标题：过表达ABAD对Aβ1-42刺激的293T细胞线粒体功能和自噬水平的影响

      作者：洪婷婷，张宇，崔理立    广东医科大学 广东省衰老相关心脑疾病重点实验室，广东 湛江 524001

      卷次： 2021年32卷11期

      【摘要】 目的 探讨β淀粉样蛋白 1-42 (Aβ1-42)刺激下，在 293T细胞中过表达淀粉样蛋白结合醇脱氢酶(ABAD)对线粒体功能及自噬相关标记蛋白水平的影响。方法 将正常培养的293T细胞随机分为：(1) Control组、过表达ABAD组、Control给药组(Aβ1-42 5 μmol/L、10 μmol/L、20 μmol/L)、过表达ABAD给药组(Aβ1-42 5 μmol/L、10 μmol/L、20 μmol/L)；(2) Control组、sh-ABAD组；(3) Control+DMSO组、Control+CCCP组、过表达ABAD+DMSO组、过表达ABAD+CCCP组(WT/Aβ 10 μmol/L)。三磷酸腺苷(ATP)试剂盒测定Control组、过表达ABAD组、Control给药组、过表达ABAD给药组细胞的ATP含量；活性氧(ROS)试剂盒测定活性氧生成量。Western blot法检测Control+DMSO组、Control+CCCP组、过表达ABAD+DMSO组、过表达ABAD+CCCP组线粒体自噬相关蛋白表达水平。通过标准曲线换算ATP细胞含量和活性氧生成量，采用 Image J对Western blot条带进行量化。结果 Control组与过表达ABAD组ATP含量和活性氧生成量变化不明显，差异无统计学意义(P>0.05)；与Control给药组相比，在Aβ1-42 (10 μmol/L)刺激后，过表达ABAD给药组的ATP含量减少，差异具有统计学意义(P<0.05)，但两组的活性氧生成量变化不明显，差异无统计学意义(P>0.05)；与Control+CCCP组相比，过表达ABAD+CCCP组在Aβ1-42 10 μmol/L诱导后线粒体自噬相关蛋白 P62降低、LC3B升高，差异均有统计学意义(P<0.05)。结论 过表达ABAD不影响293T细胞的线粒体功能，但在特定浓度Aβ1-42刺激下可影响线粒体ATP产生及促进线粒体自噬发生。
      【关键词】 淀粉样蛋白结合醇脱氢酶；β-淀粉样蛋白；三磷酸腺苷；活性氧；线粒体自噬
      【中图分类号】 R329.2+6 【文献标识码】 A 【文章编号】 1003—6350（2021）11—1365—05

Effects of overexpression of ABAD on mitochondrial function and autophagy of 293T cells stimulated by Aβ 1-42.HONG Ting-ting, ZHANG Yu, CUI Li-li.
Guangdong Provincial Key Laboratory of Age Related Heart And Brain Diseases,Guangdong Medical University, Zhanjiang 524001, Guangdong, CHINA
【Abstract】 Objective To investigate the effect of overexpression of ABAD on mitochondrial function and thelevel of mitophagy related protein in 293T cells stimulated by Aβ1-42. Methods 293T cells were randomly divided into:(1) control group, overexpression of ABAD group, control group (Aβ1-42 5 μmol/L, 10 μmol/L, 20 μmol/L), overexpres-sion of ABAD group (Aβ1-42 5 μmol/L, 10 μmol/L, 20 μmol/L); (2) control group, sh-ABAD group; (3) control + DMSOgroup, control + CCCP group, overexpression of ABAD + DMSO group, overexpression of ABAD + CCCP group (WT/Aβ 10 μmol/L).Adenosine triphosphate (ATP) content of control group, overexpression of ABAD group, control group(Aβ1-42 5 μmol/L, 10 μmol/L, 20 μmol/L) and overexpression of ABAD group (Aβ1-42 5 μmol/L, 10 μmol/L, 20 μmol/L) wasmeasured by ATP kit, and the production of reactive oxygen species (ROS) was measured by ROS kit. Western blot wasused to detect the expression of mitophagy related proteins in control + DMSO group, control+CCCP group, overexpres-sion of ABAD + DMSO group and overexpression of ABAD+CCCP group. The content of ATP and reactive OxygenSpecies (Ros) were calculated by standard curve, and the Western blot was quantified by Image J. Results Comparedwith the control group, ATP content and reactive oxygen species production in the control group and overexpressionABAD group were not significantly changed (P>0.05); Compared with the control group, the ATP content of 293T cellsoverexpression ABAD was decreased after Aβ1-42 (10 μmol/L) stimulation, and the difference was statistically significant(P<0.05), but there was no significant difference in the production of reactive oxygen species between the two groups(P>0.05); Compared with the control + CCCP group, the mitophagy related protein p62 decreased and LC3 increased inthe overexpression ABAD+CCCP group after Aβ1-42 10 μmol/L induction, and the difference was statistically significant(P<0.05). Conclusion Overexpression of ABAD does not affect the mitochondrial function of 293T cells, but can af-fect the production of mitochondrial ATP and promote the development of mitophagy under the stimulation of Aβ1-42.
      【Key words】 Amyloid binding alcohol dehydrogenase (ABAD); β-amyloid; Adenosine triphosphate (ATP); Re-active oxygen species (ROS); Mitophagy