机械拉伸对成纤维细胞生物学行为的影响及其机械生物学和神经炎症机理的实验研究

Pathological scars are typical fibroproliferative skin disorders. Mechanobiology and neuroinflammation have increasingly been recognized showing close relationship to the development and progression of pathological scars. Purpose of this study is to explore the mechanisms of mechanotransduction, neuroinflammation, the potential direct gap-junction communication, and their potential communications related to formation of pathological scars.Based on the former trials of cDNA microarray analysis on in vivo keloid samples to screen out the potential mechanotransduction pathways, in this main study, the in vitro cellular stretching model will be set up to explore the effects of cellular stretch for dermal fibroblasts on their cellular behaviors of morphology and functions such as proliferation, differentiation, synthesis and migration. Furthermore, the expressions of the intracellular mechanotransduction pathway molecues will be examined at mRNA and protein levels through real-time RT-PCRA and ELISA methods, respectively. After that, the in vitro co-culture system of dermal fibroblasts and sympathetic nerve cells will be developed in vitro for the following celluar stretching. The sympathetic nerve cell-derived neuropeptides in this system and the potential direct gap junction between fibroblasts and sympathtic nerve cell will be examined after cellular stretch, by real-time RT-PCR, ELISA, and fluorescence recovery after photo-bleaching methods. Thus, the mechantransduction mechanism, the neuroinflammation mechanism, and the potential direct cellular communication mechanism though gap junction will be put forward, together with their interactions, if there can by any, to show their roles in the development and progression of patholgocial scars in vivo under the chronic cellular stretch. A better understanding of those mechanisms will facilitate the bilateral feedback between clinic and experimental explorations of those fibroproliferative skin disorders and thereby lead to potential development of new interventions to prevent, reduce, or even reverse formation and/or progression of pathological scars.

机械生物学信号转导和神经炎症与病理性瘢痕的发生发展密切相关。本研究在前期对瘢痕疙瘩标本进行cDNA基因微阵列分析，筛选体内瘢痕疙瘩在所处慢性机械刺激微环境下相对于自体正常皮肤机械生物学信号转导通路表达的变化。在此基础上，拟在体外构建成纤维细胞拉伸模型，观察拉伸刺激对成纤维细胞的形态影响及成纤维细胞增殖、分化、合成、运动等细胞功能的变化。并进一步在mRNA和蛋白质水平检测靶机械传导通路分子的表达。进而建立体外成纤维细胞与交感神经细胞的共培养体系，检测拉伸刺激下交感神经细胞来源的神经肽的mRNA和蛋白质表达及其对成纤维细胞的影响。同时观察交感神经细胞与成纤维细胞间的直接缝隙连接变化情况。通过从机械生物学膜途径、神经炎症旁分泌途径及可能的缝隙连接直接途径揭示体外拉伸刺激下成纤维细胞形态和功能改变的作用机理及三者之间的相互关系，阐明病理性瘢痕发生发展的机理，以期为病理性瘢痕的防治和干预提供靶分子。

项目背景：瘢痕疙瘩是软组织纤维增殖性疾病的典型代表，亚洲人群多见，发病机理尚不清。近来，力学和炎症因素备受关注。探讨细胞外力学信号转化为细胞内分子生物学信号并进而引发细胞行为改变过程的力生物学发展为瘢痕疙瘩机理研究提供了新手段。此外，我们前期提出的神经炎性假说认为交感神经细胞分泌的神经肽可促进病理性瘢痕形成。故本研究拟对体内瘢痕疙瘩样本做基因芯片检测，并在体外建立成纤维细胞与神经细胞的共培养体系，施以机械拉伸以模仿体内瘢痕局部力学环境，观察形态与功能变化，探索力生物学信号转导通路与神经炎症在瘢痕疙瘩形成与发展中的作用。..主要研究内容：体外建立大鼠真皮成纤维细胞细胞和交感神经元共培养体系，模拟体内环境完成机械拉伸刺激。实验组机械拉伸刺激为周期性单周正弦拉伸，阴性对照组为连续型拉伸，持续时间为24小时。空白对照组为无拉伸刺激。观察拉伸刺激下细胞形态和功能影响。各组取材进行实时PCR(qPCR)等检测，观察细胞内力生物学信号转导通路关键分子、神经肽、缝隙连接等的变化。同时，对人瘢痕疙瘩切除后的标本进行cDNA微列阵分析。 ..重要结果数据及科学意义：体外拉伸刺激参数优化为周期性单周正弦拉伸，强度为110%，频率为10 cylces/分钟，持续时间为24小时。拉伸刺激下，形态上观察到原随机方向排列的细胞改变运动轨迹及方向为与拉伸方向成90°垂直排列。功能上，(1) I型胶原：体内标本cDNA水平上COL1A1表达上调；体外拉伸下COL1A1在mRNA水平下调，提示拉伸外存在刺激胶原产生的其他因素；(2) 炎症反应：非特异性炎症方面体现为体外拉伸下IL-6升高，高于持续性拉伸中的IL-6水平；神经炎症方面，CGRP、Substance P等神经肽的mRNA水平拉伸后只降不增；而体内标本IL-6表达未见上升，体内神经纤维的组成蛋白和神经肽受体却大幅提高。(3) 细胞内力生物学信号机细胞间的信息沟通：体内TGF-b3表达大幅提高，体外拉伸下MAP3K7的mRNA表达下调。缝隙连接蛋白在体内无表达升降变化，体外拉伸下周期性拉伸组<持续拉伸组<空白对照组。(4) 血管内皮细胞功能变化：拉伸后VEGF在mRNA水平表达下调，周期性拉伸组低于持续拉伸组；拉伸后HIF-1a在mRNA水平表达在周期性拉伸组显著下调，而在持续拉伸组出现显著上调。提示瘢痕疙瘩中存在内皮细胞功能参与。