摘要
目的探讨miR-34a通过下调Bcl-2表达,介导原代听觉皮层神经元凋亡在年龄相关性聋中枢发病机制中的可能作用。方法采用C57BL/6乳鼠,定位获得听觉皮层脑组织,进行原代神经元培养,并通过对原代神经元进行转染,过表达miR-34a或干扰下调miR-34a表达后,采用western blot进行靶基因Bcl-2蛋白水平检测,采用Hoechst染色行细胞凋亡检测。结果酶消化法可分离并获得原代皮层神经元,将原代神经元进行转染,采用western blot检测转染过表达miRNA-34a后Bcl-2表达情况,转染48 h和72 h后Bcl-2表达均下降,5 nmol/L浓度与阴性对照比较,差异有统计学意义(t=5.127,P<0.05),10 nmol/L与20 nmol/L浓度比较,差异有统计学意义(t=6.379,P<0.05)。抑制表达mi RNA-34a,Bcl-2表达均升高,5 nmol/L浓度与阴性对照比较,差异有统计学意义(t=4.926,P<0.05);10 nmol/L与20 nmol/L浓度比较,差异有统计学意义(t=5.821,P<0.05),免疫荧光检测Bcl-2表达也显示相似结果。细胞凋亡染色在过表达mi RNA-34a后,神经元核固缩,凋亡程度增加。结论 miR-34a通过下调Bcl-2表达,介导原代听觉皮层神经元凋亡,可能是年龄相关性聋中枢发病机制之一。
OBJECTIVE To explore the role of miR- 34a on upregulating the expression of Bcl-2, which induces the apoptosis of primary auditory cortex neuron in the central mechanism of age-related hearing loss. METHODS Using C57BL/6 suckling mouse to obtain the brain tissue from auditory cortex according to the Location map, and to primary culture the neurons. After transfection on primary neurons, western blot and were used to detect the expression of Bcl-2 and then hoechst staining was used to detect the apoptosis after transfection. RESULTS primary culture of auditory cortex neurons abtained from enzyme digestion were transfected with miR-34a mimic and miR-34a inhibitor to upregualte or downregulate the expression of miR-34a, the results showed that the expression of Bcl-2 was decreased after upregulation of miR-34a with the concentration of 5 nmol/L(t=5.127, P〈0.05), there was significant difference between the concentration of 10 and 20 nmol/L(t=6.379, P〈0.05), while increased after downregulation of miR-34a with concentration of 5 nmol/L(t=4.926, P〈 0.05), there was significant difference between the concentration of 10 and 20 nmol/L(t=5.821, P〈 0.05). Hoechst staining showed that the apoptotic neurons was increased after transfection of miR-34a mimic. CONCLUSION miR-34a induce the auditory cortex neuron apoptosis through downregulates Bcl-2, which central mechanism of age-related hearing loss.
出处
《中国耳鼻咽喉头颈外科》
CSCD
2017年第12期625-628,共4页
Chinese Archives of Otolaryngology-Head and Neck Surgery
基金
国家自然科学基金青年基金资助项目(81400456)