe the relationship between hsp70 and Toll-like receptor 4(TLR4) antibody , is to elaborate the possible pathogenesis and provide theory prooves for manufacture receptor blocker and new drug to blok inflammation.Partâ… Establishment of Autoimmune Inner Ear Disease Animal ModelObjective: To establish an AIED animal model which causes high positive hearing impairment and prove the animal model is successful.Methods: A mixture of membranous proteins of inner ear was separated by preparative SDS-PAGE and purified hsp70 furthermore. The auditory brainstem response(ABR) thresholds,serum hsp70 antibody and the concentration of immune serum globulin,C3 ,circulation immune compound(CIC) were detected in different time after the pretreatment of the guinea pigs with the antigens and complete Freund's adjuvant of equivalent volum.Results: Guinea pigs were vaccinated 7 days later, inflammatory cell infiltration was seen in the cochlea of the animals, the value of ABR increased greatly , the concentration of immune serum globulin,C3 , CIC were elevated obviously,there were not obvious changes when vaccinated 14 days later until vaccinated 28 days later each target above-mentioned decreased and the hearing function come back to nomal little by little.Conclusion: The value of ABR ,the concentration of immune serum globulin,C3 , CIC were elevated obviously after guinea pigs were vaccinated by inner ear antigens which proved the AIED animal model was succeeful,especially guinea pigs were vaccinated 7 days later.Partâ…¡Relationship Between TLR4 Mediate Hsp70 Singal Pathway and ABR Value in Different Time in Autoimmune Inner Ear DiseaseObjective: To detect the expression of TLR4,hsp70 ,NF-κв(P65), TNF-α, ICAM-1 and ABR value in different time in inner ear and to demonstrate the expression of ABR increased followed the incease of TLR4/hsp70 and proved that TLR4/hsp70 is positive correlation to the development of AIED.Methods: Guinea pigs were killed after vaccinated 7, 28 days, section were made and inner ear proteins were extracted. Observed the expression of hsp70 in inner ear with immunohistochemistry ,in situ hybridization(ISH) and western blot technique.Results: Guinea pigs were vaccinated 7 days later,the value of ABR increased greatly ,the expression of hsp70 in inner ear elevated obviously, when vaccinated 28 days later each target above-mentioned decreased and the hearing function come back to nomal little by little.Conclusion: Guinea pigs were vaccinated 7 days later,the value of ABR and The expression of TLR4, hsp70, NF-κв(P65), TNF-α, ICAM-1 in inner ear elevated obviously , vaccinated 28days later each target above-mentioned decreased in our research, which proved that TLR4/hsp70 is positive correlation to the development of AIED.Partâ…¢Section 1 The Expression of Hsp70 Intervened by Injecting Various Doses of TLR4 Antibody Through IntratympanicObjective: To probe for the best intratympanic injection time and the most suitable doses of TLR4 antibody by injecting various doses of TLR4 antibody.Methods: Each ear was injected 50,100,150,200,250μg TLR4 antibody Through Intratympanic respectively after Guinea pigs were vaccinated 2,5,7 days,observed the expression of hsp70 in inner ear with immunohistochemistry , ISH and western blot technique, then selected the best intratympanic injection time and the most suitable doses of TLR4 antibody.Results: The expression of hsp70 in inner ear decreased little by little after intratympanic injection 50,100,150,200,250μg TLR4 antibody when Guinea pigs were vaccinated 2 days later,especially in 200μg dose , the expression of hsp70 decreased greatly,while there were no obvious changes in 250μg dose. When Guinea pigs were vaccinated 5,7days, the expression of hsp70 increased slightly. Conclusion: Intratympanic injection 200μg TLR4 antibody when Guinea pigs were vaccinated 2 days later coud inhibit the expression of hsp70 in great extent.Section 2 The expression of ICAM-1,TNF-αNF-κв(P65)Objective: To observe the expression of ICAM-1,TNF-α,NF-κв(P65) and discuss the possible etogy.Methods: Guinea pigs were divided into three groups randomly,one is experimental group,one is intervene group and the other is control group. After guinea pigs were vaccinated 2 days, the animals of intervene group were injected TLR4 antibody with 200μg dose through intratympanic in each ear, 7days later, all animals were detected ABR before killed,then detected the expression of ICAM-1,T NF-αNF-κв(P65) with immunohistochemistry , ISH ,western blot and ELISA technique .Results: After animals were vaccinated 7 days ,ABR and the expression of ICAM-1, TNF-α, NF-κв(P65) were inreased, when TLR4 abtibody was injected,all targets above-mentioned were decreased and hearing function elvelated.Conclusion: The expression of hsp70 in inner ear increased obviously in AIED, when TLR4 antibody injected ,the expression of hsp70 in inner ear could be inhibited in great extent and elevate hearing function which suggested TLR4/Hsp70 play important roll in AIED .Inccording to this ,we can furthermore elaborate the possible pathogenesis of AIED and provide theory prooves for manufacture receptor blocker and new drug to blok inflammation. |
