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Abstract：
UNASSIGNED: The circulating progenitor cells of fibroblasts (fibrocytes) have been shown to infiltrate the airway smooth muscle compartment of asthma patients; however, the pathological significance of this discovery has yet to be elucidated. This study established a co-culture model of airway smooth muscle cells (ASMCs) and fibrocytes from asthmatic or normal subjects to evaluate innate cytokine production, corticosteroid responses, and signaling in ASMCs.
UNASSIGNED: CD34+ fibrocytes were purified from peripheral blood of asthmatic (Global Initiative for Asthma treatment step 4-5) and normal subjects and cultured for 5∼7 days. In a transwell plate, ASMCs were co-cultured with fibrocytes at a ratio of 2:1, ASMCs were cultured alone (control condition), and fibrocytes were cultured alone for 48 h. Measurements were obtained of interleukin-8 (IL-8), IL-6, IL-17, thymic stromal lymphopoietin, and IL-33 levels in the supernatant and IL-33 levels in the cell lysate of the co-culture. Screening for intracellular signaling in the ASMCs after stimulation was performed using condition medium from the patients\' co-culture (PtCM) or IL-8. mRNA and western blot analysis were used to analyze AKT/mTOR signaling in ASMCs stimulated via treatment with PtCM or IL-8.
UNASSIGNED: Compared with ASMCs cultured alone, IL-8 levels in the supernatant and IL-33 levels in the ASMCs lysate were significantly higher in samples co-cultured from asthmatics, but not in those co-cultured from normal subjects. Corticosteroid-induced suppression of IL-8 production was less pronounced in ASMCs co-cultured with fibrocytes from asthma patients than in ASMCs co-cultured from normal subjects. ASMCs stimulated using PtCM and IL-8 presented elevating activated AKT substrate PRAS40. Treatment with IL-8 and PtCM increased mRNA expression of mTOR and P70S6 kinases in ASMCs. Treatment with IL-8 and PtCM also significantly increased phosphorylation of AKT and mTOR subtract S6 ribosomal protein in ASMCs.
UNASSIGNED: The interaction between ASMCs and fibrocytes from asthmatic patients was shown to increase IL-8 and IL-33 production and promote AKT/mTOR signaling in ASMCs. IL-8 production in the co-culture from asthmatic patients was less affected by corticosteroid than was that in the co-culture from normal subjects. Our results elucidate the novel role of fibrocytes and ASMCs in the pathogenesis of asthma.
UNASSIGNED: CD34+ fibrocytes were purified from peripheral blood of asthmatic (Global Initiative for Asthma treatment step 4-5) and normal subjects and cultured for 5∼7 days. In a transwell plate, ASMCs were co-cultured with fibrocytes at a ratio of 2:1, ASMCs were cultured alone (control condition), and fibrocytes were cultured alone for 48 h. Measurements were obtained of interleukin-8 (IL-8), IL-6, IL-17, thymic stromal lymphopoietin, and IL-33 levels in the supernatant and IL-33 levels in the cell lysate of the co-culture. Screening for intracellular signaling in the ASMCs after stimulation was performed using condition medium from the patients\' co-culture (PtCM) or IL-8. mRNA and western blot analysis were used to analyze AKT/mTOR signaling in ASMCs stimulated via treatment with PtCM or IL-8.
UNASSIGNED: Compared with ASMCs cultured alone, IL-8 levels in the supernatant and IL-33 levels in the ASMCs lysate were significantly higher in samples co-cultured from asthmatics, but not in those co-cultured from normal subjects. Corticosteroid-induced suppression of IL-8 production was less pronounced in ASMCs co-cultured with fibrocytes from asthma patients than in ASMCs co-cultured from normal subjects. ASMCs stimulated using PtCM and IL-8 presented elevating activated AKT substrate PRAS40. Treatment with IL-8 and PtCM increased mRNA expression of mTOR and P70S6 kinases in ASMCs. Treatment with IL-8 and PtCM also significantly increased phosphorylation of AKT and mTOR subtract S6 ribosomal protein in ASMCs.
UNASSIGNED: The interaction between ASMCs and fibrocytes from asthmatic patients was shown to increase IL-8 and IL-33 production and promote AKT/mTOR signaling in ASMCs. IL-8 production in the co-culture from asthmatic patients was less affected by corticosteroid than was that in the co-culture from normal subjects. Our results elucidate the novel role of fibrocytes and ASMCs in the pathogenesis of asthma.
摘要：
未经证实：成纤维细胞的循环祖细胞已被证明渗入哮喘患者的气道平滑肌室；然而，这一发现的病理意义尚未阐明。本研究建立了哮喘或正常受试者的气道平滑肌细胞（ASMCs）和纤维细胞的共培养模型，以评估先天性细胞因子的产生，皮质类固醇反应，和ASMC中的信令。
UNASSIGNED：从哮喘患者（全球哮喘治疗倡议第4-5步）和正常人外周血中纯化CD34+纤维细胞，培养5~7天。在transwell平板中，ASMC与纤维细胞以2:1的比例共培养，ASMC单独培养（对照条件），和纤维细胞单独培养48小时。测量得到的白细胞介素-8（IL-8），IL-6，IL-17，胸腺基质淋巴细胞生成素，和上清液中的IL-33水平和共培养物的细胞裂解物中的IL-33水平。使用来自患者共培养物(PtCM)或IL-8的条件培养基进行刺激后ASMC中细胞内信号传导的筛选。mRNA和蛋白质印迹分析用于分析通过用PtCM或IL-8处理刺激的ASMC中的AKT/mTOR信号传导。
UNASSIGNED：与单独培养的ASMC相比，在哮喘患者共培养的样品中，上清液中的IL-8水平和ASMC裂解物中的IL-33水平明显更高，但不是从正常受试者共培养的。与正常受试者共培养的ASMC相比，与哮喘患者的纤维细胞共培养的ASMC中皮质类固醇诱导的IL-8产生抑制不那么明显。使用PtCM和IL-8刺激的ASMC呈现升高的活化AKT底物PRAS40。用IL-8和PtCM处理增加了ASMC中mTOR和P70S6激酶的mRNA表达。用IL-8和PtCM处理还显著增加了ASMC中AKT和mTOR减去S6核糖体蛋白的磷酸化。
UNASSIGNED：哮喘患者的ASMC和纤维细胞之间的相互作用被证明可以增加ASMC中IL-8和IL-33的产生并促进AKT/mTOR信号传导。哮喘患者共培养物中IL-8的产生受皮质类固醇的影响小于正常受试者共培养物中的IL-8的产生。我们的结果阐明了纤维细胞和ASMC在哮喘发病机理中的新作用。
UNASSIGNED：从哮喘患者（全球哮喘治疗倡议第4-5步）和正常人外周血中纯化CD34+纤维细胞，培养5~7天。在transwell平板中，ASMC与纤维细胞以2:1的比例共培养，ASMC单独培养（对照条件），和纤维细胞单独培养48小时。测量得到的白细胞介素-8（IL-8），IL-6，IL-17，胸腺基质淋巴细胞生成素，和上清液中的IL-33水平和共培养物的细胞裂解物中的IL-33水平。使用来自患者共培养物(PtCM)或IL-8的条件培养基进行刺激后ASMC中细胞内信号传导的筛选。mRNA和蛋白质印迹分析用于分析通过用PtCM或IL-8处理刺激的ASMC中的AKT/mTOR信号传导。
UNASSIGNED：与单独培养的ASMC相比，在哮喘患者共培养的样品中，上清液中的IL-8水平和ASMC裂解物中的IL-33水平明显更高，但不是从正常受试者共培养的。与正常受试者共培养的ASMC相比，与哮喘患者的纤维细胞共培养的ASMC中皮质类固醇诱导的IL-8产生抑制不那么明显。使用PtCM和IL-8刺激的ASMC呈现升高的活化AKT底物PRAS40。用IL-8和PtCM处理增加了ASMC中mTOR和P70S6激酶的mRNA表达。用IL-8和PtCM处理还显著增加了ASMC中AKT和mTOR减去S6核糖体蛋白的磷酸化。
UNASSIGNED：哮喘患者的ASMC和纤维细胞之间的相互作用被证明可以增加ASMC中IL-8和IL-33的产生并促进AKT/mTOR信号传导。哮喘患者共培养物中IL-8的产生受皮质类固醇的影响小于正常受试者共培养物中的IL-8的产生。我们的结果阐明了纤维细胞和ASMC在哮喘发病机理中的新作用。
