全反式视黄酸通过RARα抑制缺氧缺糖引起的原代神经元凋亡  

All-trans retinoic acid(ATRA) inhibits apoptosis of primary neurons induced by oxygen and glucose-deprivation via retinoic acid receptor alpha

作　　者：郭敏[1] 江伟[1] 侯娜丽 谷燕[1] 黄鸿眉[1] 陈洁[1] 李廷玉[1] 

机构地区：[1]重庆医科大学附属儿童医院营养研究中心儿童发育疾病研究教育部重点实验室认知发育与学习记忆障碍转化医学重庆市重点实验室儿科学重庆市重点实验室,重庆400014

出　　处：《重庆医科大学学报》2016年第1期8-13,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81161120498;81100454;81100476)

摘　　要：目的 :探讨全反式视黄酸(all-trans retinoic acid,ATRA)是否能够抑制缺氧缺糖损伤(oxygen and glucose-deprivation,OGD)诱导的原代神经元凋亡。方法 :采用原代神经元作为研究对象,分为对照组、OGD组、OGD+ATRA组;体外分离培养原代神经元,用神经元特异性烯醇化酶(neuron-specific enolase,NSE)鉴定神经元;倒置显微镜观察OGD损伤前后细胞形态变化;流式细胞技术研究OGD损伤后各组原代神经元凋亡水平;Real-time PCR检测视黄酸核受体α(retinoic acid receptor alpha,RARα)、转录生长因子β1(transforming growth factor-β1,TGF-β1)、转录生长因子β2(transforming growth factor-β2,TGF-β2)、含半胱氨酸的天冬氨酸蛋白水解酶-3(cysteinyl aspartate specific proteinase,Caspase-3)mRNA表达水平;Western blot印迹检测RARα、TGF-β、Caspase-3蛋白表达水平。结果 :成功培养原代神经元;原代神经元OGD损伤后,倒置显微镜观察发现OGD组死亡的原代神经元较OGD+ATRA组明显增多,细胞更加杂乱;流式细胞计数发现OGD+ATRA组(26.11±2.96)%细胞凋亡率较OGD组(52.14±5.23)%明显降低(P=0.025);OGD+ATRA组RARαmRNA表达水平(1.52±0.43)和蛋白表达水平(0.22±0.02)较OGD组的m RNA表达水平(0.49±013)和蛋白表达水平(0.10±0.02)均明显增高(P=0.006,P=0.005),而OGD+ATRA组TGF-β1、TGF-β2 m RNA表达水平(0.45±0.10,0.41±0.08)分别较OGD组(0.81±0.15,0.98±0.15)明显降低(P=0.034,P=0.018),而且OGD+ATRA组TGF-β、Caspase-3蛋白表达水平(0.22±0.04,0.18±0.03)分别较OGD组(0.39±0.05,0.47±0.06)也明显降低(P=0.016,P=0.002),OGD+ATRA组(1.08±0.49)与OGD组(1.00±0.15)Caspase-3 m RNA表达水平无明显差异(P=0.148)。结论 :ATRA可以激活其受体RARα进而下调TGF-β,抑制凋亡因子Caspase-3的表达,最终抑制OGD损伤引起的原代神经元凋亡。Objective：To investigate whether all-trans retinoic acid（ATRA）can inhibit apoptosis effects on primary neurons caused by oxygen and glucose-deprivation（OGD）. Methods：Primary neurons were isolated and cultured in vitro,and were determined for neuron-specific enolase（NSE） by immunofluorescence technique. Primary neurons were divided into three groups：control group,OGD group and OGD＋ATRA group. Cell morphology before and after OGD injury was observed by inverted microscopy. Apoptosis rate of primary neurons after OGD injury was measured by flow cytometry. The gene transcription levels of retinoic acid receptor alpha（RARα）,transforming growth factor-β1（TGF-β1）,transforming growth factor-β2（TGF-β2）,cysteinyl aspartate specific proteinase（Caspase-3）were examined via real-time PCR,while the protein expressions of RARα,TGF-β,Caspase-3 were determined by Western blot. Results：Successfully cultured primary neurons,neuron-specific enolase was observed in primary neurons obviously. After OGD injury,the cell death of OGD group was more obvious than OGD＋ATRA group. Flow cytometry suggested that the apoptosis rate of OGD＋ATRA group（26.11±2.96）% significantly decreased when compared with that of OGD group（52.14±5.23）%（P=0.025）. The m RNA and protein levels of RARα in OGD＋ATRA group（1.52±0.43,0.22±0.02）were significantly higher than those in OGD group（0.49±013,0.10±0.02）（P=0.006,P=0.005）. The m RNA transcription levels of TGF-β1,TGF-β2 were significantly lower in OGD＋ATRA group（0.45±0.10,0.41±0.08）than in OGD group（0.81±0.15,0.98±0.15）（P=0.034,P=0.018）. The protein expressions of TGF-β,Caspase-3 were significantly lower in OGD＋ATRA group（0.22±0.04,0.18±0.03）than in OGD group（0.39±0.05,0.47±0.06）（P=0.016,P=0.002）. And the m RNA transcription of Caspase-3 in OGD ＋ATRA group（1.08 ±0.49） and OGD group（1.00 ±0.15） were not significantly different（P =0.148）.Conclusion：ATRA inhibits the apoptosis of

关 键 词：全反式视黄酸 缺氧缺糖损伤 原代神经元 转录生长因子β 含半胱氨酸的天冬氨酸蛋白水解酶-3 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R361.3[医药卫生—基础医学]