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沉默beclin1基因对缺氧缺糖/复氧复糖HT22细胞凋亡的影响
引用本文:张怡,靳晓飞,周晓红,董贤慧,张颖,于文涛,成媛,高维娟. 沉默beclin1基因对缺氧缺糖/复氧复糖HT22细胞凋亡的影响[J]. 解剖学报, 2020, 51(1): 3-8. DOI: 10.16098/j.issn.0529-1356.2020.01.001
作者姓名:张怡  靳晓飞  周晓红  董贤慧  张颖  于文涛  成媛  高维娟
作者单位:河北中医学院,河北省心脑血管病中医药防治研究重点实验室,石家庄 050091
基金项目:河北省研究生创新项目;国家自然科学基金;河北省中医药局科研项目;河北省教育厅项目;河北省政府资助临床医学优秀人才培养和基础课题研究项目
摘    要:目的 探讨beclin1基因对缺氧缺糖/复氧复糖小鼠海马神经元细胞系HT22细胞凋亡的影响。方法 取对数生长期的HT22细胞,随机分为4组:正常组(normal)、缺氧缺糖/复氧复糖(OGD/R)模型组(model)、beclin1基因沉默组(beclin1-/-)、转染对照组(control)。除normal组外,其余各组细胞均在缺氧缺糖6 h后进行复氧复糖。利用RNAi技术,针对小鼠cDNA序列设计beclin1干扰序列,用脂质体Lipo2000包裹后转染至HT22细胞。于转染48 h后用荧光显微镜观察转染效率,Western blotting检测细胞beclin1 表达情况。各组细胞均于复氧复糖24 h后采用CCK-8法检测细胞活力,乳酸脱氢酶(LDH)法检测细胞损伤情况,免疫荧光染色检测Bax、Bcl-2表达及比值变化,Western blotting检测LC3、P62及Caspase-3表达。SPSS 19.0 统计学软件进行数据分析。结果 与normal组相比,model组细胞活力及P62蛋白表达显著降低(P<0.01),乳酸脱氢酶(LDH)漏出率、LC3Ⅱ/LC3Ⅰ、Caspase-3表达及Bax/Bcl-2均显著升高(P<0.01)。与model组相比,beclin1-/-组细胞活力及LC3Ⅱ/LC3Ⅰ 表达显著降低(P<0.01),LDH漏出率、Bax/Bcl-2及P62、Caspase-3表达显著升高(P<0.01);control组与model组比较差异无显著性。结论 沉默beclin1抑制细胞自噬可使缺氧缺糖/复氧复糖处理的HT22细胞损伤加重,细胞凋亡进一步增加。

关 键 词:Beclin1   基因沉   氧缺糖/复氧复糖   自噬   免疫印迹法  
收稿时间:2019-01-02
修稿时间:2019-02-09

Effect of silencing beclin1 gene on apoptosis of HT22 cells after oxygen and glucose deprivation/reoxygenation
ZHANG Yi,JIN Xiao-fei,ZHOU Xiao-hong,DONG Xian-hui,ZHANG Ying,YU Wen-tao,CHENG Yuan,GAO Wei-juan. Effect of silencing beclin1 gene on apoptosis of HT22 cells after oxygen and glucose deprivation/reoxygenation[J]. Acta Anatomica Sinica, 2020, 51(1): 3-8. DOI: 10.16098/j.issn.0529-1356.2020.01.001
Authors:ZHANG Yi  JIN Xiao-fei  ZHOU Xiao-hong  DONG Xian-hui  ZHANG Ying  YU Wen-tao  CHENG Yuan  GAO Wei-juan
Affiliation:Hebei University of Chinese Medicine, Heibei Key Laboratory of Chinese Medicine Research on Cardio-cerebrovascular Disease, Shijiazhuang 050091, China
Abstract:Objective To investigate the effect of Beclin1 gene on apoptosis of HT22 of mouse hippocampus neuron treated with oxygen and glucose deprivation/reoxygenation( OGD/R). Methods HT22 cells in logarithmic growth phase were randomly divided into 4 groups: normal,model,beclin1^-/- and control. All groups were reoxygenated after 6 hours of oxygen and glucose deprivation except for normal group. The beclin1 interference sequence was designed for mouse cDNA sequence using RNAi technology,and was transfected into HT22 cells by liposome Lipo2000. The transfection efficiency was observed under fluorescence microscope and Western blotting after 48 hours of transfection.Cell viability was detected by CCK-8 method. Cell damage was detected by lactate dehydrogenase( LDH) method,Bax and Bcl-2 were detected by immunofluorescence staining and the expression of LC3,P62 and Caspase-3 were tested by Western blotting after 24 hours of reoxygenation. SPSS 19. 0 statistical software was used for data analysis. Results Compared with the normal group,the cell viability and P62 expression decreased significantly( P < 0. 01),the LDH leakage rate,LC3Ⅱ/LC3Ⅰ,Caspase-3 expression and Bax/Bcl-2 increased significantly in model group( P < 0. 01).Compared with the model group,the cell viability and LC3 Ⅱ/LC3 Ⅰ decreased significantly( P < 0. 01),the LDH leakage rate,Bax/Bcl-2,P62 and Caspase-3 expression increased significantly in beclin1^-/- group( P<0. 01). There was no difference between the control group and the model group. Conclusion Silencing beclin1 inhibites autophagy,which aggravates the damage of OGD/R HT22 cells and further increases apoptosis.
Keywords:Beclin1  Gene silencing  Oxygen and glucose deprivation/reoxygenation  Autophagy  Western blotting
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