大鼠大脑皮质神经细胞氧糖剥夺再复氧(OGD/R)后自噬对凋亡的抑制作用  

Inhibitory effect of autophagy on apoptosis of rat cerebral cortical neurons after oxygen-glucose deprivation and reoxygenation (OGD/R)

作　　者：张磊[1,2] 李阳 胡玉奇[1] 许晖 朱超[1] 王旭 赵冬 贺昕[3] ZHANG Lei;LI Yang;HU Yuqi;XU Hui;ZHU Chao;WANG Xu;ZHAO Dong;HE Xin(Department of Neurosurgery,First Affiliated Hospital,School of Medicine,Shihezi University,Shihezi 832000;Department of Neurosurgery Xinjang Production and Construction Corps Tenth Division Beitun Hospial,Beitun 836000;Department of Maxilofacial Surgery,Urumqi Stomatological Hospital,Xinjiang Province,Urumqi 830000,China)

机构地区：[1]石河子大学第一附属医院神经外科,新疆石河子832000 [2]新疆生产建设兵团第十师北屯医院神经外科,新疆北屯836000 [3]新疆乌鲁木齐市口腔医院颌面外科,新疆乌鲁木齐830000

出　　处：《细胞与分子免疫学杂志》2022年第7期590-597,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81960222);石河子大学国际科技合作促进计划(GJHZ201704);兵团中青年科技创新领军人才(2020CB011)。

摘　　要：目的 观察大脑皮质神经元氧糖剥夺再复氧(OGD/R)后自噬对凋亡的影响。方法 取18只出生在24 h内的新生SD大鼠,采用机械分离胰蛋白酶消化法提取大脑皮质神经元培养7 d后,建立OGD/R模型,随机分为3组,正常对照组、OGD/R组、OGD/R联合巴弗洛霉素A1(BafA1)组。采用CCK-8法和乳酸脱氢酶(LDH)实验检测细胞活力,用异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测细胞凋亡,采用反转录PCR法检测微管相关蛋白1轻链3(LC3)、 P62及胱天蛋白酶3(caspase-3)的mRNA表达,Western blot法检测LC3、 P62及caspase-3的蛋白表达。采用红色荧光蛋白-绿色荧光蛋白-LC3(RFP-GFP-LC3)腺病毒转染神经细胞检测自噬荧光强度,透射电子显微镜观察自噬体的超微结构变化。结果 与对照组相比,OGD/R组神经细胞活力明显降低,LDH释放水平明显升高,凋亡增加;LC3、 P62、 caspase-3的mRNA和蛋白表达增强;RFP荧光强度增加,自噬体增加且自噬溶酶体更加聚集,加用自噬晚期抑制剂BafA1后RFP荧光强度显著降低。结论 OGD/R诱导了神经元细胞的自噬和凋亡,抑制自噬后加重了神经细胞的损伤和凋亡。Objective To observe the effect of autophagy on apoptosis of cerebral cortical neurons after oxygen-glucose deprivation and reoxygenation(OGD/R). Methods Eighteen newborn SD rats born within 24 hours were selected. The cortical neurons were extracted by mechanical trypsin digestion and cultured for 7 days. The OGD/R model was established. The rats were randomly divided into 3 groups: control group, OGD/R group, and OGD/R combined with Bafilomycin A1(BafA1) group. Cell viability was detected by CCK-8 assay and lactate dehydrogenase(LDH) assay, and apoptosis was detected by flow cytometry combined with fluorescein isothiocyanate labeled annexin V/propidium iodide(annexin Ⅴ-FITC/PI) double labeling. The mRNA expressions of microtubule associated protein 1 light chain 3(LC3), P62 and cystatin 3(caspase-3) were detected by reverse transcription PCR, and the protein expressions of LC3, P62 and caspase-3 were detected by Western blot analysis. The autophagy fluorescence intensity of neurons infected with red fluorescent protein-green fluorescent protein-LC3(RFP-GFP-LC3) adenovirus was measured, and the ultrastructural changes of autophagosomes were observed by transmission electron microscopy. Results Compared with the control group, the activity of nerve cells in OGD/R group decreased significantly while the release level of LDH increased significantly and the apoptosisincreased significantly;It was also detected the increased mRNA and protein expressions of LC3, P62 and caspase-3, increased fluorescence intensity of RFP, increased autophagosomes and more gathered autophagy lysosomes. The fluorescence intensity of RFP decreased significantly after adding late autophagy inhibitor BafA1. Conclusion OGD/R induces autophagy and apoptosis of neurons, and aggravates neuronal injury and apoptosis after inhibition of autophagy.

关 键 词：皮质神经元细胞 氧糖剥夺再复氧(OGD/R) 细胞凋亡 自噬 巴弗洛霉素A1(BafA1) 

分 类 号：R965[医药卫生—药理学] R392-33[医药卫生—药学] R364.4[生物学—细胞生物学] Q255