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锌指 Krüppel 样转录因子2对慢性阻塞性肺疾病支气管上皮细胞中γ谷氨酰半胱氨酸合成酶的作用

Effect of Krüppel like zinc finger transcription factor 2 onγ-glutamylcysteine synthetase in bronchial epithelial cells of chronic obstructive pulmonary disease

摘要:

目的:探讨锌指Krüppel样转录因子2( KLF2)对慢性阻塞性肺疾病(简称慢阻肺)支气管上皮细胞中γ-谷氨酰半胱氨酸合成酶(γ-GCS )的调控作用。方法(1)临床试验:收集湖南省肿瘤医院胸外科2008年12月—2009年12月肺癌伴或不伴慢阻肺肺叶切除患者临床肺组织标本,分为慢阻肺组和对照组,应用原位杂交及免疫组化检测两组患者肺组织中KLF2、γ-GCS mRNA及蛋白的表达情况;并对KLF2 mRNA及蛋白与γ-GCS mRNA及蛋白, KLF2及γ-GCS 蛋白与吸烟指数、第1秒用力呼气容积( FEV1)占预计值百分比( FEV1%)、FEV1与用力肺活量( FVC)的比值( FEV1/FVC)进行相关分析。(2)动物实验:提取大鼠原代支气管上皮细胞,加入10%烟草烟雾提取物(TSE)培养6 h后,用酶联免疫吸附(ELISA)法检测细胞谷胱甘肽(GSH)含量;将KLF2的特异性抑制剂用脂质体转染至细胞中抑制KLF2蛋白表达;再分为KB组(不予任何处理的空白对照组)、KB+TSE组(加入TSE处理)、NC组(转染miRNA的对照组)、NC+TSE组(miRNA转染和TSE处理)、92a组(转染KLF2抑制剂)、92a+TSE组(KLF2抑制剂转染和TSE处理),用逆转录-PCR、Western印迹法观察各组细胞中KLF2、γ-GCS的mRNA及蛋白质表达变化。结果(1)临床试验:慢阻肺组肺组织中KLF2mRNA、蛋白和γ-GCS mRNA、蛋白呈强阳性表达,均显著高于对照组(0.32±0.04比0.19±0.03、0.35±0.05比0.22±0.03和0.28±0.03比0.16±0.03、0.31±0.05比0.21±0.03,均P<0.01),且两者表达部位一致;KLF2蛋白与γ-GCS mRNA及蛋白表达均呈正相关(r=0.705、0.722,均P<0.01),KLF2及γ-GCS蛋白与吸烟指数、FEV1%、FEV1/FVC 均呈正相关( r=0.552、0.728、0.670及r=0.631、0.727、0.657,均P<0.01)。(2)动物实验:大鼠支气管上皮细胞中GSH含量KB+TSE组显著高于KB组[(28.05±2.04)比(7.27±0.33) nmol/mg,P<0.01];KLF2 mRNA、蛋白和γ-GCS mRNA、蛋白表达KB+TSE组(1.715±0.026、1.842±0.028和2.117±0.067、1.879±0.065)均显著高于KB组(1.130±0.017、1.177±0.033和1.378±0.053、1.177±0.042),92a组(0.472±0.028、0.634±0.025和0.582±0.025、0.554±0.021)均显著低于KB 组、NC 组(1.047±0.056、1.092±0.045和1.303±0.037、1.252±0.037),92a+TSE组(0.262±0.017、0.288±0.017和0.337±0.022、0.321±0.022)均显著低于 KB +TSE 组、92a 组、NC +TSE 组(1.576±0.036、1.646±0.066和1.948±0.093、1.843±0.078)(均P<0.05)。结论 KLF2通过调控慢阻肺支气管上皮细胞中γ-GCS的表达而发挥抗氧化作用。

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abstracts:

Objective To research the regulation effects of Kr üppel like zinc finger transcription factor 2 (KLF2) onγ-glutamylcysteine synthetase (γ-GCS) in airway epithelial cells of chronic obstructive pulmonary disease ( COPD ).Methods ( 1 ) Human specimen experiment: lung tissue of pulmonary lobectomy patients with lung cancer with or without COPD was collected from Department of Thoracic Surgery of Hunan Cancer Hospital from December 2008 to December 2009.The patients were divided into COPD group and control group without COPD.The levels of KLF2,γ-GCS mRNA and protein expression in lung tissues were measured by immunohistochemistry and in situ hybridization ( ISH ).Then, the correlation between KLF2 and γ-GCS mRNA and protein expression levels were analyzed , as well as the correlation between KLF2 orγ-GCS protein and smoking index , percentage of forced expiratory volume in one second to predicted value ( FEV1%) , percentage of forced expiratory volume in one second to forced vital capacity ( FVC/FEV1 ).( 2 ) Animal experiment: the primary bronchial epithelial cells of rats were extracted by enzyme digestion.After 6 hours of incubation with 10%tobacco smoke extract (TSE), cellular glutathione ( GSH) was measured by enzyme linked immunosorbent assay ( ELISA) method.The cells were transfected by specific inhibitor of KLF2 through the liposom, which inhibited the protein expression of KLF2.Then, the cells were divided into KB group ( blank control group without any treatment ) , KB+TSE group ( treated with TSE), NC group (control group transfected with miRNA ), NC+TSE group (treated with miRNA and TSE), 92a group (transfected with KLF2 inhibitor), 92a +TSE group (treated with KLF2 inhibitor transfection and TSE ) based in the treatment.After that, the changes of KLF2 and γ-GCS mRNA and protein expression in the cells of each group were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot method.Results (1) Human specimen experiment:The expressions of KLF2 mRNA, protein andγ-GCS mRNA, protein in the lung tissue of COPD patients were strong positive and higher than those in control group (0.32 ±0.04 vs 0.19 ±0.03, 0.35 ±0.05 vs 0.22 ±0.03;0.28 ± 0.03 vs 0.16 ±0.03, 0.31 ±0.05 vs 0.21 ±0.03;all P<0.01).Linear correlation analysis showed that KLF2 mRNA and protein were positively correlated with γ-GCS mRNA and protein (r=0.705, 0.722;both P<0.01).The KLF2 and γ-GCS protein were positively correlated with smoking index , FEV1% and FEV1/FVC (r=0.552, 0.728, 0.670, and r=0.631, 0.727, 0.657; all P<0.01).(2) Animal experiment:The level of GSH in KB+TSE group was significantly higher than that in KB group [(28.05 ± 2.04) vs (7.27 ±0.33) nmol/mg, P<0.01].The KLF2 mRNA, protein and γ-GCS mRNA, protein in KB+TSE group (1.715 ±0.026, 1.842 ±0.028 and 2.117 ±0.067, 1.879 ±0.065) were higher than those in KB group (1.130 ±0.017, 1.177 ±0.033 and 1.378 ±0.053, 1.177 ±0.042; all P<0.05), and those in 92a group ( 0.472 ±0.028, 0.634 ±0.025 and 0.582 ±0.025, 0.554 ±0.021 ) were significantly lower than those in KB group , NC group (1.047 ±0.056, 1.092 ±0.045 and 1.303 ±0.037, 1.252 ±0.037), and those in TSE +92a group (0.262 ±0.017, 0.288 ±0.017 and 0.337 ±0.022, 0.321 ±0.022) were significantly lower than those in KB +TSE group, 92a group and NC +TSE group (1.576 ±0.036, 1.646 ±0.066 and 1.948 ±0.093, 1.843 ±0.078 ) ( all P<0.05 ).Conclusion KLF2 exerts antioxidative effect by regulating the expression of γ-GCS in the bronchial epithelial cells of chronic obstructive pulmonary disease .

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作者: 李洁 [1] 戴爱国 [2] 黄畅宇 [1] 胡瑞成 [1]
作者单位: 湖南省人民医院 湖南师范大学第一附属医院呼吸内科,长沙,410016 [1] 长沙医学院 湖南省人民医院呼吸疾病研究所 [2]
期刊: 《中华医学杂志》2017年97卷2期 112-118页 MEDLINEISTICPKUCSCD
栏目名称: 临床研究
DOI: 10.3760/cma.j.issn.0376-2491.2017.02.007
发布时间: 2017-03-09
基金项目:
湖南省科技计划重点资助项目(2008FJ4206) 湖南省医药卫生科研课题( B2009068)Fund program Key Program of Science and Technology Planning Project of Hunan Province (2008FJ4206) Medical and Health Research Project of Hunan Province
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