增生性玻璃体视网膜病变中GSK-3β对视网膜色素上皮细胞胶原合成的作用及机制研究

In previous work, we found that the expression of Glycogen synthase kinase 3β (GSK-3β) in human retinal pigment epithelium (RPE) in proliferative vitreoretinopathy (PVR) model was significantly down-regulated. The sugar metabolism in RPE was changed,with more collagen produced, compared to the controls. All those results indicate that GSK-3β may involve in collagen production by RPE. There has been reported that RPE and its produced collagen is the major factor in PVR process. And GSK-3β was the key regulatory factor of Wnt signalling pathway, which may affect collagen production, but the mechanism remains unclear. To investigate the effect and mechanism of GSK-3β on collagen production in PVR, first we will evaluate the effect of GSK-3β on metabolism and collagen production in RPE, by siRNA and gene overexpression. Then we will discover the mechanism of GSK-3β regulation on Wnt signalling pathway in RPE, by transcriptional detecting technology. After that, we will clarify the molecular mechanism of Wnt signalling pathway on coallgen production in PVR,by bi-directional regulation. And finally, we will use animal model to verify our hypothesis. This study will contribute to further understanding of collagen production by RPE in PVR. And it will provide a new target, as well as the theoretical basis, for PVR clinical intervention.

在前期工作中，课题组发现增生性玻璃体视网膜病变（PVR）模型中人视网膜色素上皮细胞（RPE）的糖原合成酶激酶3β（GSK-3β）的表达下调，RPE的糖代谢改变，胶原合成增加，提示GSK-3β可能与PVR中RPE的胶原合成有关。研究发现RPE及其合成的胶原是PVR发展的重要因素；GSK-3β是Wnt信号通路的关键调控因子；Wnt通路可能影响胶原合成，但具体机制不清。为明确PVR中GSK-3β对RPE胶原合成的作用和机制，本课题拟首先用RNA干扰和基因过表达技术，明确GSK-3β的表达改变对RPE代谢和胶原合成的作用；其次用转录检测等技术揭示在RPE中GSK-3β对Wnt通路的调控机制；然后通过拮抗剂和激动剂双向调节Wnt通路及下游的关键信号分子，阐明其影响RPE胶原合成的机制；最后使用动物模型加以验证。本课题的完成将有助于深入理解PVR中RPE胶原合成的机制，也为临床干预PVR提供了新靶点。

增生性玻璃体视网膜病变（proliferative vitreoretinopathy, PVR）是视网膜脱离发生、发展过程中的常见并发症，也是手术失败的主要原因。视网膜色素上皮细胞（retinal pigment epithelium, RPE）及其合成的胶原在PVR的发展中发挥着重要的作用，但胶原的具体合成机制及其影响因素尚未明确。课题组在前期实验中，在PVR体外模型中通过分子生物学实验发现人RPE的糖原合成酶激酶3β（glycogen synthase kinase 3β, GSK-3β）的表达明显下调；使用代谢组学检测和分析后发现人RPE的细胞代谢发生明显变化（主要为糖代谢，包括糖酵解和糖异生减弱，三羧酸循环和糖原合成加强）；通过光学二次谐波（second harmonic-generation, SHG）显微镜发现人RPE的胶原合成增加，其三维结构也发生改变。.在上述研究基础上，本项目进一步研究发现：经过PI3K/AKT抑制剂wortmannin的预处理有效抑制了TGF-β1诱导的α-平滑肌肌动蛋白和波形蛋白的表达，而闭锁小带1和紧密连接蛋白的表达明显上升，上述结果显示PI3K/AKT信号通路参与了TGF-β1诱导ARPE-19细胞EMT的过程。通过划痕实验发现，Wortmannin预处理后，TGF-β1(-)组未发生明显变化，而TGF-β1(+)组迁移能力与未经Wortmannin处理组相比有明显降低趋势(P<0.05); GSK-3β抑制剂LiCl预处理后，与对照组相比，明显增强了TGF-β1(+)诱导的细胞迁移能力(P<0.05)。而TGF-β1(+)组中wortmannin预处理组胶原收缩程度与未经wortmannin处理组相比有明显降低趋势(P<0.01); GSK-3β抑制剂LiCl预处理后，与对照组相比，明显增强了TGF-β1(+)诱导的细胞胶原收缩(P<0.05)。这些结果说明Wnt/β-catenin和PI3K/Akt信号通路均参与到PVR进展过程。上调GSK-3β可以抑制PVR的进展。GSK-3β通过Wnt/β-catenin和PI3K/Akt信号通路抑制实验性增殖性玻璃体视网膜病变。通过本项目的研究确立了GSK-3β是PVR的有效靶点。