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胸腺基质淋巴细胞生成素在A549细胞中的表达及药物对其的影响

作者: 专业:呼吸内科 导师:殷凯生 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

Thymic stromal lymphopoietin, A549, NF-κB, Budesonide, Astragaloside IV

        第一部分胸腺基质淋巴细胞生成素在A549细胞中的表达目的观察气道上皮细胞A549细胞中胸腺基质淋巴细胞生成素(TSLP)的表达及与核因子-κB(NF-κB)的关系。方法A549细胞与细胞因子白介素1β(IL-1β)、IL-4或NF-κB抑制剂PDTC共同孵育,采用RT-PCR、细胞免疫荧光方法测定TSLP mRNA及蛋白的表达情况。结果A549细胞胞浆里表达TSLP蛋白,用IL-1β联合IL-4刺激4 h ,TSLP mRNA的表达量相对于β-actin为0.93,达到峰值。随着时间的推移,18 h时,TSLP mRNA的表达量相对于β-actin为0.31,基本降至0 h时水平。PDTC可明显降低TSLP mRNA的峰值水平。结论正常气道上皮细胞A549表达TSLP mRNA及蛋白,细胞因子促进气道上皮细胞诱导性表达TSLP,并受NF-κB信号通路调控。第二部分药物对A549细胞胸腺基质淋巴细胞生成素表达的影响目的探讨布迪奈德和黄芪甲苷对A549细胞TSLP表达和NF-κB的影响。方法A549细胞与细胞因子IL-1β、IL-4或药物(布迪奈德、黄芪甲苷)共同孵育,以不加任何细胞因子或药物培养的A549细胞为对照组,采用RT-PCR方法测定TSLP mRNA表达,细胞免疫荧光方法观察NF-κB(p65)的表达情况,westernblot方法检测胞浆IκBα的表达。结果布地奈德能够显著抑制TSLP mRNA表达,黄芪甲苷抑制作用不明显(P>0.05);黄芪甲苷及布地奈德均能够显著抑制A549细胞NF-κB ( p65)的核转位;黄芪甲苷可明显升高A549细胞胞浆IκBα的蛋白水平。结论抑制TSLP表达和NF-κB信号通路激活可能是布地奈德治疗哮喘的重要机制;黄芪甲苷能够明显抑制NF-κB信号通路,但对TSLP的基因表达没有抑制作用。
    Part 1 The expression of thymic stromal lymphopoietin in A549 cellsObjective To investigate the expression of TSLP mRNA in A549 cells, and explore whether nuclear factorκB (NF-κB) is involved. Methods A549 cells were cultured with interleukin1β(IL-1β) and IL-4, or PDTC (NF-κB inhibitor). The expression of TSLP mRNA and protein were detected by RT-PCR and immunofluorescence assays.Results The expression of TSLP mRNA was significantly increased in A549 cells stimulated with IL-1βand IL-4 at 4h, and decreased at 10h, almost reduced to the baseline at 18h. PDTC greatly inhibited the level of TSLP mRNA. PDTC had inhibitory effects on the expression of TSLP mRNA. Conclusions Cytokines enhanced the inducible expression of TSLP in bronchial epithelial cells, and which was associated with NF-κB activation.Part 2 Effect of drugs on the expression of thymic stromal lymphopoietin in A549 cellsObjective To investigate the effect of the drugs (astragaloside IV and budesonide) on the expression of TSLP mRNA in A549 cells, and explore whether nuclear factorκB (NF-κB) is involved. Methods A549 cells were cultured with IL-1β, IL-4, or the drugs (astragaloside IV and budesonide). The effect of astragaloside IV or budesonide on the expression of TSLP mRNA were also quantified by RT-PCR. The cytoplasm protein expression of IκBαwas evaluated by western blot and the nuclear translocation of NF-κB P65 was detected by immunofluorescence assays respectively. Results The expression of TSLP mRNA and the nuclear translocation of NF-κB P65 were greatly abolished in A549 cells pretreated with budesonide. Astragaloside IV had little inhibitory effects on the expression of TSLP mRNA (P>0.05). However, Astragaloside IV suppressed the nuclear translocation of NF-κB P65, enhanced the protein level of IκBαin the cytoplasm.Conclusions: Inhibition of NF-κB activation and TSLP expression might be an essential therapeutic mechanism of budesonide for asthma therapy. Astragaloside IV had little inhibitory effects on the expression of TSLP mRNA, but astragaloside IV could suppress the activation of NF-κB.
        

胸腺基质淋巴细胞生成素在A549细胞中的表达及药物对其的影响

主要英文缩略语词表4-5
中文摘要5-7
Abstract7-8
引言9-12
第一部分 胸腺基质淋巴细胞生成素在A549 细胞中的表达12-21
    材料和方法12-16
    结果16-18
    讨论18-21
第二部分 药物对气道上皮细胞A549 细胞胸腺基质淋巴细胞生成素表达的影响..21-36
    材料和方法21-28
    结果28-34
    讨论34-36
小结36-37
参考文献37-41
附录41-42
发表的文章42-64
致谢64
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