目的 探讨大鼠心脏微血管内皮细胞的原代培养技术,以获取纯化的大鼠心脏微血管内皮细胞.方法 应用体质量80 g的雄性Wistar大鼠左心室的微血管内皮细胞进行原代培养并传代培养；通过倒置显微镜扫描观察其形态,同时对培养细胞采用免疫组织化学荧光染色法进行内皮表面因子(vWF)鉴定,并用Hoechst染色,于荧光倒置显微镜下观察.结果 体外培养的大鼠微血管内皮细胞呈梭形,形成单层后呈典型的鹅卵石样或铺路石样排列.采用Hoechst染色后,可见90％以上的细胞胞浆和核膜周围被染成红色,细胞核呈蓝色荧光；merge 3染色见细胞胞浆和核膜周围被染成黑色,细胞核呈蓝色；cy-3染色见细胞胞浆和核膜周围被激发出红色光.证明培养细胞为血管内皮细胞.结论 通过改进的微血管内皮细胞分离方法培养获得的细胞,生长状态良好、纯度高,且能够稳定地传代培养.%Objective To study the primary culture methods for obtain purified cardiac microvascular endothelial cells in rats. Methods Microvascular endothelial cells of male rat's left ventricle were performed with primary culture and serial subcultivation. The cellular morphous was observed by inverted microscope scanning, vascular hemophilia factor was identified by immunofluorescence, and the result of Hoechst staining was observed by inverted microscope. Results The primary cultured cardiac endothelial cells in vitro were fusiform, and formed a monolayer of typical cobblestone or slabstone array. After Hoechst staining,above 90% of the cells were positive staining, red fluorescence existed in cytoplasm, and blue fluorescence existed in cell nucleus. The results of merge 3 staining showed black in cytoplasm and around caryotheca, and blue in cell nucleus. The result of cy-3 staining showed that the red fluorescence existed in cytoplasm and caryotheca. These results revealed that the cells were vascular endothelial cells. Conclusion The improvement methods can obtain the microvascular endothelial cells with good growth,high purity,and stable serial subcultivation.
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