光动力学疗法作用于瘢痕疙瘩成纤维细胞TGF-β1/Smad3信号通路的分子机制

Keloid are pathologic proliferations of the dermal skin layer resulting from excessive collagen deposition during the healing process of cutaneous wounds. Many factors such as skin injury or inflammation, wound tension, racial difference and genetic predisposition have been implicated in the aetiology of keloid formation. However, the underlying mechanisms of keloid pathogenesis remain unclear. There is no succussful treatment exists currently, and they often recur following traditional treatment. Photodynamic therapy (PDT) is carried out using photosensitizer and an adequate light source, which produce singlet oxygen and free radicals. Our studies have demonstrated that PDT may lead to beneficial outcome. The biological effects may include changing signal pathway, inhibiting cell proliferation, or increasing apoptosis. Interest in photodynamic therapy has grown in recent years based on new kind of photosensizer drugs, and it has gained increasing attention as a new and promising approach for keloid. While there is no basic study about this treatment for keloid, and little has been written about its clinical evaluation and basic mechnism. The transforming growth factor (TGF)- β1/Smad signalling plays a central role in the pathogenesis of keloid. To more clearly define the potential effect that regulate TGFβ1/ Smad activation and expression after PDT, a three-dimensional culture model will be used, where in normal and keloid fibroblasts will be physically cultured by collagen inserts while allowing cytokine diffusion. That is to say, the present study will analyse the effect of PDT combined with a hematoporphyrin derivative photosensitizer (Hematoporphrin monomethyl ether, HMME) in fibroblast-populated collagen lattices (FPCLs) in vitro model. We will investigate the role of transforming growth factor (TGF)- β1, TGβR, Smad3 and the role of downstream Smad cascade induction in this interaction, and the mechanisms involved in the PDT that mediate TGFβ1/Smad signalling were investigated by blocking the pathway with specific inhibitiors: ERK inhibitor(PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580). The method include immunohistochemistry, immunofluorescence, RNAi, flow cytometry, Western-blot and real-time reverse transcriptasepolymerase chain reaction analysis. A better understanding of the underlying molecular mechanisms will greatly improve our current knowledge of keloid therapy after PDT, which in turn could also help to develop an effective therapy for KELOID. Meanwhile, the present study will provide meaningful reference research route about HMME for other indications.

瘢痕疙瘩是临床上常见的病理性瘢痕，其生发机制与外伤、炎症等诸多因素相关，但仍然不明，目前缺乏有效的治疗手段，且经常规治疗后极易复发。采用光动力学疗法治疗瘢痕疙瘩是新的、具有潜力的研究方向，我们既往的研究表明光动力学疗法通过改变信号通路、抑制细胞增殖或诱导凋亡来发挥抑制瘢痕增生的生物学效应。但是，目前的研究仅限于有效性验证，缺乏针对临床应用的机理研究。本课题通过在三维培养瘢痕疙瘩成纤维细胞的基础上，建立成纤维细胞胶原网架，并进行光动力治疗，利用RNAi、流式细胞术以及免疫蛋白印记、RT-PCR等，探求光动力治疗对转化生长因子β1/Smad信号通路的分子作用，并通过特异性蛋白抑制子，研究该信号通路与ERK、JNK和P53蛋白的交叉效应，从而掌握光动力学疗法对瘢痕疙瘩成纤维细胞的作用机理，为该新技术应用于瘢痕疙瘩的治疗奠定理论基础。

瘢痕疙瘩是临床上常见的病理性瘢痕，其生发机制与外伤、炎症等诸多因素相关，但仍然不明，目前缺乏有效的治疗手段，且经常规治疗后极易复发。采用光动力学疗法治疗瘢痕疙瘩是新的、具有潜力的研究方向，我们既往的研究表明光动力学疗法通过改变信号通路、抑制细胞增殖或诱导凋亡来发挥抑制瘢痕增生的生物学效应。但是，目前的研究仅限于有效性验证，缺乏针对临床应用的机理研究。本研究的目的旨在初步揭示PDT对瘢痕疙瘩成纤维细胞TGF-β1/Smad3信号分子的作用机制，深入探讨该方法诱导的细胞效应，进一步阐明PDT治疗瘢痕疙瘩的可能作用机制。通过采用小分子RNA干扰(RNAi)技术，转染体外原代培养的人瘢痕疙瘩成纤维细胞(KFB)；同时培养正常皮肤成纤维细胞，将细胞分为NFB组、KFB组、siRNA组、NFB+PDT组、KFB+PDT组、siRNA+PDT组,倒置相差显微镜下观察细胞形态学变化；采用逆转录聚合酶链反应(RT-PCR)方法检测各组细胞中TGF-β1、Smad3、α-SMA基因水平的变化；采用免疫印迹分析试验（Western blot analysis）检测TGF-β1、Smad3、P-Smad3、α-SMA蛋白水平的变化。研究结果表明RNAi技术可有效干扰KFB中Smad3mRNA和蛋白的表达，抑制α-SMA基因和蛋白的表达，但TGF-β1基因和蛋白水平反馈性升高；PDT不能改变Smad3基因和蛋白的表达，但抑制Smad3的磷酸化水平；KFB经RNAi干扰后再进行PDT治疗，可显著抑制TGF-β1/Smads信号分子中TGF-β1、Smad3和α-SMA的表达。通过本研究表明PDT可抑制瘢痕疙瘩TGF-β1/Smads信号通路中TGF-β1、α-SMA基因和蛋白表达，降低Smad3的磷酸化水平, 达到RNAi技术的抑制效应，PDT有可能成为治疗瘢痕疙瘩的新方法。