大鼠肺微血管内皮细胞原代培养方法的改进

Improving Primary Culture of Pulmonary Microvascular Endothelial Cells of Rats

中文关键词:  肺微血管内皮细胞 原代培养 鉴定

英文关键词:Pulmonary microvascular endothelial cells Primary culture Identification

中文摘要:

      目的 改进SD大鼠肺微血管内皮细胞(PMVECs)原代培养方法，以获得纯化的大鼠PMVECs。方法 对组织块法培养PMVECs过程中的组织取材、植块贴壁、培养基配制、传代培养进行改进包括：在麻醉前提前注射肝素钠；利用腹腔放血，待大鼠停止呼吸后剪开胸腔；改进操作以及试剂中加入双抗以降低污染率；原代培养48 h即取出组织块;进行再次消化去除成纤维细胞；异硫氰酸标记的植物凝集素（FITC-BSI）鉴定培养的PMVECs。培养后在倒置显微镜下观察细胞形态。结果 原代培养的细胞呈现为多角形或短梭形，呈单层铺路石样排列的典型结构。随着细胞传代或培养条件的改变，细胞形态发生变化，可呈长梭形，漩涡状排列。FITC-BSI结合实验呈绿色荧光，阳性率达到90%以上。结论 通过改进分离及培养方法可获得生长状态良好、纯度高且能够稳定传代培养的PMVECs。

英文摘要:

      Objective To improve the culturing method of pulmonary microvascular endothelial cells (PMEVCs) of SD rats.Methods The culturing processes in regard to obtaining peripheral lung tissue, attaching tissue block,preparing medium and subculturing were modified.These included an injection of heparin sodium before anesthesia, abdominal bleeding, opening of chest when breathing stopped, improvement of operational details, reduction of pollution by adding penicillin and streptomycin, discard of tissues after 48 h of primary culturing, remove of fibroblasts by a second digestion, and identification of cells using a fluorescence microscope for binding with lectin from BSI (FITC-BSI).An inverted microscope was used to observe the morphological characteristics of PMEVCs. Results Purified PMEVCs were obtained,which displayed a polygon or short fusiform, exhibiting a typical cobblestone-like morphology. The morphology of PMVECs turned into swirling or long fusiform following subculture or changes in culture conditions. The results of FITC-BSI assay showed that more than 90% cells were stained with green fluorescence．Conclusion Purified PMEVCs with a good growth state and subculture stability can be obtained using the modified method.