NF-κB对哮喘气道重塑平滑肌细胞表型转化的作用及PDTC干预研究

发布时间：2018-04-30 06:28

本文选题：哮喘 + 气道平滑肌细胞　；参考：《暨南大学》2016年硕士论文

【摘要】：目的:气道平滑肌表型转换是哮喘气道重塑的重要特征,而前期体外研究发现NF-k B信号通路对其表型转化起到关键性作用。在此基础上,本研究通过在体检测哮喘气道平滑肌细胞表型标志物的改变特征及相关功能改变,探讨NF-kB信号通路对其影响,同时明确干预NF-k B信号通路对哮喘气道重塑、气道高反应性及慢性炎症的作用,为寻找新的哮喘治疗方法提供理论及实验依据。方法:采用腹腔注射和重复雾化吸入卵清白蛋白的方法建立急性及慢性哮喘小鼠模型,NF-kB特异性抑制剂PDTC腹腔注射对哮喘小鼠进行干预;采用BUXCO无创肺功能仪测定各组小鼠气道高反应性;HE染色观察组织大体改变及炎症浸润度;Masson trichrome染色检测胶原沉积情况;免疫组织化学检测气道平滑肌层厚度;通过形态学定量分析对比各组胶原面积及气道平滑肌层厚度;α-SMA和PCNA免疫荧光检测增殖细胞数量;RT-PCR检测肺支气管表型标志物以及COL1A1mRNA表达量;Western-Blot检测磷酸化P65和α-SMA蛋白表达量;RayBiotech蛋白芯片检测血清中炎症因子表达水平。结果:(1)急性及慢性哮喘小鼠气道高反应性指标Penh值较对照组均显著增高,而干预组较哮喘组显著降低;(2)慢性哮喘小鼠平滑肌层面积较对照组显著增高,干预组则有显著缓解;急性哮喘小鼠平滑肌层增厚不显著;(3)气道胶原面积定量急性哮喘各组定量比较亦无显著差别,慢性哮喘模型中,形态定量学分析发现哮喘组胶原面积显著高于其对照组,而PDTC干预后较哮喘组胶原面积显著减少;(4)免疫荧光染色未检出气道平滑肌与PCNA共表达,可见急性哮喘肺内PCNA+的细胞数量较正常组明显增多,主要位于气道以及血管周围,慢性哮喘小鼠未检出增殖细胞;(5)通过qPCR发现慢性哮喘模型中,哮喘组α-SMA、calponin和SM-MHC较对照组显著下降。急性哮喘小鼠模型中,只有calponin在哮喘组中显著下调,PDTC干预组在慢性和急性哮喘模型中均有缓解表型转化的作用,然而calponin在慢性哮喘模型中的下降不能被PDTC干预逆转。Western blot显示PDTC干预后哮喘小鼠phospho-P65表达量显著降低,同时α-SMA蛋白升高。(6)急性哮喘模型中,哮喘组血清G-CSF、IL-6、和IL-17相比正常对照组显著增高,PDTC干预显著降低了G-CSF和IL-6水平,但不影响IL-17。慢性哮喘模型中,哮喘组中只有IL-17和TNF-α显著增高,TNF-α在干预组中显著降低。结论:(1)哮喘模型小鼠气道平滑肌表型转化过程具有时序性,在急性哮喘期,收缩型物质即开始减少,同时伴随细胞功能的早期变化;慢性哮喘则减少更为显著。(2)干预NF-kB通路一定程度上逆转了哮喘气道平滑肌细胞表型转化;(3)干预NF-kB信号通路能一定程度缓解哮喘模型小鼠气道重塑、气道高反应性及慢性炎症等哮喘病理生理特征,可望成为哮喘治疗的新靶点。
[Abstract]:Objective: phenotypic transformation of airway smooth muscle is an important feature of airway remodeling in asthma. In vitro studies have found that NF-k B signaling pathway plays a key role in phenotypic transformation. On this basis, the changes of phenotypic markers and related functional changes of airway smooth muscle cells of asthma were detected in vivo to investigate the effects of NF-kB signaling pathway on airway remodeling of asthma. At the same time, the effects of NF-k B signaling pathway on airway remodeling of asthma were investigated. The effects of airway hyperresponsiveness and chronic inflammation provide theoretical and experimental basis for finding new treatment methods for asthma. Methods: acute and chronic asthma models were established by intraperitoneal injection and repeated atomization inhalation of ovalbumin (ovalbumin). PDTC, a specific inhibitor of NF-kB, was injected intraperitoneally to treat asthmatic mice. BUXCO noninvasive pulmonary function instrument was used to detect airway hyperreactivity and HE staining in each group to observe the gross changes of the tissue and the degree of inflammatory infiltration. The collagen deposition was detected by Masson trichrome staining, and the thickness of airway smooth muscle layer was detected by immunohistochemistry. The area of collagen and thickness of airway smooth muscle in each group were compared by morphological quantitative analysis, the number of proliferating cells was detected by 伪 -SMA and PCNA immunofluorescence, the phenotype markers of lung bronchi were detected by RT-PCR and the expression of COL1A1mRNA was detected by Western-Blot phosphorylated p65 and 伪 -SMA protein were detected by Western-Blot. The expression of inflammatory factors in serum was detected by RayBiotech protein chip. Results (1) the airway hyperresponsiveness (Penh) of acute and chronic asthma mice was significantly higher than that of control group, while the area of smooth muscle layer in intervention group was significantly lower than that in asthma group) the area of smooth muscle layer in chronic asthmatic mice was significantly higher than that in control group, while that in intervention group was significantly alleviated. There was no significant difference in airway collagen area in acute asthma mice. In chronic asthma model, morphological quantitative analysis showed that the collagen area in asthma group was significantly higher than that in control group. However, compared with the control group, the area of collagen in PDTC group decreased significantly (P < 0.05). No co-expression of airway smooth muscle and PCNA was detected by immunofluorescence staining. It was found that the number of PCNA cells in the lung of acute asthma was significantly higher than that in the normal group, mainly located around the airway and blood vessels. In chronic asthma model, 伪 -SMAcalponin and SM-MHC in asthma group were significantly lower than those in control group. In acute asthma mice, only calponin significantly decreased phenotypic transformation in both chronic and acute asthma models. However, the decrease of calponin in chronic asthma model could not be reversed by PDTC intervention. Western blot showed that the expression of phospho-P65 in asthmatic mice after PDTC intervention was significantly decreased, while 伪 -SMA protein increased. Compared with the normal control group, the serum levels of G-CSF and IL-6 in asthma group were significantly increased. PDTC intervention significantly decreased the levels of G-CSF and IL-6, but did not affect IL-17. In chronic asthma model, only IL-17 and TNF- 伪 increased significantly in asthma group. Conclusion the phenotypic transformation of airway smooth muscle in asthmatic model mice has a time series. During the acute asthma period, the contractile substances begin to decrease and the cell function changes at the same time. On the other hand, the decrease of chronic asthma was more significant. (2) intervention of NF-kB pathway reversed the phenotypic transformation of airway smooth muscle cells in asthmatic mice to a certain extent. Intervention of NF-kB signaling pathway could alleviate airway remodeling in asthmatic mice to a certain extent. Airway hyperresponsiveness, chronic inflammation and other pathophysiological features of asthma are expected to be a new target for asthma treatment.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R562.25

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