神经生长因子通过c-fos促进支气管上皮细胞NK-1R表达

doi:10.3877/cma.j.issn.1674-6902.2017.02.007

中华肺部疾病杂志(电子版) ›› 2017, Vol. 10 ›› Issue (02) : 146 -150. doi: 10.3877/cma.j.issn.1674-6902.2017.02.007

所属专题：文献；

论著

神经生长因子通过c-fos促进支气管上皮细胞NK-1R表达

李志芳¹, 秦岭¹, 贺若曦¹, 胡新月¹, 汤渝玲², 胡成平¹^,^†(

), 冯俊涛¹

^1. 410008　长沙，中南大学湘雅医院呼吸科
^2. 410005　长沙，长沙市第一医院呼吸科

收稿日期:2017-01-17 出版日期:2017-04-20
通信作者: 胡成平
基金资助:
国家自然科学基金资助项目(81400022，81270080)

Nerve growth factor stimulates the expressions of NK-1R in bronchial epithelial cells via c-fos

Zhifang Li¹, Ling Qin¹, Ruoxi He¹, Xinyue Hu¹, Yuling Tang², Chengping Hu¹^,^†(), Juntao Feng¹

^1. Department of Respiratory Medicine, Xiangya Hospital, Central South University, Changsha 410008, China
^2. Department of Respiratory Medicine, the First Hospital of Changsha, Changsha 410005, China

Received:2017-01-17 Published:2017-04-20
Corresponding author: Chengping Hu
About author:
Corresponding author: Hu Chengping, Email: huchengp28@126.com

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目的

探讨神经生长因子(NGF)通过c-fos影响支气管上皮细胞NK-1R的表达。

方法

以人支气管上皮细胞(NHBEC)为研究对象，通过阳离子脂质体将c-fos siRNA转染至细胞内，然后用NGF干预。实验设五组：对照组、NGF干预组、NGF＋c-fos siRNA组、NGF＋空白脂质体组、NGF＋非特异性dsRNA组。转染12 h后，用NGF干预60 min，采用Western blot法测定c-fos蛋白表达，采用免疫细胞化学法和RT-PCR从蛋白和mRNA水平观察细胞中NK-1R的表达。

结果

与对照组比，NGF组c-fos蛋白、NK-1R蛋白和NK-1RmRNA表达均明显增加(P<0.01)；与NGF组比，NGF＋c-fos siRNA组中c-fos蛋白、NK-1R蛋白和NK-1RmRNA表达均明显减少(P<0.01)，而NGF组、NGF＋空白脂质体组、NGF＋非特异性dsRNA组之间的c-fos蛋白、NK-1R蛋白和NK-1RmRNA表达无显著差异。

结论

NGF可通过c-fos促进支气管上皮细胞中NK-1R的表达，介导哮喘神经源性炎症，RNAi技术可以减少NGF诱导的人支气管上皮细胞c-fos蛋白及NK-1R的表达。

关键词: 支气管哮喘, 神经生长因子, c-fos, NK-1R

Objective

To investigate whether the effect of nerve growth factor on NK-1R expression of bronchial epithelial cells is via c-fos.

Methods

Human bronchial epithelial cells (NHBEC) were taken as the research object, the c-fos siRNA was transected into cells through the positive ion fat plastid, and then the NGF intervention was carried on. The control group, NGF intervention group, NGF+ c-fos siRNA group, NGF+ liposomes group, NGF+ non-specific dsRNA group were decided. After trans-infection for 12 h and then intervention for 60min, the protein expression of c-fos was detected by western-blot. The protein and mRNA expression of NK-1R was determined by RT-PCR and immune cell chemical definite quality respectively.

Results

Compared with the control group, the expressions of c-fos protein, NK-1R protein and NK-1RmRNA were significantly increased in NGF group (P<0.01); compared with NGF group, the expressions of c-fos protein, NK-1R protein and NK-1RmRNA were significantly reduced in NGF+ c-fos siRNA group (P<0.01), NGF intervention group, NGF+ liposomes group, NGF+ non-specific dsRNA group had no significant difference in the expression of them.

Conclusion

NGF stimulates the expressions of NK-1R in bronchial epithelial cells via c-fos. and then mediate neurogenic inflammation of asthma. RNAi could reduced the of c-fos protein and NK-1R induced by NGF in human bronchial epithelial cells.

Key words: Bronchial asthma, Nerve growth factor, C-fos, NK-1R

图1 Western-blot半定量检测c-fos蛋白表达；注：1和2：NGF组；3和4：NGF＋siRNA组；5和6：对照组

表1 Western-blot半定量检测c-fos蛋白表达

分　组	复孔数	c-fos/GAPDH吸光度比值
NGF组	6	1.1861±0.1427^a
NGF＋siRNA组	6	0.6574±0.1373^b
对照组	6	0.3773±0.136
NGF＋空白脂质体组	3	0.9827±0.1244
NGF＋dsRNA组	3	1.165±0.1789

表1 Western-blot半定量检测c-fos蛋白表达

分　组	复孔数	c-fos/GAPDH吸光度比值
NGF组	6	1.1861±0.1427^a
NGF＋siRNA组	6	0.6574±0.1373^b
对照组	6	0.3773±0.136
NGF＋空白脂质体组	3	0.9827±0.1244
NGF＋dsRNA组	3	1.165±0.1789

图2 RT-PCR半定量检测NK-1R mRNA表达；注：与对照相比^*P<0.01；与NGF组相比^#P<0.01

图3 免疫细胞化学法观察NK-1R表达；注：A:对照组(SABC×200)；B: NGF组(SABC×200)；C：NGF＋空白脂质体组(SABC×200)；D：NGF＋siRNA组(SABC×200)；E：NGF＋dsRNA组(SABC×200)

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