C1Q-INDUCED CA2+ RESPONSES IN FIBROBLAST HETEROGENEITY

在牙周炎中，支持牙齿的连续组织会被长期感染系统地破坏。如果治疗成功，通常会导致疤痕修复，因为肉芽细胞取代了正常的成纤维细胞。牙周修复不如完全功能牙周组织的再生对患者有利。为了促进临床再生治疗，我们研究了从人牙龈培养的正常和肉芽成纤维细胞的不同群体增殖的分子和细胞机制。这些研究让我们仔细研究了在正常和炎症组织中大量存在的抗微生物补体成分C1q是如何抑制宿主细胞的生长的。C1q起到压力激活剂的作用。而正常成纤维细胞对C1q的反应是通过激活与细胞延迟相关的细胞内应激蛋白来选择肉芽成纤维细胞而不是正常成纤维细胞的生长，从而阻碍牙周再生。这一假说的验证可以为更有效地预防和治疗人类牙周病提供基础，通过对受影响的组织进行药物和遗传操作，以增强正常的成纤维细胞功能和牙周器的再生。具体目标是：目的I：评估C1q是否通过阻止细胞周期进程(细胞停滞)或程序性细胞死亡(细胞凋亡)来改变成纤维细胞群体的生长反应。细胞停滞是可逆的，而细胞凋亡是不可逆的。这一信息将为评估疾病在细胞组成上的结果，从而为牙周组织的功能提供更可预测的基础。目的II：确定保护肉芽细胞免受C1q诱导的应激的分子事件，并将这些信息用于未来的临床应用，旨在提高正常成纤维细胞群体的存活率。我们的策略包括表征C1q诱导的正常成纤维细胞应激途径，以确定由高水平的蛋白激酶A激活的生化步骤和仅发生在肉芽细胞中的蛋白激酶C转位，以响应C1q。实现这些目标的方法应用了已建立的药理生物测定、组织学和流式细胞术技术。这些信息将为药物的设计提供见解，这些药物中和C1q对正常成纤维细胞生长的不利影响，同时保留补体级联的有益抗菌活性。虽然这项工作的重点是牙周炎，但其结果应该适用于各种炎症性疾病以及伤口愈合。

In periodontitis, the consecutive tissues supporting the teeth are systematically destroyed by long term infections. Treatment, if successful, usually leads to repair by scarring as a result of the replacement of normal fibroblasts with granulation cells. Periodontal repair is not as advantageous to the patient as regeneration of a fully functional periodontium. In order to facilitate clinical regenerative therapies, we studied molecular and cellular mechanisms that control proliferation of distinct populations of normal and granulation fibroblasts cultured from human gingiva. These studies led us to scrutinize how the anti-microbial complement component C1q, which occurs in high quantities in normal and inflamed tissues, inhibits the growth of the host's cells. C1q acts as a stress activator. While normal fibroblasts respond to C1q by activating intracellular stress proteins associated with delays in cell select growth of granulation fibroblasts over normal fibroblasts, thus hampering periodontal regeneration. Verification of this hypotheses could provide a basis for more effective prevention and treatment of human periodontal disease through pharmacological and genetic manipulations of the affected tissues to enhance normal fibroblast function and regeneration of the periodontal apparatus. Specific goals are: Aim I: To assess whether C1q modifies the growth response of the fibroblast populations by a block in cell cycle progression (cytostasis) or programmed cell death (apoptosis). Whereas cytostasis is reversible, apoptosis is irreversible. This information will provide a more predictable basis for evaluating the outcome of the disease on cellular composition, and therefore functionality, on the periodontium. Aim II: To identify the molecular events that protect the granulation cells from C1q- induced stress, and use this information for future clinical applications aimed at improving the survival of normal fibroblast populations. Our strategy consists of characterizing C1q-induced stress pathways of normal fibr9oblast for identification of the biochemical steps activated by high levels of protein kinase A and by translocation of protein kinase C, which occurs in granulation cells only, in response to C1q. The methods for achieving these goals apply established pharmacological bioassays, histology, and flow cytometry techniques. This information will provide insights for the design of drugs that neutralize the detrimental effect of C1q upon the growth of normal fibroblasts, while retaining the beneficial antimicrobial activity of the complement cascade. While this work focuses on periodontitis, its results should be applicable to a variety of inflammatory diseases as well as wound healing.