TGF-β3对角膜基质体外三维培养模型中纤维化细胞外基质合成的影响

Scarring formation during wound healing of corneal stroma is one of the major causes of cornea blindness. Investigate this process and find ways to reduce scarring are important in this research area. The phynotype of the keratocytes changed and fibrotic extracellular matrix (ECM) deposition are the basis of the corneal stroma scarring formation. Three dimension (3D) culture provides new technique for investigation of ECM assemble, which is beyond of two dimension (2D) culture. We created an 3D culture system by bovine keratocytes in vitro, which with abundant collagen fibrilla deposited. This 3D model is easy to be created and without disturbance from the scaffold material when investigating protein expression. In this project, we will apply the Pellet model with different culture system containing transforming growth factor(TGF) beta 1 to looking for the culture condition which can induce the keratocytes to adopt fibroblast or myofibroblast phynotype and produce fibrotic ECM. This model can should be useful for investigating fibrotic ECM synthesis during corneal stroma wound healing process. We will further evaluate the effect of the three isoforms of TGF on the ECM deposition and investigate whether the TGF beta3 can reduce fibrotic ECM assemble. These results may discover anti-scarring formation reagents with great potential for clinical useage.

角膜基质创伤修复中瘢痕形成所致视力损伤是角膜盲的主要原因之一。研究角膜基质瘢痕形成过程并寻找抗瘢痕形成手段是角膜病研究领域需解决的重要问题之一。角膜基质细胞表型改变及纤维化细胞外基质（Extracellular Matrix, ECM）的合成是角膜基质瘢痕形成的基础。三维培养为研究ECM沉积提供了可能。我们在前期的研究工作中成功地摸索到将Pellet模型研发成具有大量胶原纤维的ECM的三维培养模型。该模型具有操作简单、不受支架材料影响目标蛋白评估等优越性。本项目拟应用该模型，结合不同的TGFβ1培养体系，探索将Pellet引导成具有纤维化ECM的培养条件，为研究角膜基质创伤修复中纤维化ECM合成提供三维培养模型。项目将进一步评估TGFβ的三个亚型（TGFβ1、TGFβ2、 TGFβ3）对ECM合成的影响，明确TGFβ3是否可以减少纤维化ECM的合成，为探寻抗角膜基质瘢痕筛选可能化学物质。

本研究中我们首先构建了TGF-β1介导的角膜基质细胞外基质纤维化的体外三维模型，可作为研究角膜基质ECM纤维化的体外三维培养模型；再对该培养体系中添加两种不同浓度的TGF-β1，探索TGF-β1如何影响角膜基质ECM的合成及构筑；我们还为研究了低浓度TGF-β1和血清对Pellet体外三维培养模型中角膜基质细胞生长状况及ECM合成的影响，发现低剂量TGF-β1具有维持该培养模型中牛角膜基质细胞生长及缓解ECM纤维化的作用；我们研究了不同浓度的TGF-β1、TGF-β2、TGF-β3对Pellet的ECM表达的影响， 发现三者均具有促进Pellet体外三维培养模型中牛角膜基质细胞合成Col Ⅰ、Col Ⅲ、FN、α-SMA，且呈浓度-相关性；TGF-β1组和TGF-β2组Col Ⅰ、Col Ⅲ、FN、α-SMA合成量较多且均合成大量的胶原纤维，而TGF-β3组胶原纤维合成较少且排列紊乱、稀疏。在通过应用real-time PCR、IF及TEM对TGF-β3 做进一步研究后，我们发现TGF-β3能够减少Pellet体外三维培养模型中角膜基质纤维化ECM的合成并影响胶原纤维的构筑，使角膜呈少或无瘢痕化修复趋势。