摘要
目的 探讨CXC趋化因子配体10(CXCL10)基因对人神经母细胞瘤细胞SH-SY5Y氧化损伤、钙稳态失衡及核因子E2相关因子2(Nrf2)、BCL2相互作用蛋白3(BNIP3)表达的影响。方法 采用H2O2损伤SH-SY5Y细胞,分为对照组(未经H2O2损伤的SH-SY5Y细胞)、模型组(H2O2干预的SH-SY5Y细胞)、NC-siRNA组(H2O2干预的SH-SY5Y细胞转染NC-siRNA)及CXCL10-siRNA组(H2O2干预的SH-SY5Y细胞转染CXCL10-siRNA),peDNA3.1.D组(H2O2干预的SH-SY5Y细胞转染空载体)及CXCLl0/peDNA3.1组(H2O2干预的SH-SY5Y细胞转染CXCL10/peDNA3.1)。采用实时荧光定量(qRT-PCR)检测各组细胞CXCL10 mRNA、MTT检测各组细胞活性、相关试剂盒检测氧化应激指标、流式细胞仪检测各组细胞Ca2+荧光强度和免疫印迹检测各组细胞Nrf2、BNIP3表达。结果 对照组、模型组、NC-siRNA组、CXCL10-siRNA组、peDNA3.1.D组和CXCLl0/peDNA3.1组中CXCL10 mRNA水平分别为1.02±0.08、1.44±0.14、1.40±0.17、0.73±0.05、1.49±0.18和2.20±0.16,差异有统计学意义(F=77.400,P<0.05);与对照组相比,模型组细胞活性、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、过氧化氢酶(CAT)、Nrf2、BNIP3降低,丙二醛(MDA)、Ca2+荧光强度升高(P<0.05);模型组、NC-siRNA组、peDNA3.1.D组上述指标比较无意义(P>0.05),与模型组相比,CXCL10-siRNA组细胞活性、GSH、SOD、CAT、Nrf2、BNIP3升高(P<0.05),MDA、Ca2+荧光强度升降低(P<0.05),CXCL10/peDNA3.1组细胞活性、GSH、SOD、CAT、Nrf2、BNIP3降低(P<0.05),MDA、Ca2+荧光强度升高(P<0.05)。结论 下调CX-CL10基因可提高H2O2损伤后SH-SY5Y细胞活性,并可抗氧化应激,降低细胞内游离Ca2+水平,这可能与激活Nrf2/BNIP3信号通路相关。
Objective To investigate the effect of CXC chemokine ligand⁃10(CXCL10)gene on oxidative damage,calcium homeostasis imbalance,and expression of nuclear factor⁃E2⁃related factor 2(Nrf2)and BCL2/ade⁃novirus E1B 19kDa interacting protein 3(BNIP3)in human neuroblastoma cells(SH⁃SY5Y).Methods H 2 O 2 was used to induce damage to SH⁃SY5Y cells,which were divided into control group(SH⁃SY5Y cells without H 2 O 2⁃in⁃duced damage),model group(H 2 O 2 intervention in SH⁃SY5Y cells),NC⁃siRNA group(H 2 O 2 intervention in SH⁃SY5Y cells transfected with NC⁃siRNA),CXCL10⁃siRNA group(H 2 O 2 intervention in SH⁃SY5Y cells transfected with CXCL10⁃siRNA),peDNA3.1.D group(H 2 O 2 intervention in SH⁃SY5Y cells transfected with empty vector)and CXCL10/peDNA3.1 group(H 2 O 2 intervention in SH⁃SY5Y cells transfected with CXCL10/peDNA3.1).The quantitative real⁃time PCR(qRT⁃PCR)was used to detect the CXCL10 mRNA in each group of cells.MTT assay was used to detect cell viability.Relevant reagent kits were used to detect oxidative stress markers.The flow cytometry was used to detect the fluorescence intensity of calcium ions in each group of cells.The western blot was used to detect the ex⁃pression of Nrf2 and BNIP3 in each group of cells.Results The CXCL10 mRNA in the control group,model group,NC⁃siRNA group,CXCL10⁃siRNA group,peDNA3.1.D group and CXCLl0/peDNA3.1 group were 1.02±0.08,1.44±0.14,1.40±0.17,0.73±0.05,1.49±0.18 and 2.20±0.16,respectively,and the difference was statistical⁃ly significant(F=77.400,P<0.05).Compared with the control group,cell viability,glutathione(GSH),superoxide dismutase(SOD),catalase(CAT),Nrf2 and BNIP3 decreased(P<0.05),malondialdehyde(MDA)and Ca 2+fluo⁃rescence intensity increased in the model group(P<0.05).There was no significant difference in the above indicators within the model group,NC⁃siRNA group and peDNA3.1.D group(P>0.05).Compared with the model group,the cell viability,GSH,SOD,CAT,Nrf2 and BNIP3 increased,MDA and Ca 2+fluorescence intensity
作者
刘琳
马倩
万光宇
田玲
LIU Lin;MA Qian;WAN Guang-yu;TIAN Ling(Department of Health Medicine,the Eighth Medical Center of the PLA General Hospital,Beijing 100091,China;Department of Neurology,the Eighth Medical Center of the PLA General Hospital,Beijing 100091,China;Department of Geriatrics,the Eighth Medical Center of the PLA General Hospital,Beijing 100091,China)
出处
《解剖学研究》
CAS
2023年第6期511-517,522,共8页
Anatomy Research
基金
北京市海淀区卫生健康发展科研培育计划(HP2021-03-10101)。
