| 徐晨,王胜,李春颖.基于 Toll样受体 4/核因子 κB通路观察香烟烟雾溶液刺激对人支气管上皮样细胞相关基因表达的影响[J].安徽医药,2022,26(12):2416-2421. |
| 基于 Toll样受体 4/核因子 κB通路观察香烟烟雾溶液刺激对人支气管上皮样细胞相关基因表达的影响 |
| Effects of cigarette smoke solution stimulation on gene expression in human bronchial epithelial-like cells based on the Toll-like receptor 4/nuclear factor κB pathway |
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| DOI：10.3969/j.issn.1009-6469.2022.12.018 |
| 中文关键词: 烟雾吸入损伤 Toll样受体 4 核因子 κB 肺疾病，慢性阻塞性 上皮细胞 香烟烟雾溶液 人支气管上皮样细胞 黏蛋白 信使 RNA |
| 英文关键词: Smoke inhalation injury Toll-like receptor 4 Nuclear factor-kappa B Pulmonary disease, chronic obstructive Epithelial cells Cigarette smoke solution 16HBE Mucin Messenger RNA |
| 基金项目:安徽省自然科学基金项目( 1908085MH288) |
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| 中文摘要: |
| 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(siR. NA)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1，6HBE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。 |
| 英文摘要: |
| 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, con. ventionally cultured human bronchial epithelial-like cells with 16HBE 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+16HBE), siRNA TLR4-2 group (hs-TLR4-si-2+16HBE), siRNA TLR4-3 group (hs TLR4-si-3+16HBE), and another negative control group (NC+16HBE). Real-time fluorescence quantitative reversetranscription polymerase chain reaction (qRT.PCR) was used to determine the best silencing effect of the TLR4 gene. Homemade CSEwas applied to 16HBE cells, and experiments were performed with conventional cultured 16HBE cells as a blank control group, CSEgroup (CSE+16HBE), siRNA TLR4 group (CSE+hs-TLR4-si-2+16HBE), siRNA TLR4-NC group (CSE+NC+16HBE), Lv-TLR4 group (CSE+Lv-TLR4-NC+16HBE), Lv-TLR4 group (CSE+Lv-TLR4-NC+16HBE), and Lv-TLR4-NC group (CSE+Lv-TLR4-NC+16HBE). qRT.PCR was performed to detect the expression of TLR4, nuclear factor-κB (NF-κB), and mucin MUC5AC, 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), MUC5AC (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), MUC5AC (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), MUC5AC (2.09±0.24 vs. 2.56±0.19), MUC7 (2.21±0.26 vs. 2.72±0.33), and MUC8 (1.75±0.06 vs. 2.10±0.10) mRNA expression was elevated in the CSE+Lv-TLR4 group (P<0.05).Conclusions TLR4/NF-κB signaling pathway is related to airway inflammation produced by CSE, and its downstream related genes and mucin expression are regu.lated by the TLR4 gene. TLR4 overexpression can aggravate airway inflammation and chronic obstructive pulmonary disease (COPD)caused by CSE, and inhibition of the TLR4/NF-κB signaling pathway can reduce airway inflammation and COPD caused by CSE to some extent. |
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