胚胎期干预Cx26条件基因敲除小鼠治疗感音神经性耳聋的研究

Mutations in the GJB2 gene, which encodes the gap junction (GJ) protein connexin26 (Cx26), are the most common cause of inherited non-syndromic hearing loss (NSHL). In our previous study, we found that Cx26 conditional knockout could induce apoptosis of cochlear cell, and miR-27a overexpression may promote cell apoptosis in part by inhibiting sgk1 and FKHRL1 phosphorylation. When inoculated modified adeno-associated viral (AAV) vectors into the scala media of early postnatal conditional Cx26 knockout mice to drive exogenous Cx26 expression, we found that both cell apoptosis in the organ of Corti and degeneration of spiral ganglion neurons in the cochlea of mutant mice were substantially reduced. However, auditory brainstem responses did not show significant hearing improvement. The reason may be that Cx26 plays essential roles in embryonic inner ear development. Therefore, we proposed to carry out the following research: 1) Verify whether miR - 27 a act as an apoptotic molecule through regulation its target gene sgk1 and thus affecting FKHRL1 phosphorylation to participated in Cx26 knockout-induced apoptosis of cochlear sensory epithelium; 2) Inoculate modified adeno-associated viral (AAV) and lentivirus (LV) into otocyst of conditional Cx26 knockout mice in E11.5 respectively and make a comparsion, to establish a safe and high-efficiency embryonic stage otocyst virus transfection technique; 3）overexpress GJB2 gene of conditional Cx26 knockout mice in E11.5 to drive exogenous Cx26 expression, and test the cochlear sensory epithelium pathologic morphology, function of new formed gap junction channels, lymph potential within the inner ear and auditory function in the different time points after transfection.

GJB2（Connexin26，Cx26）基因突变是人类常见致聋因素。我们前期研究发现：Cx26基因条件敲除小鼠出生后基底膜细胞发生凋亡；miR-27a可能通过下调靶基因sgk1进而抑制FKHRL1蛋白磷酸化促进其凋亡；出生后内耳基因治疗可抑制基底膜细胞的凋亡，但未能恢复其正常形态和听觉功能，推测GJB2突变可能影响到胚胎期耳蜗发育和功能。因此我们拟展开以下研究：1）验证miR-27a对靶基因sgk1的调控是否通过影响FKHRL1蛋白磷酸化水平这一机制参与耳蜗基底膜细胞的凋亡路径；2）采用Cx26条件基因敲除小鼠耳蜗模型，比较腺相关病毒和慢病毒载体对于胚胎期听囊转染的异同，建立高效安全的胚胎内耳转染技术；3）利用该技术过表达E11.5天条件基因敲除小鼠耳蜗中的Cx26基因，研究转染后不同时间点耳蜗感觉上皮病理形态学变化，新形成的缝隙连接通道的功能以及内耳内淋巴电位和听觉功能的修复情况。

GJB2（Connexin26，Cx26）基因突变是人类常见致聋因素。我们前期研究发现：Cx26 基因条件敲除小鼠出生后基底膜细胞发生凋亡；miR-27a可能通过下调靶基因sgk1进而抑 制FKHRL1蛋白磷酸化促进其凋亡；出生后内耳基因治疗可抑制基底膜细胞的凋亡，但未能 恢复其正常形态和听觉功能，推测GJB2突变可能影响到胚胎期耳蜗发育和功能。因此我们 拟展开以下研究：1）验证miR-27a对靶基因sgk1的调控是否通过影响FKHRL1蛋白磷酸化水 平这一机制参与耳蜗基底膜细胞的凋亡路径；2）采用Cx26条件基因敲除小鼠耳蜗模型， 比较腺相关病毒和慢病毒载体对于胚胎期听囊转染的异同，建立高效安全的胚胎内耳转染 技术；3）利用该技术过表达E11.5天条件基因敲除小鼠耳蜗中的Cx26基因，研究转染后不 同时间点耳蜗感觉上皮病理形态学变化，新形成的缝隙连接通道的功能和听觉功能的修复情况

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