【作者】 张天杰；

【导师】 罗自强； 汉建忠；

【作者基本信息】 中南大学 ， 生理学， 2009， 硕士

【摘要】 革兰氏阴性菌感染是引起急性肺损伤的临床常见病因,革兰氏阴性菌内毒素主要成分脂多糖(lipopolysaccharide,LPS)与体内相应受体结合后,在肺内启动多种信号级联放大系统,导致前炎因子、炎症介质、化学趋化因子、黏附分子等产生和释放。炎症因子大量释放入血,诱发炎症瀑布级联效应,导致炎症继续发展和放大；但在上述过程中,同时有抗炎介质如白细胞介素10(interleukin-10, IL-10)等的合成和释放,它们之间构成复杂的网络,调控炎症的发生、发展和转归。而IL-10在其中发挥着重要的病理生理学作用。IL-10是一种具有抗炎作用的细胞因子,其主要生物活性表现为直接抑制炎症细胞活化,进而抑制细胞因子的产生。适时的、适度的IL-10的产生可以调节炎症的程度,并保护正常组织免受炎症损伤,在机体对抗感染的过程中发挥重要的保护性作用。肺泡巨噬细胞(Alveolar Macrophage)是肺内重要的免疫防御细胞,具有趋化、吞噬和杀菌作用,是炎症反应中炎症因子及IL-10的重要来源细胞。Clara细胞是指衬覆于远端细支气管的非纤毛上皮细胞,其分泌蛋白(Clara cell secretory protein, CCSP,又称CC1O)属于子宫珠蛋白(uteroglobin,UG)家族；子宫珠蛋白是一种进化过程中高度保守的分泌性多功能非类固醇类激素蛋白,具有抗炎、抗氧化、免疫调节、肿瘤抑制及刺激胚胎生长的作用。AF-1 (Antiflammin-1, AF-1)是来源于子宫珠蛋白C末端第三个α螺旋的九肽,已有的研究证明AF-1具有明显的抗炎作用。但关于AF-1的抗炎作用是否与IL-10有关却未见报道。目的：1.观察AF-1对LPS诱导的巨噬细胞IL-10分泌及表达的影响。2.检测巨噬细胞子宫珠蛋白结合蛋白(uteroglobin-binding protein, UGBP)的表达。3.探讨UGBP在介导AF-1对LPS诱导的巨噬细胞IL-10分泌及表达影响中的作用,初步揭示AF-1的抗炎机制,为急性肺损伤的治疗提供新的线索和思路。方法：1.应用ELISA和RT-PCR法分别检测巨噬细胞培养上清液中IL-10蛋白含量和细胞IL-10mRNA表达,观察AF-1对LPS诱导的巨噬细胞IL-10表达的影响。2.应用细胞免疫荧光技术及RT-PCR法检测UGBP在巨噬细胞的表达。3.通过应用抗-UGBP抗体预处理巨噬细胞,观察抗-UGBP多抗对于AF-1引起的LPS诱导的巨噬细胞IL-10分泌及表达的影响。结果：1.LPS可诱导巨噬细胞IL-10的分泌和表达增加。AF-1对正常巨噬细胞IL-10的分泌和mRNA表达无影响,但可有效促进内毒素诱导的巨噬细胞IL-10的分泌和mRNA表达,并具有一定的时间、剂量依赖性。2.免疫荧光及RT-PCR实验均证实在巨噬细胞上有UGBP的表达。3.抗-UGBP抗体预处理可抑制AF-1促进内毒素诱导的巨噬细胞IL-10的分泌及mRNA表达增加的作用。结论：1.AF-1对正常巨噬细胞IL-10的分泌和mRNA表达无影响,但可有效促进内毒素诱导的巨噬细胞IL-10的分泌和mRNA表达,并具有一定的时间、剂量依赖性,提示AF-1抗炎作用的产生与调节IL-10的分泌和mRNA表达有关。2.本实验首次证实巨噬细胞上有UGBP的表达。3.本实验首次证实AF-1促进内毒素诱导的巨噬细胞IL-10的分泌及mRNA表达的效应有赖于UGBP的介导。更多还原

【Abstract】 Gram-negative bacterium is the main cause resulting in the acute lung injury clinically. Lipopolysaccharide(Lps), the components of Gram-negative bacteria, combining with its receptors on the host cells is the first step in a multi-step sequence leading to the activation of a plethora of singal transduction cascades in lung, which result in the production of proinflammatory cytokines and inflammatory mediators, chemical-chemotatic factors, adhesion molecules. These secreted factors initiated the inflammatory reaction of the body, leading to eliminate of infected microbes, meanwhile may even destruct the organs. Studies have proved that in the process of inflammation development, some anti-inflammatory media, such as interleukin-10(IL-10) is also secreted. The interactions between inflammatory factors and anti-inflammatory ones constitute a complicated network by which the onset of inflammatory reaction, and its development as well as its resolvation were regulated.Interleukin-10 is a potent anti-inflammatory molecule that has been shown to directly suppress the activation of inflammatory cells and then to decrease the production of cytokines. Timely and moderate production of IL-10 can regulate the extent of inflammation, and play an important protection role in against inflammattion. Alveolar macrophage(AM) are important immune defence cells with several functions of chemotaxis, phagocytosis and bactericidal, and AM is the important source of inflammatory factor includeing IL-10 in the process of inflammation.Clara cells are non-ciliated lining epithelium of distal bronchiolar, Clara cell secretory protein (CCSP) belongs to uteroglobin families. Uteroglobin (UG) is an evolutionarily conserved, no-steroid induciblehomodimeric secretory protein with multiple physiological functions. In a series of experiments it was demonstrated that UG has potent anti-inflammatory, anti-oxydation, immunomodulatory, anti-tumorigenic and embryonic growth-stimulatory properties.Antiflammin-1 (MQMKKVLDS, AF-1) is equivalent to the 9 C-terminal amino acids of a-helix 3 of uteroglobin and has been showed to have potent anti-inflammatory effects. It is not yet established whether the mechanism of AF-1 induced anti-inflammatory effects are related with IL-10 secretion and expression.Objective:1.To investigate the effect of Antiflammin-1 (AF-1) on LPS induced IL-10 secretion and gene expression of macrophages。2. To determine the expression of uteroglobin-binding protein (UGBP) on macrophage。3.To observe the effect of anti-UGBP antibody on AF-1 induced change of LPS induced IL-10 secretion and expression in macrophages.Methods:1. The amount of IL-10 in the macrophage culture supernatant was determined by ELISA and the macrophage mRNA expression of IL-10 was measured by RT-PCR.2. The expression of UGBP on macrophage was investigated by Immunofluorescence and RT-PCR.3. The effect of anti-UGBP antibody on AF-1 induced increase of IL-10 secretion and gene expression of macrophage induced by LPS was observed by pretreatment with anti-UGBP antibody on the macrophages.Results:1. LPS induced the increase of IL-10 expression and secrettion in macrophage. AF-1 had no effect on IL-10 mRNA expression and IL-10 secretion in normal macrohpage, but significantly increased the IL-10 expression and secretion of macrophages induced by LPS, and this effect was dose and time dependent.2. The UGBP expression was detected in macrophages expressed UGBP protein detected by Immunofluorescence and mRNA detected by RT-PCR.3. Pretreatment with anti-UGBP antibody attenuated the enhenced effect of AF-1 on LPS induced IL-10 secretion and gene expression in macrophages.Conclusions:1. AF-1 had no effect on IL-10 mRNA expression and IL-10 secretion in normal macrohpages, but increased the IL-10 expression and secretion of macrophages induced by LPS, and this effect was dose and time dependent, which suggested that AF-1 may play the anti-inflammatory role by modulating IL-10 gene expression and secretion.2. UGBP expression was at the first time detected in macrophages.3. We firstly revealed that the increased effect ofAF-1 on LPS induced IL-10 gene expression and secretion in macrophage was mediated by UGBP.更多还原