【摘要】 目的 探究Toll样受体7(TLR-7)在阿尔茨海默病人神经母细胞瘤细胞（SH-SY5Y）模型中对β淀粉样蛋白表达及神经炎症反应的调控作用及机制。方法 构建稳定表达β淀粉样前体蛋白瑞典型突变（APPswe）的阿尔茨海默病模型细胞SH-SY5Y/APPswe，将细胞分为SH-SY5Y组、SH-SY5Y/APPswe组、TLR-7敲低SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-TLR-7)组、敲低对照SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-NC)组、TLR-7敲低+miR-15b抑制SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-TLR-7+miR-15b inhibitor)组和TLR-7敲低+微小RNA-15b(miR-15b)抑制对照SH-SY5Y/APPswe(SH-SY5Y/APPswe+sh-TLR-7+inhibitor NC)组。蛋白质印迹法检测β淀粉样前体蛋白（APP）的表达；实时荧光定量PCR实验检测TLR-7、miR-15b、β淀粉样前体蛋白裂解酶1(BACE1)、白介素-1β(IL-1β)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)m RNA的表达；酶联免疫吸附实验检测β淀粉样蛋白42(Aβ42)以及IL-1β、IL-6和TNF-α的分泌；荧光素酶报告基因实验检测核因子κB(NF-κB)活性。结果 与SH-SY5Y组比较，SH-SY5Y/APPswe组细胞APP表达水平上调，Aβ42分泌水平增加[（11.24±0.76）ng/m L比（2.09±0.17）ng/m L,P<0.01）]；与SH-SY5Y/APPswe+sh-NC组比较，SH-SY5Y/APPswe+sh-TLR-7组miR-15b表达水平上调[（2.98±0.21）比（1.10±0.05）,P<0.01],BACE1 m RNA表达水平下降[（0.72±0.06）比（1.02±0.03）,P<0.01],Aβ42分泌水平下降[（6.09±0.42）ng/m L比（10.52±0.67）ng/m L,P<0.01],IL-1β、IL-6和TNF-αm RNA表达水平下降[IL-1β:(3.34±0.25)比（4.62±0.41）,P<0.01;IL-6:(3.75±0.34)比（5.43±0.31）,P<0.01;TNF-α:(1.25±0.11)比（2.07±0.18）,P<0.01],IL-1β、IL-6和TNF-α分泌水平下降[IL-1β:(61.40±7.76)pg/m L比（101.76±7.95）pg/m L,P<0.01;IL-6:(547.29±39.11)pg/m L比（893.35±101.43）pg/m L,P<0.01;TNF-α:(256.17±24.32)pg/m L比（375.28±33.99）pg/m L,P<0.01],NF-κB活性下降[（3.25±0.31）比（5.01±0.52）,P<0.01]。与SH-SY5Y/APPswe+sh-TLR-7+inhibitor NC组比较，SH-SY5Y/APPswe+sh-TLR-7+miR-15b inhibitor组miR-15b表达水平下降[（0.67±0.13）比（2.96±0.35）,P<0.01],BACE1 m RNA表达水平升高[（1.39±0.42）比（0.71±0.05）,P<0.05],Aβ42分泌水平升高[（19.54±3.01）ng/m L比（7.47±1.58）ng/m L,P<0.01],IL-1β、IL-6和TNF-α m RNA表达水平升高[IL-1β:(6.07±0.43)比（3.55±0.33）,P<0.01;IL-6:(5.98±0.28)比（4.02±0.22）,P<0.01;TNF-α:(1.88±0.21)比（1.36±0.20）,P<0.01],IL-1β、IL-6、TNF-α分泌水平升高[IL-1β:(155.32±13.67)pg/m L比（68.62±5.41）pg/m L,P<0.05;IL-6:(1255.49±119.79)pg/m L比（439.73±60.01）pg/m L,P<0.05;TNF-α:(423.66±34.32)pg/m L比（227.82±26.53）pg/m L,P<0.05],NF-κB活性升高[（5.91±0.66）比（3.58±0.27）,P<0.01]。结论 TLR-7可能通过miR-15b调控NF-κB信号通路，并影响阿尔茨海默病模型细胞β淀粉样蛋白的分泌及神经炎症反应。更多还原

【Abstract】 Objective To investigate the regulation and mechanism of TLR-7 on amyloid β protein expressionand neuroinflammatory response in SH-SY5Y cell model of Alzheimer’s disease.Methods An Alzheimer’s diseasecell model SH-SY5Y/APPswe with stable expression of APPswe was constructed. The cells were divided into SH-SY5Y, SH-SY5Y/APPswe, SH-SY5Y/APPswe+sh-TLR-7, SH-SY5Y/APPswe+sh-NC, SH-SY5Y/APPswe+sh-TLR-7+miR-15b inhibitor and SH-SY5Y/APPswe +sh-TLR-7 +inhibitor NC groups. The expression of APP protein was de-tected using western blot, the m RNA expression levels of TLR-7, miR-15b, BACE1, IL-1β, IL-6, and TNF-αwere detected using real-time fluorescent quantitative PCR, and the levels of secreted amyloid β protein 42(Aβ42), IL-1β, IL-6, and TNF-α were detected using enzyme-linked immunosorbent assay. NF-κB activity wasdetected using luciferase reporter gene assay.Results Compared with the SH-SY5Y group, the SH-SY5Y/APPswegroup showed upregulated cellular APP expression and increased secretion levels of amyloid β protein [(11.24 ±0.76)ng/m Lv s.(2.09 ±0.17)ng/m L,P<0.01)]. Compared with the SH-SY5Y/APPswe +sh-NC group, the SH-SY5Y/APPswe+sh-TLR-7 group exhibited upregulated miR-15b expression[(2.98±0.21)v s.(1.10±0.05),P<0.01], decreasedBACE1 m RNA expression [(0.72 ±0.06)v s.(1.02 ±0.03),P<0.01], reduced secreted Aβ42 level [(6.09 ±0.42)ng/m Lv s.(10.52±0.67)ng/m L,P<0.01] and decreased m RNA expression of IL-1β [IL-1β:(3.34 ±0.25)v s.(4.62±0.41),P<0.01], IL-6[(3.75±0.34)v s.(5.43±0.31),P<0.01], and TNF-α[(1.25±0.11)v s.(2.07±0.18),P<0.01]. Additional-ly, the SH-SY5Y/APPswe+sh-TLR-7 group also exhibited decreased secreted levels of IL-1β [(61.40 ±7.76)pg/m Lv s.(101.76 ±7.95)pg/m L,P<0.01], IL-6 [(547.29 ±39.11)pg/m Lv s.(893.35 ±101.43)pg/m L,P<0.01], and TNF-α[(256.17 ±24.32)pg/m Lv s.(375.28 ±33.99)pg/m L,P<0.01], as well as decreased NF-κB activity [(3.25 ±0.31)v s.(5.01±0.52),P<0.01]. Compared with SH-SY5Y/APPswe+sh-TLR-7+inhibitor NC group, the SH-SY5Y/APPswe+SH-TLR-7+miR-15b inhibitor group showed decreased miR-15b expression[(0.67 ±0.13)v s.(2.96±0.35),P<0.01], in-creased BACE1 m RNA expression level[(1.39±0.42)v s.(0.71±0.05),P<0.05], increased secreted Aβ42 level [(19.54 ±3.01)ng/m Lv s.(7.47±1.58)ng/m L,P<0.01], and increased m RNA expression of IL-1β[(6.07±0.43)v s.(3.55±0.33),P<0.01], IL-6[(5.98±0.28)v s.(4.02±0.22),P<0.01], and TNF-α[(1.88±0.21)v s.(1.36±0.20),P<0.01]. Additional-ly, the SH-SY5Y/APPswe +SH-TLR-7 +miR-15b inhibitor group also showed increased secreted levels of IL-1β[(155.32±13.67)pg/m Lv s.(68.62±5.41)pg/m L,P<0.05], IL-6 [(1255.49 ±119.79)pg/m Lv s.(439.73 ±60.01)pg/m L,P<0.05], and TNF-α[(423.66±34.32)pg/m Lv s.(227.82±26.53)pg/m L,P<0.05], as well as increased NF-κB activi-ty[(5.91±0.66)v s.(3.58±0.27),P<0.01].Conclusion TLR-7 may regulate the NF-κB signaling pathway throughmiR-15b and influence the secretion of amyloid beta protein and neuroinflammatory response in Alzheimer’s diseasemodel cells.更多还原