血-视网膜内屏障相关细胞系的建立:视网膜血管性疾病研究和药物筛选的新工具

Impairment of inner blood-retinal barrier (iBRB) and neovascularization is the major cause that lead to severe vision loss and even blindness for patients with retinal vascular diseases. Retinal microvascular endothelial cells (RMEC), retinal microvascular pericytes (RMP) and Müller cells compose iBRB and involve in retinal abnormal new vessel formation. Purification，culture and co-culture model of the above cells are the fundament of in-vitro and drug screening study for retina vascular diseases. However, isolation and primary culture of RMEC and RMP are very difficult, and no immortalized cell lines can be obtained inland at present. In the study, we plan to purify RMEC and RMP with immunomagnetic beads, and inhibit p53 expression by lentivirus-mediated RNA interference (RNAi), to make cell bypass M1 phase (senescence phase), then introduce human telomerase reverse transcriptase (hTERT) cDNA into these cells to lead to cells bypass M2 phase (crisis phase) by maintaining telomerase activity, so to get immortalized cell lines. Different pattern of co-culture of these immortalized cells will be used to set up in-vitro models of iBRB and retinal neovasularization. These immortalized cell lines will provide a very useful platform for in-vitro and drug-screening study, and guide the clinical treatment for retinal vascualr diseases.

内层血-视网膜屏障（iBRB）损害和新生血管是视网膜血管性疾病患者失明的主要原因。视网膜微血管内皮细胞（RMEC）、周细胞（RMP）和Müller细胞构成iBRB，并参与血管新生。对iBRB和新生血管的体外研究及药物筛选基于这些细胞的纯化培养和体外模型，但RMEC和RMP的分离培养十分困难，目前我国还没有这三种细胞的永生化细胞系。本研究中我们拟通过免疫磁珠法纯化培养人RMEC和RMP，然后采用慢病毒介导的RNA干扰技术沉默细胞内p53基因表达，使细胞绕过M1期（衰老期）后，再以脂质体转染法将外源性人端粒酶逆转录酶(hTERT) 基因导入细胞，通过保持细胞端粒酶活性使细胞进一步绕过M2期（危机期），从而建立以上三种细胞的永生化细胞系。并利用这些细胞系在体外建立iBRB和新生血管模型。这些细胞系的建立将为视网膜血管性疾病的体外研究和药物筛选提供实用的操作平台，具有很重要的实际工作意义。

因获得小额资助，时间仅有一年，我们根据时间对研究目标和内容进行了调整。目前完成的内容有：① 构建了含针对人p53 基因有效的vshRNA 慢病毒重组体，并从外源和内源进行RNAi 有效靶点的筛选，将含有最有效靶点的慢病毒重组体命名为p53-RNAi-LV。② 完成了人视网膜Müller 细胞的分离培养； ③ 将构建好的慢病毒重组体p53-RNAi-LV感染人视网膜Müller 细胞，嘌呤霉素筛选并传代，获得抗性细胞，目前细胞正在进一步传代和纯化中。④应用免疫磁珠法分离培养人视网膜微血管内皮细胞和人视网膜微血管周细胞，但是纯化培养的结果不太理想，成功率较低，因目前角膜移植的供体眼球较少，短时间内无法进行及时重复实验来摸索和更正实验条件，待有供体眼球后，将继续重复该部分实验。