摘要
目的 观察Toll样受体4(TLR4)基因沉默或过表达后香烟烟雾溶液(CSE)对人支气管上皮样细胞(16HBE)相关基因表达的影响。方法 2020年8—12月,用常规培养的人支气管上皮样细胞16HBE作为空白对照组,实验设干扰小RNA(siRNA)TLR4-1组(hs-TLR4-si-1+16HBE)、siRNA TLR4-2组(hs-TLR4-si-2+16HBE)、siRNA TLR4-3组(hs-TLR4-si-3+16HBE),另设阴性对照组(NC+16HBE),实时荧光定量逆转录聚合酶链反应(qRT-PCR)筛选TLR4基因最佳沉默效果;自制CSE作用于16HBE,实验设常规培养的16HBE作为空白对照组、CSE组(CSE+16HBE)、siRNA TLR4组(CSE+hs-TLR4-si-2+16HBE)、siRNA TLR4-NC组(CSE+NC+16HBE)、Lv-TLR4组(CSE+Lv-TLR4+16HBE)、Lv-TLR4-NC组(CSE+Lv-TLR4-NC+16HBE),qRT-PCR检测TLR4、核因子κB(NF-κB)以及黏蛋白MUC5AC、MUC7、MUC8基因mRNA的表达。结果 与空白对照组相比,siRNA TLR4-2组TLR4 mRNA表达显著降低(1.00±0.22比0.47±0.04,P<0.05),因此选择hs-TLR4-si-2作为TLR4基因沉默的最佳siRNA。与空白对照组相比,CSE组TLR4(1.00±0.09比1.81±0.18)、NF-κB(1.00±0.04比1.65±0.11)、MUC5AC(1.00±0.12比2.09±0.24)、MUC7(1.00±0.20比2.21±0.26)、MUC8(1.00±0.11比1.75±0.06)mRNA表达均升高(均P<0.05);与CSE组相比,CSE+siRNA TLR4组TLR4(1.81±0.18比1.43±0.17)、NF-κB(1.65±0.11比1.12±0.05)、MUC5AC(2.09±0.24比1.38±0.13)、MUC7(2.21±0.26比1.46±0.30)、MUC8(1.75±0.06比1.23±0.03)mRNA表达均降低(均P<0.05),CSE+Lv-TLR4组TLR4(1.81±0.18比2.42±0.06)、NF-κB(1.65±0.11比1.94±0.07)、MUC5AC(2.09±0.24比2.56±0.19)、MUC7(2.21±0.26比2.72±0.33)、MUC8(1.75±0.06比2.10±0.10)mRNA表达均升高(均P<0.05)。结论 TLR4/NF-κB信号通路与CSE所产生的气道炎症有关,且其下游相关基因及黏蛋白表达受TLR4基因调控,TLR4的过度表达会加重CSE导致的气道炎症及慢性阻塞性肺疾病(COPD),抑制TLR4/NF-κB信号通路可在一定程度上减轻CSE所导致的气道炎症及COPD。
Objective To observe the effect of cigarette smoke solution(CSE) on the expression of related genes in human bronchial epithelial-like cells after Toll-like receptor 4(TLR4) gene silencing or overexpression.Methods From August to December 2020,conventionally cultured human bronchial epithelial-like cells with 16 HBE were used as a blank control group,and experiments were set up for the small interfering RNA(siRNA) TLR4-1 group(hs-TLR4-si-1+16 HBE),siRNA TLR4-2 group(hs-TLR4-si-2+16 HBE),siRNA TLR4-3 group(hs TLR4-si-3+16 HBE),and another negative control group(NC+16 HBE).Real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR) was used to determine the best silencing effect of the TLR4 gene.Homemade CSE was applied to 16 HBE cells,and experiments were performed with conventional cultured 16 HBE cells as a blank control group,CSE group(CSE+16 HBE),siRNA TLR4 group(CSE+hs-TLR4-si-2+16 HBE),siRNA TLR4-NC group(CSE+NC+16 HBE),Lv-TLR4 group(CSE+Lv-TLR4-NC+16 HBE),Lv-TLR4 group(CSE+Lv-TLR4-NC+16 HBE),and Lv-TLR4-NC group(CSE+Lv-TLR4-NC+16 HBE).qRT-PCR was performed to detect the expression of TLR4,nuclear factor-κB(NF-κB),and mucin MUC5 AC,MUC7,and MUC8 mRNAs.Results Compared with the blank control group,TLR4 mRNA expression was significantly lower in the siRNA TLR4-2 group(1.00±0.22 vs.0.47±0.04)(P<0.05),so hs-TLR4-si-2 was selected as the best siRNA for TLR4 gene silencing.Compared with the blank control group,TLR4(1.00±0.09 vs.1.81±0.18),NF-κB(1.00±0.04 vs.1.65±0.11),MUC5 AC(1.00±0.12 vs.2.09±0.24),MUC7(1.00±0.20 vs.2.21±0.26),and MUC8(1.00±0.11 vs.1.75±0.06) mRNA expression were elevated(P<0.05).Compared with the CSE group,TLR4(1.81±0.18 vs.1.43±0.17),NF-κB(1.65±0.11 vs.1.12±0.05),MUC5 AC(2.09±0.24 vs.1.38±0.13),MUC7(2.21±0.26 vs.1.46±0.30),and MUC8(1.75±0.06 vs.1.23±0.03) mRNA expression was reduced in the CSE+siRNA TLR4 group(P<0.05),and TLR4(1.81±0.18 vs.2.42±0.06),NF-κB(1.65±0.11 vs.1.94±0.07),MUC5 AC(2.09±0.24 vs.2.56±0.19),MUC7(2.21±0.26 vs
作者
徐晨
王胜
李春颖
XU Chen;WANG Sheng;LI Chunying(Graduate School of Anhui University of Chinese Medicine,Hefei,Anhui 230000,China;Department of Respiratory Medicine,Geriatrics Center,The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei,Anhui 230000,China)
出处
《安徽医药》
CAS
2022年第12期2416-2421,共6页
Anhui Medical and Pharmaceutical Journal
基金
安徽省自然科学基金项目(1908085MH288)。
