Notch/alpha-SMA信号通路调控人Tenon's囊成纤维细胞向肌成纤维细胞分化的机制研究

Background and Hypothesis: .The transdifferentiation of human Tenon's fibroblasts to myofibroblasts is a critical step in filtering bleb scarring. This transdifferentiation is characterized by de novo expression of α-Smooth muscle actin (α-SMA), but the signaling pathway that mediates α-SMA induction of myofibroblast differentiation remains unknown. Notch is an evolutionarily conserved receptor that regulates cell fate, including events such as cell differentiation and apoptosis. Notch1 signaling has previously been shown to regulate myofibroblast differentiation in pulmonary fibrosis and renal disease; we hypothesize that Notch signaling also plays a pivotal role in myofibroblast transdifferentiation during glaucomatous filtration scarring, and we hypothesize that Notch signaling achieves this by speeding up the expression of histone acetyltransferases (HATs), which in turn activates α-SMA gene promoter...Methods: .(1) In vitro study: Human Tenon's fibroblasts will be grown in culture, stimulated with transforming growth factor-β1, or pretreated with Notch inhibitor (DAPT), Notch stimulator (siRNA), or Trans-differentiation inhibitor (KLF-4). Expression of Notch-1 to -4 will be detected by Western blot. α-SMA transcription will be assessed by real-time PCR; α-SMA protein expression and localization will be measured by Western blot and confocal immunofluorescence microscopy. The role of HATs/histone deacetylases (HDACs) in the regulation of α-SMA gene promoter will be determined by RT-PCR and chromatin immunoprecipitation (ChIP) assay. Cell contractility will be determined in collagen gel contraction assay. (2) In vivo study: In a randomized, controlled, masked-observer study, 48 rabbits will undergo trabeculectomy in the right eye and randomly receive postoperative administration of Notch inhibitor (DAPT), Notch stimulator (siRNA), Trans-differentiation inhibitor (KLF-4), or no treatment. Bleb characteristics and functions will be evaluated over a period of 4 weeks. The animals will be killed on days 1, 7, 14, and 28. Histopathology and immunohistochemistry will determine the amount of scarring...Significance of Research: .Our experiments will determine whether Notch signaling plays key roles in myofibroblast transdifferentiation and contributes to fibrosis development after glaucomatous filtration survey. Insights into the role of Notch/α-SMA signaling pathway should advance understanding of the pathogenesis of wound healing process after glaucoma filtering surgery, and help develop new therapeutic strategy to reduce fibrosis development.

人Tenon's囊成纤维细胞特征性表达α-平滑肌肌动蛋白（α-SMA）进而转化为肌成纤维细胞的过程是滤过区瘢痕化的关键步骤，但α-SMA基因启动子被激活的机制尚未明确。既往的研究发现Notch信号通路是肺纤维化和肾小管纤维化的必经步骤,并且在这些组织中Notch1受体/Jagged1配体信号通过激活α-SMA基因促进了瘢痕化的发展。我们推测在青光眼滤过术术区中，Notch通路也通过激活α-SMA蛋白的表达促进了人Tenon's囊成纤维细胞向肌成纤维细胞的转化从而促进了术区的瘢痕化。我们前期研究发现已活化的Tenon's囊成纤维细胞表面出现Notch1受体的高表达，在本研究中将通过体外细胞和体内动物模型阐明Notch信号对α-SMA基因启动子的调控修饰机制及其对术区瘢痕化的促进作用。本研究有望明确青光眼滤过术后术区瘢痕化的必经通路，为青光眼滤过区瘢痕化的调控提供新的思路和新靶点。

Notch通路在人体多种组织细胞的纤维化病变中发挥关键的调控作用，但尚未明确其是否在抗青光眼滤过术中调控滤过泡术区的瘢痕化。有研究发现人Tenon’s囊成纤维细胞特异性表达α-平滑肌肌动蛋白(α-SMA)进而转化为肌成纤维细胞的过程是滤过区瘢痕化的关键步骤。我们的前期研究表明在青光眼滤过术术区中，Notch通路可通过激活α-SMA蛋白的表达促进人Tenon's囊成纤维细胞向肌成纤维细胞的转化，从而导致术区的瘢痕化。通过开展本项目，我们明确了Notch1受体和配体在Tenon’s囊成纤维细胞表面的表达情况；通过体外细胞实验阐明了阻滞Notch1信号通路对人Tenon’s囊成纤维细胞转化为肌成纤维细胞（细胞内α-SMA蛋白的表达是转化的标记物）的影响；通过构建动物滤过手术模型明确了Notch1信号对术区瘢痕化发生发展的影响。本研究有望明确青光眼滤过术后术区瘢痕化的必经通路,为青光眼滤过区瘢痕化的调控提供新的思路和新靶点。