THROMBIN IN WOUND HEALING--MULTIPLE FUNCTIONS

凝血酶在伤口愈合中的多种功能

基本信息

批准号：
2545649
负责人：
ROLAND R ARNOLD
金额：
$ 16.74万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-12-01 至 1999-09-29
项目状态：
已结题

项目摘要

Periodontal diseases are the pathologic outcome of infection with specific pathogens that possess virulence factors that contribute either directly or indirectly to the destruction of the supporting structures of teeth. There is overwhelming evidence that host response especially to the lipopolysaccharide of these pathogens makes a primary contribution to the bone and connective tissue destruction that define periodontal diseases. Most research clearly indicates that the polymorphonuclear leukocyte is central to the defense against periodontopathogens and that the bacterial evasion and/or neutralization of PMN anti-bacterial function are common virulence traits of the successful pathogen. Most forms of periodontal disease are characteristically episodic and represent the result of an iterative process of tissue destruction and wound healing in response to chronic exposure to noxious bacterial products. It is the overall objective of the proposed studies to begin to understand the interactions between pathogenic bacteria, and/or their metabolic products, inflammatory mediators and proinflammatory factors, both acute and chronic inflammatory cells, hemostatic and thrombic components and the initiating factors of wound healing in periodontal diseases. Specifically the subcellular localization of kallikrein in neutrophils will be determined. The signal transduction and second messengers involved in thrombin stimulation of PMN and the up regulation of kallikrein synthesis and release will be determined. The granule content of human neutrophils will be fractionated and the distribution of kallikrein determined relative to that of characterized neutrophil granule markers. Kallikrein synthesis, compartmentalization and release will be characterized following the stimulation of neutrophils with thrombin, as well as, with other well characterized agonists. Release of granule markers, respiratory burst, chemotaxis, change in cell morphology and adherence will be characterized following thrombin stimulation of neutrophils and the promyelocytic cell line, HL60 undifferentiated and differentiated to either neutrophilic or monocytic cells. Pulse-chase experiments will be used to monitor kallikrein synthesis by both human neutrophils and HL6O cells. The influence of thrombin on the phagocytosis and killing of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis will be determined using a dual fluorescent microscopy assay. Influence of proinflammatory and prothombic components on neutrophil function will be examined in admix experiments using purified cytokines and inflammatory mediators, as well as platelets, monocytes, and extracellular matrix surfaces. Parallel studies will examine the kinetics of appearance of cells types and the appearance and influence of selected biologically relevant molecules in fluid collected from a murine subcutaneous chamber model for the evaluation of host/periodontopathogen interaction in vivo. These studies will provide insight into the complex interactions that occur between host cells, pathogenic bacteria, and biologically active molecules. Such information is essential to the development of interventive clinical approaches for bacterial mediated tissue destructive processes such as periodontal diseases.

牙周疾病是特定感染的病理结局具有毒力因子的病原体直接贡献或间接地破坏牙齿的支撑结构。有大量证据表明，主机的反应尤其是对这些病原体的脂多糖是对定义牙周疾病的骨骼和结缔组织破坏。大多数研究清楚地表明，多形核白细胞是防御牙周病原和细菌的核心 PMN抗细菌功能的逃避和/或中和很常见成功病原体的毒力特征。大多数牙周疾病是特征性的，代表了组织破坏和伤口愈合的迭代过程长期暴露于有害细菌产物中。这是总体拟议的研究的目的开始了解相互作用在致病细菌和/或其代谢产物之间，炎症急性和慢性炎症的介体和促炎因子细胞，止血和血栓成分以及启动因子牙周疾病中的伤口愈合。特别是亚细胞将确定kallikrein在中性粒细胞中的定位。信号介导和第二个使者参与PMN的凝血酶刺激 Kallikrein合成和释放的UP调节将是决定。人类嗜中性粒细胞的颗粒含量将被分馏 Kallikrein的分布相对于表征中性粒细胞颗粒标记。 Kallikrein合成，隔间化和释放将在用凝血酶以及其他井刺激中性粒细胞特征激动剂。释放颗粒标记，呼吸爆发，趋化性，细胞形态的变化和依从性将被表征凝血酶刺激嗜中性粒细胞和临时细胞细胞后线，HL60未分化并与中性粒细胞或区分单核细胞。脉冲练习实验将用于监测人类嗜中性粒细胞和HL6O细胞的Kallikrein合成。这凝血酶对吞噬作用和杀死肌动杆菌的影响将确定放线菌和卟啉单胞菌。使用双荧光显微镜测定法。促炎的影响在Ambix中将检查有关中性粒细胞功能的原栓成分也使用纯化的细胞因子和炎症介质的实验如血小板，单核细胞和细胞外基质表面。平行线研究将检查细胞类型外观的动力学和选定的生物学相关分子在中的外观和影响从鼠皮下腔室模型中收集的流体体内宿主/牙周病相互作用的评估。这些研究将洞悉主机之间发生的复杂交互细胞，致病细菌和生物活性分子。这样的信息对于开发干预临床至关重要细菌介导的组织破坏性过程的方法，例如牙周疾病。