中文摘要：

目的：研究神经生长因子（NGF）介导哮喘神经源性炎症的信号转导通路。方法：选用参与哮喘神经源性炎症的重要效应细胞-支气管气道上皮细胞株（NHBEC）作为研究对象。NHBEC于含体积分数为10%胎牛血清的达尔伯克（氏）改良伊格尔（氏）培养基（DMEM）中培养。实验分8组：①A组：正常对照组;②B组：NGF组;③C组：NGF＋丝裂素活化蛋白激酶抑制剂（PD98059）组;④D组：PD98059组;⑤E组：NGF＋佛波酯（PMA）组;⑥F组：PMA组;⑦G组：NGF＋酪氨酸磷酸化抑制剂（AG490）组;⑧H组：AG490组。分别进行细胞干预。干预后免疫印迹法半定量测定细胞原癌基因（c-fos）、磷酸化胞外信号调节激酶（p-ERK）蛋白含量。逆转录聚合酶链反应（RT-PCR）方法半定量检测各组细胞神经激酶1受体（NK-1R）mRNA含量。并应用免疫细胞化学定性观察各组细胞表达NK-1R的情况。结果：在NGF未处理的PD98059组及AG490组c-fos蛋白水平、磷酸化的ERK1/2蛋白水平几乎检测不到;而在NGF组,经NGF作用30 min后,可见c-fos蛋白水平及磷酸化的ERK1/2蛋白水平显著增高,c-fos/3-磷酸甘油醛脱氢酶（GAPDH）及p-ERK/GAPDH光密度比值分别为（0.835 4±0.101 2）及（1.318 7±0.2078）,与正常对照组相比P〈0.01。PD98059可明显抑制NGF诱导NHBEC表达c-fos蛋白及磷酸化的ERK1/2蛋白,c-fos/GAPDH及p-ERK/GAPDH光密度比值分别为（0.301 2±0.103 4）及（0.314 6±0.095 7）,与NGF组相比P〈0.01。PMA可刺激NGF诱导NHBEC表达c-fos及磷酸化的ERK1/2蛋白。而转录信号转导子与激活子3（Signaltransducers and activators of transcription 3,STAT3）传导途径特异性抑制剂AG490则无此作用。免疫细胞化学研究结果显示：正常对照组及PD98059组NK-1R表达呈阴性反应;NGF组及NGF＋PMA组呈强阳性反应;PMA组呈阳性反应;而NGF＋PD98059组呈弱阳性反应。RT-PCR结果显示：与正常对照组相比,NK-1RmRNA在NGF未处理

英文摘要：

Aim：To investigate the regulatory effect of NGF on Ras-MAPK signal transduction pathway in neurogenic inflammation of asthma. Methods： The NHBEC was cultured in the DMEM medium containing 10% calf blood serum.NHBEC were divided into 8 groups： ① A group： the control group;② B group： NGF group;③ C group： NGF＋PD98059 group;④ D group： PD98059 group;⑤ E group： NGF＋PMA group;⑥ F group： PMA group;⑦ G group： NGF＋AG490 group;⑧ H group： AG490 group.The cells intervention by NGF,PD98059,PMA and AG490 was carried on separately.After intervened,Weston-blot was taken to detect the protein content in cells c-fos,p-ERK and while the RT-PCR method to each group of cell NK-1RmRNA.And immune cells chemical qualitative applied to observe each group of cell express NK-1R.Results： Compared with the control group,the level of NHBEC c-fos protein and phosphorylized ERK1/2 protein affected at 30 min by NGF rose to the peak value remarkably.But,in the NHBEC of the PD98059 and the AG490 groups,the level didn＇t be detected nearly;the level rose to the peak value remarkably in the NGF group affected at 30 min by NGF,and c-fos/GAPDH and pERK/GAPDH light density ratio was（0.835 4±0.101 2）and（1.318 7±0.207 8）respectively,compared with the control,P0.01.PD98059 might suppress NGF to induce the NHBEC to express the level completely,and c-fos/GAPDH and pERK/GAPDH light density ratio were（0.301 2±0.103 4）and（0.314 6±0.095 7）respectively,compared with the NGF group,P0.01.PMA might stimulate NGF to induce the NHBEC to express c-fos and the phosphorylized ERK1/2 protein.But STAT3 conduct way specific inhibitor AG490 could not suppress NGF to do that.The immune cell chemistry findings showed that NK-1R expression assumed negative reaction in the control and the PD98059 group;strong positive in the NGF and the NGF＋PMA groups;positive in the PMA group;and weak positive in the NGF＋PD98059 group.The RT-PCR result showed that,the NK-1R mRNA level expressed few in the PD98059 and the AG4