小鼠角膜新生血管内皮细胞的分离培养及其趋化因子受体的表达

Isolation and culture of corneal neovascular endothelial cells and expression of chemokine receptors

背景研究角膜新生血管生成的病理机制对角膜新生血管的治疗具有重要意义，但目前的相关研究多采用非角膜新生血管来源的血管内皮细胞，研究结果的可靠性受到一定影响。目的探索从碱烧伤诱导实验性小鼠角膜新生血管组织中分离和培养血管内皮细胞的方法，并检测新生血管内皮细胞中趋化因子受体的表达，为角膜新生血管内皮细胞的生物学特性研究奠定基础。方法7—8周龄SPF级健康雄性BALB／c小鼠10只，采用NaOH碱烧伤法构建小鼠实验性角膜新生血管模型，采用免疫荧光技术检测CD31在角膜新生血管中的表达。在碱烧伤后2周摘取眼球分离角膜组织，眼科手术剪剪碎角膜组织后应用胶原酶D消化获取单个细胞，使用包被CD31抗体的磁珠分选获取血管内皮细胞。采用免疫组织化学染色法检测CD31在细胞中的表达以鉴定培养的细胞，采用逆转录PCR法检测血管内皮细胞中趋化因子受体基因的表达。结果碱烧伤后7d裂隙灯显微镜下可见小鼠左眼角膜出现新生血管，碱烧伤后2周角膜新生血管的形成达高峰，免疫荧光检测可见角膜组织内特异性CD31染色的血管网。角膜新生血管血管内皮细胞培养后2h贴壁生长，形态呈扁平多边形，体积较大，传代培养后细胞生长速度明显加快。培养的角膜新生血管内皮细胞CD31表达阳性，细胞质中呈棕黄色染色，而角膜基质细胞中CD31表达阴性。逆转录PCR结果显示分离培养的血管内皮细胞中CCR1、CCR2、CCR3和CCR4mRNA相对表达量最高，CXCR3、CCR6、CCR10和CX3CRlmRNA呈中等强度表达，CCR5、CCR8mRNA相对表达量较低，而CCR9、CXCR4和CXCR5mRNA表达未检测到。结论CD31抗体的磁珠分选法可有效分离纯化小鼠角膜新生血管内皮细胞，并可进行体外培养且培养的细胞可表达趋化因子受体。

Background The pathogenesis and mechanism research of corneal neovaseularization is of important significance for the prevention and management of corneal neovascularization. Some relative researches are being performed on non-corneal neovaseularization-derived vascular endothelial cells, so the results are affected to a certain extent. Objective This study was to isolate and culture vascular endothelial cells from experimental corneal neovaseularization tissue and detect the expression of ehemokine receptors in vitro. Methods Corneal neovascnlarization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas,and then the immunofluoreseenee technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling. Corneal pieces were made in 2 weeks after alkali burn and then were digested by eollagenase type D. Vascular endothelial cells were isolated from neovaseularized tissue by affinity purification using magnetic beads coated with anti-CD31. The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry. Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells. The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee. Results Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescenee exhibited the green network-like fluorescence for CD31 antibody in corneas. The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage. The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm, in contrast, the expression of CD31 was absent in corneal stromal cells. Chemokine receptors were positively expressed in the ceils with the strongest expression levels in CCR1, CCR2,CCR3 and CCR4 mRNA and the weakest expression levels in CCR9, CXCR4 and CXCR5 mRNA,while CXCR3, CCR6, CCRIO and CX3CR1 mRNA were expressed with the moderate intensity. Conclusions Vascular endothelial ceils can be obtained from experimental neovaseularized corneas by affinity purification and express ehemokine receptors,which facilitate the study of their biological properties.