中文摘要：

目的 量化AIF基因在细胞能量代谢中的功能特性,探讨该基因相关听神经病的分子致病机制。方法 选择第三代基因编辑工具CRISPR-Cas9技术结合阳离子脂质体转染技术,快速实现对HELA细胞的AIF基因进行敲除,并通过Western blot进行蛋白表达分析,Sanger测序进行基因型的分析,然后利用Oxygraph-2K对多个AIF基因敲除细胞系以及野生型细胞系进行氧化供能的检测,最后采用Graph Pad prism5.00进行数据分析。结果 通过该方法获得了多株AIF基因敲除细胞系,并发现野生型细胞系耗氧功能明显强于AIF基因敲除细胞系。结论 通过CRISPR-Cas9技术的应用,本研究快速实现了AIF蛋白表达缺失细胞系和野生型细胞系在氧化呼吸方面的差异比较,间接量化了该基因缺失所导致的能量损失,能量供应障碍可能是听神经病的致病机制之一。

英文摘要：

Objective To know of the functional features ofAIF gene in cell metabolism process, and to further explore molecular mechanism of AIF related auditory neuropathy. Methods For the aim of obtaining AIF Knock out cell line, the Lipofectamine 2000 was used to transfect CRISPR-Cas9 in HELA cells. Western blot and Sanger sequence were performed to test protein expression and genotype analysis, respectively. Oxygraph-2K was chosen to detect the oxidation function of multiple AIF knockout cell line and wild-type cell line, GraphPad prism5 was used to analyze data. Results Multiple strains ofAIF gene knockout cell lines were obtained by the methods, and the oxygen consumption function of wild type cell lines were significantly stronger than the AIF gene knockout cell lines. Conclusion This study compared the oxygen consumption difference between AIF-deficient cells lines and wild-type cells lines by applying CRISPR-Cas9 technology, indicating that the protein expression of AIF plays a key role in maintaining cells energy metabolism, which may be an incentive mechanism of auditory neuropathy.