课题背景：表皮角质形成细胞是皮肤创面愈合的主要修复细胞之一，创面愈合过程中表皮角质形成细胞增殖、迁移和分化，最终完成再上皮化过程。激活一些细胞的Toll样受体可以促进细胞的增殖。实验通过检测激活表皮角质形成细胞表面Toll样受体后对创面愈合的影响，对其促进创面愈合的机制作初步探讨。

偏倚或不足：实验只是课题研究的初级阶段，关于激活Toll样受体促进创面愈合的研究国内外均少见，主要集中于Toll样受体与炎症反应的关系，促进创面愈合是一个复杂的过程，可能涉及到多个信号转导通路，对组织再生的影响也是多方面的，这方面的机制仍不清楚，需进一步研究才能明确。

术语解析：Toll样受体家族属于白细胞介素1受体/ Toll样受体超家族，主要分布于免疫细胞以及同外界相通的腔道上皮细胞表面，在巨噬细胞、树突状细胞等专职抗原提呈细胞表面的表达尤其丰富。不同的Toll样受体分子识别不同的病原体相关分子模式，其配体几乎涵盖了人类所能遇到的所有病原体及其产物，从而赋予人类先天性抵抗感染的能力。

摘要
目的：细胞表面Toll样受体对激发先天性免疫、抵抗微生物感染有重要作用。最近研究发现激活细胞表面Toll样受体不仅可以预防微生物感染，还可以促进组织再生。实验通过脂多糖激活体外培养的角质细胞株Hacat细胞表面Toll样受体，观察其促进创面愈合的可能机制。
方法：实验于2007-07在解放军第四军医大学西京医院烧伤与皮肤外科完成。①实验分组及方法：角质细胞株Hacat由解放军第四军医大学西京医院皮肤科馈赠；脂多糖为Sigma公司产品；小鼠抗人Toll样受体2单克隆抗体、兔抗人Toll样受体4多克隆抗体购于ebioscience公司。体外培养Hacat细胞，根据脂多糖(500 ng/L)与Hacat细胞共培养24，48，72 h将细胞随机分为脂多糖作用24 h 组、脂多糖作用48 h 组、脂多糖作用72 h 组，另设对照组，只加溶剂二甲基亚枫。②实验评估：蛋白免疫印迹技术检测脂多糖对Hacat细胞Toll样受体2、Toll样受体4及核因子-κB表达的影响；荧光定量聚合酶链反应检测脂多糖作用后Hacat细胞内基质金属蛋白酶3、基质金属蛋白酶9及血管内皮生长因子的表达。
结果：光学显微镜下正常人皮肤表皮及角质细胞Toll样受体2、Toll样受体4表达呈蓝黑色；脂多糖作用于Hacat细胞24，48，72 h 后Toll样受体2、Toll样受体4、核因子-κB表达均高于对照组（P < 0.01）。与对照组相比，其他3组基质金属蛋白酶3及血管内皮生长因子表达均增高（P < 0.01，P < 0.05）。脂多糖作用24 h 组基质金属蛋白酶9表达与对照组差异无显著性（P > 0.05），脂多糖作用48，72 h 组与对照组比较，差异显著（P < 0.05）。
结论：激活Hacat细胞表面Toll样受体有促进创面愈合作用，其机制可能与脂多糖作用于Toll样受体从而激活核因子-κB信号传导通路促进细胞释放基质金属蛋白酶3、基质金属蛋白酶9及血管内皮生长因子等因素有关。
关键词：Hacat细胞；Toll样受体；创面愈合；组织构建

韩勇彬，胡大海，杨崇志，蔡维霞，白晓智，胡晓龙. 激活Hacat细胞表面Toll样受体促进创面愈合的机制[J].中国组织工程研究与临床康复，2008，12(7):1272-1276 [www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1272(ps).pdf]

Mechanism of active Toll-like receptors on the surface of Hacat cells to promote wound healing

Abstract

AIM：Toll-like receptors (TLRs) on cell surface are essential to activate innate immune system and recognize microbial pathogens. Recent studies suggest that TLRs cannot only defend microbial immune responses, but also promote tissue regeneration after injury. This study investigated the possible mechanism of lipopolysaccharide (LPS) induced TLRs activation on the Hacat cell surface to promote wound healing.
METHODS: The experiment was performed at the Department of Burn and Skin Surgery, Xijing Hospital of Fourth Military Medical University of Chinese PLA in July 2007. ①Hacat cells were given by Department of Dermatology, Xijing Hospital of Fourth Military Medical University of Chinese PLA; LPS was the product of Sigma, and mouse anti-human TLR2 monoclonal antibody and rabbit antihuman TLR4 polyclonal antibody were purchased from Ebioscience Company. LPS (500 ng/L) and Hacat cells were co-cultured and divided into LPS 24, 48, and 72 hours groups. The cells cultured with dimethyl sulfoxide served as control. ②The influence of LPS on TLR2, TLR4 and nuclear factor-kappa B (NF-κB) expressions were detected by Western blot. The expressions of matrix metalloproteinase-3 (MMP-3), MMP-9 and vascular endothelial growth factor (VEGF) in Hacat cells cultured with LPS were detected by RT-PCR and real-time PCR.
RESULTS: Under light microscope, TLR2 and TLR4 expressions in normal epidermis and keratinocytes showed black blue. The expressions of TLR2, TLR4 and NF-κB in Hacat cells cultured with LPS for 24, 48, and 72 hours were significantly higher than control cells (P < 0.01). Moreover, the expressions of MMP-3 and VEGF in three LPS groups were significantly increased compared with control group (P < 0.01, P < 0.05). LPS 24 hours group showed similar MMP-9 secretion as the control group (P > 0.05), but in LPS 48 and 72 hours groups, the expression of MMP-9 was significantly higher (P < 0.05).
CONCLUSION: LPS can activate TLRs on the surface of Hacat cells to promote wound healing. The mechanism probably is LPS activate NF-κB signal conduction and secretion of MMP-3, MMP-9 and VEGF through TLRs.

Han YB, Hu DH, Yang CZ, Cai WX, Bai XZ, Hu XL. Mechanism of active Toll-like receptors on the surface of Hacat cells to promote wound healing.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(7):1272-1276(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1272(ps).pdf]