【摘要】 目的探讨香烟烟雾浓缩物(CSC)对人类支气管上皮细胞系(16-HBE)表达4-羟基壬烯醛(4-HNE)的影响以及银杏内酯 B 的干预作用;并观察4-HNE 对中性粒细胞的趋化作用。方法采用免疫细胞化学和 Western blot 方法。(1)不同浓度(1、10μg/ml CSC)CSC 刺激16-HBE 细胞4h后4-HNE 加成蛋白的表达水平,设正常对照组(无血清培养基代替 CSC);(2)10μg/ml CSC 刺激不同时间(1、4、8、12、24、30h)后,16-HBE 中4-HNE 加成蛋白的水平,设正常对照组(无血清培养基代替 CSC);(3)用100μmol/L 银杏内酯 B 孵育16-HBE 后,CSC 刺激1、4、8、12h,检测4-HNE 加成蛋白表达的变化,设正常对照组(无银杏内酯 B 孵育);(4)以趋化实验检测0.1、1、10μmol/L 4-HNE 对兔中性粒细胞的趋化作用。结果(1)1、10μg/ml CSC 刺激4h 后,16-HBE 细胞表达4-HNE 加成蛋白水平(免疫化学为2.12±0.38、2.69±0.42,Western blot 为100.2±6.3、72.3±6.1)均较正常对照组显著增加(免疫化学为1.25±0.37,Western blot 为122.4±4.2,P 均<0.01)。(2)10μg/ml CSC 刺激1、4、8、12、24、30h后,16-HBE 细胞表达4-HNE 加成蛋白(免疫化学为2.67±0.46、2.69±0.42、2.71±0.48、2.72±0.56、2.93±0.11、2.92±0.20,Western blot 为73.2±8.3,72.3±6.4,72.6±9.2,71.5±8.1,54.4±3.6,56.7±4.4)较正常对照组(免疫化学为1.25±0.35,Western blot 为122.4±4.1)显著增加(P 均<0.01),刺激24h 和30h,细胞内4-HNE 加成蛋白较其他时间点增加。(3)50、100μmol/L 银杏内酯 B 孵育后再以 CSC 刺激时,16-HBE 细胞内4-HNE 加成蛋白表达水平(Westernblot 为84.6±4.4、101.2±4.4)明显低于未以内酯 B 孵育组,但高于正常对照组(Western blot 为72.5±6.4,P 均<0.01)。(4)0.1、1、10μmol/L 4-HNE 对兔中性粒细胞趋化数目(92±12、104±16、131±12)较正常对照组(72±12)均显著增加,10 μmol/L 4-HNE 趋化数目显著高于0.1和1μmol/L4-HNE,差异均有统计学意义(P 均<0.01)。结论香烟所引起肺中性粒细胞趋化的结果可能与其促使4-HNE 产生增加有关,银杏内酯 B 可以抑制 CSC 对4-HNE 的诱生。更多还原

【Abstract】 Objective To explore the expression of 4-hydroxy-2-nonenal (4-HNE) in human bronchial epithelial cells(16-HBE) caused by cigarette smoke condensate(CSC),and to study the effects of ginkgolid B on 4-HNE expresssion and the role of 4-HNE on neutrophils chemotaxis.Methods Iinmunohistochemical and Western blot method were used to estimate the expression of 4-HNE-modified protein in 16-HBE of different groups(control group,1 μg/ml or 10 μg/ml CSC stimulation for 4 h,and 10 μg/ml CSC stimulation for 1,4,8,12,24 or 30 h).16-HBE were incubated with 100 μmol/L ginkgolid B for 24 h before CSC(10 μg/ml) stimulation,and the expression of 4-HNE-modified protein were recorded at 1, 4,8 and 12 h after CSC stimulation.The chemotaxis of neutrophils was measured in different concentrations of 4-HNE(0.1,1 and 10 μmol/L).Results (1)The expression of 4-HNE-modified protein was elevated significantly in CSC stimulating groups (immunihistochemistry:2.12±0.38,2.69±0.42,Western blot: 100.2±6.3,72.3±6.1) compared with the group without CSC stimulation(immunihistochemistry:1.25± 0.37,Western blot:122.4±4.2,all P<0.01).(2)The expression of 4-HNE-modified protein in groups treated with 10 μg/ml CSC in different times (1,4,8,12,24,30 h,immunochemistry:2.67±0.46,2.69± 0.42,2.71±0.48,2.72±0.56,2.93±0.11,2.92±0.20,Western blot:73.2±8.3,72.3±6.4,72.6± 9.2,71.5±8.1,54.4±3.6,56.7±4.4) were higher than the control group(immunochemistry:1.25± 0.35,Western blot:122.4±4.1,all P<0.01).The expression of 4-HNE-modified protein in the 10 μg/ml CSC for 24 and 30 h was significantly higher than other groups.(3)There was a lower expression level of 4-HNE-modified protein in the groups with ginkgolid B(50,100 μmol/L) pre-incubated for 24 h before 10 μg/ml CSC stimulation for 4 h (Western blot:84.6±4.4,101.2±4.4) than those without pre-incubated with ginkgolid B (Western blot:72.5±6.4 );all elevated significantly compared to the group without stimulating with CSC(all P<0.01 ).(4)4-HNE(0.1,1,10 μmol/L) showed higher neutrophil chemotaxic aetivity(92±12,104±16,131±12) than the control group (72±12,all P<0.01).The numbers of chemotaxic neutrophils in 10 μmol/L 4-HNE group were higher than those in the 0.1 μmol/L and 1 μmol/L 4-HNE groups.Conclusion 4-HNE might be involved in neutrophil chemotaxis caused by cigarette smoking in the lung,and ginkgolid B could inhibit the expression of 4-HNE caused by CSC.更多还原