【作者】 张蕊；

【导师】 刘邦华； 孔维佳；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉头颈外科学， 2013， 硕士

【摘要】 目的：为探索GJB2基因突变导致耳聋的机制及防治，我们拟建立Cx26基因敲除小鼠模型。同时初步探索慢性噪声暴露对某一发育阶段敲除Cx26的小鼠内耳的影响。方法：1.（1）条件诱导性Cx26基因敲除小鼠模型的建立：应用PCR技术鉴定两种基因改建的小鼠Cx26^loxP/loxP（C57/BL背景）和ROSA26^Cre-ERT，通过遗传学手段杂交建立Cx26条件诱导性基因敲除小鼠模型。这种小鼠是在Rosa26的基因位点插入了配体依赖Cre重组酶基因，人工合成的雌激素拮抗剂tamoxifen（TMX）能够激活Cre表达。从而切除GJB2基因位点上被loxp序列锚定的片段（2）条件性Cx26基因敲除小鼠模型的建立：使用PCR技术鉴定出Cx26LoxP/LoxP小鼠（CD1背景）与Pax2·Cre小鼠，Cre随Pax2的表达于特定组织中表达，根据遗传学定律两者杂交，F2代获得条件性Cx26基因敲除小鼠（Cx26-/-；Pax2·Cre）。2.慢性噪声暴露： Cx26^loxP/loxP;ROSA26^Cre-ERT小鼠在出生第15天腹腔注射TMX3mg/10g体重，小鼠出生第21天，每日暴露于100dBSPL稳态白噪声，每天8小时，连续暴露5天。听性脑干反应检测小鼠听阈，耳蜗基底膜铺片观察毛细胞情况。结果：基因分型结果显示我们通过遗传学手段将两种基因改建小鼠杂交，获得了Cx26条件诱导性基因敲除小鼠Cx26^loxP/loxP;ROSA26^Cre-ERT和条件性Cx26^loxP/loxP；Pax2·Cre基因敲除小鼠。TMX注射组慢性噪声暴露后底回毛细胞损伤较单纯噪声组严重，差异有显著性。结论：我们在本实验室中建立了两种Cx26基因敲除小鼠模型。1)时间诱导性Cx26基因敲除小鼠模型，该模型可以在耳蜗发育、内淋巴电位产生和听力形成的各个时期敲除Cx26基因。2)条件性Cx26基因敲除小鼠模型，该模型中小鼠耳蜗部分Cx26已被敲除。降低Cx26的表达增加噪声对内耳的损伤。更多还原

【Abstract】 Objective: In this study, we aimed to generate two cCx26null mouse models.and toexplore how the chronic noise exposure influence the cochlea function after knockdown theCx26at development stage.Methods:1.（1） To generate conditional inducible Cx26null transgenic mouse models：We back crossed the transgenic mouse Cx26^loxP/loxPwith a C57/BL genetic background withROSA26^Cre-ERTtransgenic mouse, based on law of inheritance, we generateCx26^loxP/loxPROSA26^Cre-ERTtransgenic mouse in F2. Ligand-dependent Cre gene sequencewas inserted into the ROSA promoter. Therefore, only exposure to tamoxifen will induceCre expression.（2） To generate conditional connexin26（Cx26） null of transgenic mousemodels：we back crossed the transgenic mouse Cx26^loxP/loxPwith a CD1genetic backgroundwith Pax2·Cre transgenic mouse, that generate Cx26-/-Pax2·Cre mouse in F2. Crerecombinase can express at specific tissue with Pax2promoter.2. Chronic noise exposure：Cx26^loxP/loxP;ROSA26^Cre-ERTmice were intraperitoneal injected TMX（3mg/10gweight） at P15,then exposured to100dBSPL noise for8hours per day,continued for five days from P21.ABR was used to detect the hearing threshold. Cochlea basilar membrane stretchedpreparation technique was used to assess hair cell loss.Results: Genotyping initially confirmed that we generated the conditional inducible Cx26null transgenic mouse models and conditional Cx26null transgenic mouse models,viacrossing bred two types of genetically engineered mice..Hair cell degeneration was severerin the basal tunnel of TMX injection with noise exposure group compared with the noiseexposure groupConclusion: We have successfully generated two types of Cx26null transgenic mousemodels.1) conditional inducible connexin26（Cx26） null of transgenic mousemodels,which can knockdown Cx26gene at each developmental stage,such as cochleadevelopment、the stage of endolymphatic potential generation or at the stage of hearingonset.2) Conditional Cx26null transgenic mouse models, that can knockdown Cx26gene at the early developmental stage. We concluded that downing--regulated Cx26acceleratednoise-induced hearing loss。更多还原