【作者】 向阳；

【导师】 秦晓群；

【作者基本信息】 中南大学 ， 生理学， 2003， 硕士

【摘要】 支气管上皮细胞（bronchial epithelial cells，BEC）的抗氧化活性对改善上皮的抗损伤能力、维持上皮的结构和功能完整具有重要意义。BEC表达的整合素分子是细胞外基质成分如纤维连接蛋白（fibronectin，Fn）的受体，整合素特异性识别纤维连接蛋白分子中的精-甘-天冬氨酸片段（Arg-Gly-Asp peptide RGD肽）并与之结合，经跨膜信号转导至胞内，与细胞的生长、分化、代谢调控有关。为验证支气管上皮细胞表达的整合素与纤维连接蛋白反应后对细胞的保护作用，本研究用臭氧（1.5ppm）攻击原代培养的兔支气管上皮细胞，测定细胞的损伤指标：³H释放率、乳酸脱氢酶（LDH）、丙二醛（MDA）含量；细胞的抗氧化酶：谷胱甘肽过氧化物酶（GSH-P_X）、过氧化氢酶（Catalase）、超氧化物歧化酶（SOD）活性及谷胱甘肽（GSH）含量：测定一氧化氮（NO）释放量及一氧化氮合酶（NOS）活性。观察整合素的配体分子Fn及人工合成的RGD肽的保护效应，并观察钙调素抑制剂N-（6-氨基己烷）-5-氯-1-萘酚-磺酰胺（N-（6-Aminohexyl）-5-chloro-1-naphthalene-sulfonamide,W₇）(10^-5M)对Fn效应的阻断；RT-PCR观察Fn对过氧化氢酶表达的影响。 结果 一、纤维连接蛋白—整合素反应对支气管上皮细胞的抗氧化损伤保护作用 臭氧攻击明显提高细胞的³H释放率（P＜0.01），Fn（10μg／ml）处理及RGD（30μg／ml）处理减轻臭氧损伤（分别为P＜0.05及P＜0.01），W₇能逆转Fn的作用（P＜0.05）；臭氧攻击提高LDH活性（P＜0.01），Fn及RGD降低臭氧毒性效应（两者均P＜0.01），W₇可阻断Fn的作用（P＜0.01）；MDA含量呈平行变化，Fn及RGD处理均降低臭氧对MDA含量的提升（P＜0.05）。 二、纤维连接蛋白—整合素反应上调兔支气管上皮细胞抗氧化能力 Fn（5～20μg／ml）处理后GSH含量呈剂量依赖性上升（r=0.82），RGD门一60 u旮ml)也有明显量一效关系（，0．84人 Fn及 RGD均呈剂量依赖性提高GSHPx活性（分别为 r＝0．93和，0．73人 Fn的上调作用被W，所逆转（P＜0．05）；Fn提高Catalase活性（P＜0＊5），W。可阻断这一效应o功．05人Fn提高SOD活性O功＊人 此效应可被W7阻断。三、纤维连接蛋白或精一甘一天冬氨酸肽促进兔支气管上皮细胞NO合成 臭氧攻击 Zh后，NO释放增多O叼．of），Ffl （ 5 11 g／ml）也能增加＊O释放（P＜0刀1人 并被W，所阻断o＜0刀1）；Fnu～20 p旮）及RGD门～60 H旮)均显示对NO释放量的剂量依赖性促进作用 （分别为 r—0．85禾 r＝0．89）；Fn（15 u g／hl）明显提高 BEC的 NOS活性(P＜0刀1人 此效应也能被w，阻断（P＜0刀1人Fn及＊＊D对＊OS活性的上调有量一效关系，分别为r—0．93及r1．90。四、纤维连接蛋白一整合素反应上调支气管上皮细胞过氧化氢酶表达 O。攻击对BEC内过氧化氢酶表达天显著影响o功“），而Ffl可明显增强过氧化氢酶表达，提示整合素一配体反应对支气管上皮细胞过氧化氢酶活性的提高与其表达增强有关如动刀1人蛋白酪氨酸激酶抑制剂Genistein和钙调素抑制剂W，可逆转该效应（两者均P＜0刀1）。结论 细胞外基质成分纤维连接蛋白通过与整合素作用可提高支气管上皮细胞的抗氧化能力，减轻损伤，提高适应、应激能力，具有保护作用，其胞内信号传递有钙调素途径参与。纤维连接蛋白一整合素反应对BEC过氧化氢酶活性的提高是由于其表达增强，其信号转导途径与整合素介导的酪氨酸磷酸化和 Ca》一钙调素信号通路有关。更多还原

【Abstract】 The antioxidant activity of bronchial epithelial cells (EEC) plays a essential role in maintenance of integrity of the airway epithelium in structure and function, and preventing the epithelial cells from damage. Integrins expressed on EEC are considered as receptors of extracellular matrix such as fibronectin (Fn). The interaction of integrins with their ligands maybe involve in many aspect of cell biology including proliferation, differentiation and metabolism, etc. In order to test the hypothesis that activation of integrin molecules may exert protection effects on bronchial epithelial cells from oxidant injury, the present study, based on a cell-injury model made with ozone (1.5ppm) exposure, investigated the protective effect of fibronectin, a kind of ligand of integrin, and it’s specific sequence Arg-Gly-Asp (ROD peptide ) which is a recognizable domain for integrin on EEC. 3H release, LDH and MDA were measured as indexes of cellular damage. The antioxidant ability has been shown as the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and the level of glutathione (GSH) in cells. Detect the NO released from primary cultured rabbit bronchial epithelial cells and the activity of NO synthesase (NOS) in cells .Use RT-PCR to investigate the gene expression of catalase. ResultsI . The protective effect of fibronectin on bronchial epithelial cellsOzone exposure induced a significant increase of H release (PO.01), pretreatment with Fn(10 U g/ml) or RGD(15 u g/ml) markedly attenuated the injury (respectively P<0.05and PO.01), and the effects were reversed by W7 ,a calmodulin inhibitor; Ozone exposure increasedLDH activity (P<0.01), treatment with Fn and RGD decreased the LDHIVactivity and the effect was abolished by W7 (P<0.01); The content of MDA was altered as similar as 3H release and LDH parallel. Treatment with Fn and ROD both decreased the elevation of MDA induced by ozone (PO.05).II. Integrin-fibronetin binding reaction upregulates the antioxidantactivity of rabbit bronchial epithelial cellsDose-dependent increase of GSH level were observed both in Fn (5-20 u g/ml) (i=0.82) and in RGD( 15-60 u g/ml)treatment (r=0.84); Fn binding increased significantly the activity unit of GSH-Px (P<0.05) while was inhibited by W7 (P<0.05).Both Fn and ROD show a dose-dependent upregulatory effect on GSH-Px activity (respectively r=0.93 and r=0.73); Catalase activity could be also stimulated by Fn (P<0.05) and reversed by W7; Treatment with Fn increased SOD activity (P <0.01) and the effect was abolished by W7(P<0.01).III. Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbitbronchial epithelial cellsOzone stress enhanced NO release from BEC(P<0.01), and treatment with Fn also promoted the NO production(P<0.01). The effect of Fn could be blocked by calmodulin inhibitor W-(P<0.01); Either Fn or RGD showed a dose-dependent promotion on NO release (respectively r=0.85 and r=0.89); The NOS activity was significantly elevated in Fn treatment group(P<0.01) and the effect of Fn was abolished by W. 7(P<0.01). A dose-response relation in NOS activity could be observed either in Fn treatment or in RGD treatment(respectively r=0.93 and r=0.90).IV.Upregulation of catalse gene expression in bronchial epithelial cells by fibronetin-integrin reactionFn could promote transcription of the catalase encoding mRNA (P<0.01). However, this effect was abolished either by inhibitor of protein-tyrosine kinase, Genistein or by the inhibitor of calmodulin, W7. Conclusion:The data suggests that the binding of integrin with Fn improve the antioxidant ability of EEC and lighten the damage induced by oxidant attack. The protective mechanism of Fn-integrin binding may be relatedvwith calmodulin. The promotion of catalase activity was due to the upregulation of catalase gene expression via Fn-integrin reaction, in which tyrosine phosphorylation and calmodulin are requisite.更多还原