MeCP2-421在增殖性玻璃体视网膜病变形成中的作用及其机制

Proliferative vitreoretinopathy (PVR) can cause serious vision loss even lead to blindness. Transforming growth factor-β (TGF-β) -induced retinal pigment epithelium (RPE) epithelial-mesenchymal transition (EMT) plays a key role in the pathogenesis of PVR. However, basic mechanisms of the RPE transdifferentiation from an epithelial cell to a myofibroblast-like cell are still under investigation. Knowledge of the cause and progression of RPE transdifferentiation is needed to support development of new therapeutic strategies based on a mechanistic understanding of the disease. In our preliminary experiment, we found that the expression of phosphorylated methylated CpG-binding protein 2-421 (MeCP2-421) is significantly high in human PVR membrane, TNF stimulates the expression of MeCP2-421 and importantly MeCP2-421 regulates TGF-β/Smad2/3 activation and its downstream gene transcription, suggesting that the expression of MeCP2-421 is closely related to the pathogenesis of PVR. Our hypothesis is that TNF/MeCP2-421/TGF-β pathway is essential in the development of EMT and PVR and inhibition of MeCP2-421 can suppress TGF-β - induced EMT and PVR. In this study, the effect of over expression (reconstructed MeCP2 vector) or knocking down MeCP2 (siRNA) on the EMT will be investigated in cultured RPE cell. In vivo, MeCP2 siRNA and Zebularine will be injected into rabbit vitreous for the inhibition of experimental PVR. Immunohistochemistry, ELISA, real-time PCR and methylation-specific PCR will be used to confirm the hypothesis, the success of the study will provide a therapeutic approach for the treatment of PVR.

增殖性玻璃体视网膜病变（PVR）可严重损害视功能甚至致盲。TGF-β诱导的RPE细胞上皮间质转化（EMT）是PVR形成的关键，但EMT发生的确切机制不清。我们的预实验发现人PVR膜磷酸化甲基化CpG结合蛋白2-421（MeCP2-421）表达显著增高，TNF能刺激人RPE细胞MeCP2-421磷酸化，进而调控TGF-β/Smad通路活化及其下游基因转录，提示PVR的形成与MeCP2-421密切相关。据此提出假设：TNF/MeCP2-421/TGF-β通路是EMT及PVR形成的关键环节，抑制MeCP2-421可抑制TGF-β诱导的EMT及PVR。本研究将体外用MeCP2过表达及基因静默、体内建立兔PVR模型玻璃体腔注射MeCP2 siRNA、Zebularine抑制PVR，用免疫组化、ELISA、real-time PCR、甲基化特异性PCR等技术证实假设，为PVR的药物治疗提供理论依据。

增生性玻璃体视网膜病变（proliferative vitreoretinopathy, PVR）是眼外伤及原发性孔源性视网膜脱离复位手术失败的严重并发症之一，是一种致盲性纤维化性眼病。RPE细胞发生上皮间质转化（epithelial–mesenchymal transition ，EMT），在PVR的发病机制中发挥重要作用。诱导RPE细胞发生EMT的主要因子是TGF-β。MeCP2是系统性疾病EMT及纤维化形成的关键诱导因子。本研究拟进行体外细胞实验及体内动物模型实验，采用免疫组化、免疫荧光双染siRNA敲减MeCP2、Western blot、Real-time PCR、IP、CCK8等方法进行研究。我们发现人PVR膜中P-MeCP2-421高表达， P-MeCP2-421与α-SMA、CK和TGF-β存在双重染色；敲减MeCP2可显著抑制TGF-β诱导的RPE细胞Smad2/3的活化及α-SMA, collagen I和FN的表达（ P<0.05）。TGF-β可抑制RPE细胞PPAR-γ的表达，然而用siRNA敲减MeCP2可使RPE细胞PPAR-γ表达增加。给予重组MeCP2处理可显著诱导RPE细胞α-SMA的表达。染色质免疫沉淀发现MeCP2能与TGF-β发生特异性的结合。Zebularine能够抑制人RPE细胞增殖，具有剂量和时间依赖性。TNF-α早期达到一定剂量可抑制炎症反应负调节因子miRNA-132-3p的表达。MeCP2对TGF-β2表达的上调呈现一定程度的时间和剂量依赖性。MeCP2、MeCP2-421和MeCP2-80在小鼠PVR膜中均呈阳性表达，TGF-β2呈强阳性表达， MeCP2-S421与TGF-β2存在明显共定位表达。因此，MeCP2 尤其是P-MeCP2-421可能在PVR的发病机制中发挥重要作用，MeCP2 可能是治疗PVR新的靶点，早期抗肿瘤坏死因子治疗对延缓PVR的进程具有重要作用，miRNA-132是早期干预纤维化疾病发展的潜在治疗靶标。