免疫磁珠法原代分离培养大鼠视网膜微血管周细胞

Primary isolation of rat retinal microvascular pericytes with immuomagnetic beads

目的建立免疫磁珠法原代分离纯化大鼠视网膜微血管周细胞,并确定其免疫组化特征.方法免疫磁珠法结合传统的胶原酶消化及筛网过滤法分离出大鼠视网膜微血管周细胞,采用含有20%胎牛血清的DMEM培养.通过周细胞的形态和生长方式初步鉴别,再进行细胞α-SMA,vWF,GFAP抗体免疫组织化学特征鉴定,及激光共聚焦显微镜行周细胞PDGFR-β和desmin抗体荧光双标观察.周细胞生长曲线通过MTT法测定.结果本法获得的周细胞纯度可达98%,并能连续传代.原代周细胞约2～3周融合,传代后生长和融合速度加快,细胞形态不规则,胞内可见丝状结构,无接触性抑制.免疫组化证实周细胞胞浆内α-SMA蛋白表达阳性,内皮细胞特异性vWF因子表达阴性,胶质细胞特异性GFAP表达阴性.激光共聚焦显示周细胞PDGFR-β和desmin抗原表达均为阳性.结论本研究首次报道应用免疫磁珠法分离培养大鼠视网膜微血管周细胞.获得高度纯化的周细胞具有明确的免疫组化特征,可用于相关视网膜血管性疾病的进一步研究.

Objective To establish immuomagnetic beads methods for primary isolating and culturing rat retinal pericytes and to identify their immuocytochemical characteristics. Methods Using immuomagnetic beads sticking combined with traditional collagenase digestion and filtration, pericytes were isolated and then cultured on non-coated dishes in Dulbecco＇ s modified Eagle＇ s medium （DMEM） containing 20% fetal bovine serum. Pericytes were identified from morphology and patterning of growth initially. The cells were further identified by α-SMA, vWF, GFAP immunocytochemical staining and by PDGFR-β with desmin double antibody labeled fluorescence staining using laser scanning cofocal microscope （LSCM）. The growth curve of pericytes was determined by MTT assay. Results With these methods, pericytes were obtained with high purity of 98% and could be passaged serially. Primary pericytes formed a confluent monolayer after 2 to 3 weeks and passaged pericytes grew accelerately. The cells presented irregular shapes with thick filament inside and overlapping growth pattern without contact inhibition. These cells were positive stained for α-SMA and negative for both endothelial cells specific vWF factor and glial cells specific GFAP. LSCM revealed expressions of PDGFR-β and desmin in the cells. Conclusion This study originally described isolation and culture of rat retinal pericyte with immuomagnetic beads method. Highly purified pericytes with clear immuocytochemical characteristics can be obtained and used for further study of related retinal vascular diseases.