IPS细胞调节培养基抑制增生性瘢痕的机制研究

The treatment of hypertrophic scars has been an urgent problem need to be solved. Excessive inflammatory response and tissue fibrosis has been confirmed to play an important role in scar formation process. We found in our previous study that iPS cell-conditioned medium (iPSCM) can obviously reduce tissue fibrosis, and inhibit the local inflammatory response. However, there is lack of mechanism studies concerned iPSCM for scar treatment currently. In this study, we plan to control the quality of the collected iPSCM by spectrometry analysis. Then the quality controled iPSCM will be applied to hypertrophic scar animal models and scar fibroblasts and monocytes in vitro co-culture system. Lentiviral MCP-1 gene overexpression and siRNA interference, microarray analysis, immunofluorescence staining, ELISA, Real time PCR, Western blot and other methods will be used to explore the mechanisms of inhibiting hypertrophic scars by use of iPSCM at cellular and molecular level. Involvement of MCP-1 will be investigated in the aspect of regulation of inflammation and fibrosis microenvironment during inhibition of hypertrophic scars in iPSCM. In this study, we hope to provide new methods and theoretical providence for treatment of hypertrophic scars.

增生性瘢痕的治疗一直是一个亟待解决的问题。过度的炎症反应与组织纤维化已被证实在瘢痕增生过程中扮演着的重要角色。我们在前期的研究中发现iPS细胞条件化培养液（iPS cell-conditioned medium , iPSCM）能减轻多种组织纤维化，并抑制局部炎症反应。然而，目前缺乏iPSCM用于瘢痕治疗的相关机制研究。本研究中，我们拟通过质谱分析对所收集的iPSCM进行质量控制。并将经质控的iPSCM用于增生性瘢痕动物模型与体外瘢痕成纤维细胞与单核细胞共培养体系，采用慢病毒MCP-1基因过表达及siRNA干扰，表达谱芯片分析、免疫荧光染色、ELISA、Real time PCR、Western blot等方法，探索MCP-1分子在iPSCM抑制增生性瘢痕发生过程中,调控微环境炎症和纤维化水平的细胞及分子机制，并为控制瘢痕增生提供新方法和理论依据。

我们在本期的研究中发现iPS细胞条件化培养液（iPS cell-conditioned medium , iPSCM）能减轻多种组织纤维化，并抑制局部炎症反应。iPSCM用于体外瘢痕成纤维细胞与单核细胞共培养体系后，瘢痕成纤维细胞与单核细胞粘附下降，采用MCP-1基因siRNA干扰，可抑制增生性瘢痕中的炎症反应。然而，当我们在瘢痕动物模型体内应用该方法，并未发现MCP-1基因能明显抑制瘢痕增生及纤维化。增生性瘢痕的过度纤维化，是增生性瘢痕的另一研究重点，研究表明促纤维化的M2巨噬细胞有助于增生性瘢痕的形成。还研究表明，chrfam7a能抑制M2型巨噬细胞的形成。通过qPCR和组织芯片检测，我们发现在增生性瘢痕临床标本中chrfam7a表达较非增生疤痕明显降低。接受皮肤移植的SCID小鼠被随机分为chrfam7a基因转染组和对照组。同时，chrfam7a基因转染组使用慢病毒系统。疤痕的形成，研究在2, 4例移植皮肤的巨噬细胞浸润和炎症相关因子，8周后。移植的皮肤从对照组发达肥厚性瘢痕而移植的皮肤从chrfam7a转染组痊愈显着减少纤维化和M2巨噬细胞浸润。M2型巨噬细胞局部注射促进瘢痕纤维化又在chrfam7a转染组。有趣的是，炎症是通过上调M1型巨噬细胞诱导TNFα，IL-6表达的提升，在chrfam7a转染组2周后。我们的研究结果表明，chrfam7a可以有效地防止通过巨噬细胞表型转化的调控增生性瘢痕的形成。免疫刺激疗法是改善疤痕质量的一个更好的选择。