三维三细胞支架培养模型中miR-126抑制糖尿病血视网膜内屏障损伤的机制研究

The inner blood retinal barrier (iBRB) plays an important role in maintaining retinal health. Chronic hyperglycemia and hypoxia can lead to the injury of iBRB and this injury is a critical pathological step for diabetic retinopathy (DR). The recent emerging three dimensional bio-scaffold cell co-culture technique can simulate the microenvironment in vivo and also has the features of the in vitro experiments which are simple and controllable. The previous co-culture system usually constructed on single or double cells. In this study, we have initially established a fluorescent probe-labelled 'Endothelial-Pericyte-Müller Cells' three dimensional tri-culture model with microenvironment reconstruction. In this project, using two-photon microscopy, protein profile array and other molecular biological methods, we aim to evaluate the advantage of this novol three dimensional tri-culture system compared with the traditional transwell model, including cell biology, barrier function and the tolerance under a high glucose/ hypoxia status mimicking diabetic condition. Based on the previous study of miR-126 in DR, we will also verify the suppression of miR-126 on DR with three dimensional tri-culture bio-scaffold model. This project would establish a novel three dimensional tri-culture model simulating the iBRB under physiological and diabetic pathological microenvironment, provide a scientific and reliable in vitro research platform for iBRB-related diseases, and also elucidate the potential role and value of miR-126 in clinical application.

血视网膜内屏障（iBRB）结构功能的完整是维护视网膜机能的首要防线，慢性高糖缺氧导致iBRB损伤是糖尿病视网膜病变（DR）的重要病理基础。在前期研究中，我们突破了以往的单/双细胞三维培养体系，初步构建“内皮-周-Müller细胞”三维三细胞支架培养模型模拟iBRB。本课题拟通过双光子显微镜活细胞实时追踪、芯片分析、荧光激活细胞分选等方法，开展两方面研究：①三维三细胞支架培养模型相对传统小室模型的优势评估，包括细胞生物学特性、屏障结构功能及对高糖缺氧状态的耐受差异；②针对我们前期发现miR-126在DR中的重要作用，利用三维三细胞支架培养模型开展miR-126抑制DR的分子机制研究。旨在提供一个科学可靠的三维支架培养平台，并阐明miR-126在DR发生发展过程中的作用及潜在的临床应用前景。

血视网膜内屏障（iBRB）结构功能的完整是维护视网膜机能的首要防线，慢性高糖缺氧导致iBRB损伤是糖尿病视网膜病变（DR）的重要病理基础。我们突破了以往的单/双细胞三维培养体系，应用多种三维细胞培养材料包括低浓度琼脂糖、高浓度琼脂糖、胶原、纤维素等材料进行三维培养细胞，反复进行各种三维培养材料的最适培养条件的摸索，同时施行多种细胞的混合培养，多种细胞可以在三维材料中生长成团网状，建立“内皮-周-Müller细胞”三细胞三维支架培养模型模拟iBRB。通过高糖或缺氧诱导后，发现三维三细胞共培养模型中屏障功能损伤，iBRB模型中,发现miR-126信号分子下调，CD31等细胞标记蛋白和ZO-1等紧密连接相关蛋白分子表达减少，应用血管光学断层扫描技术发现iBRB屏障结构损伤。本项目通过构建“内皮-周-Müller细胞”三维三细胞共培养的iBRB模型，可模拟体内iBRB及多细胞所处的屏障微环境。该模型的建立为iBRB相关性疾病的药理和病理研究提供简便、可控、科学的体外研究平台。