改良大鼠肺微血管内皮细胞原代培养技术及细胞鉴定

发布时间：2017-12-27 10:13

本文关键词：改良大鼠肺微血管内皮细胞原代培养技术及细胞鉴定　出处：《中华损伤与修复杂志(电子版)》2014年01期 　论文类型：期刊论文

【摘要】：目的改进大鼠肺微血管内皮细胞的原代培养方法,并对所培养的原代细胞进行鉴定。方法应用SD大鼠的外周肺组织,进行大鼠肺微血管内皮细胞的原代培养。从动物选取、取材、肺灌注、植块贴壁、培养基及添加物的选择等方面对肺微血管内皮细胞原代培养技术方法进行改进。从细胞形态学特征,免疫组织化学检测血管内皮细胞表面标志物(CD31抗原和Ⅷ因子相关抗原的表达),荧光显微镜观察异硫氰酸荧光标记植物凝集素与肺微血管内皮细胞特异性结合情况等方面鉴定所培养的肺微血管内皮细胞。流式细胞仪检测细胞纯度,噻唑蓝测量细胞生长曲线。结果原代培养肺微血管内皮细胞成多角形,融合为单层后呈典型的鹅卵石或铺路石样生长,传代培养后可变为梭形呈漩涡样生长或聚集生长。免疫组织化学显示细胞CD31和Ⅷ因子相关抗原的表达阳性;异硫氰酸荧光标记植物凝集素结合试验阳性。细胞生长状态良好、细胞纯度达93.2%。结论改进后的原代培养方法简便、可靠,获得的肺微血管内皮细胞污染少、纯度高,状态良好,保持了内皮细胞的结构和特性。
[Abstract]:Objective to improve the primary culture of rat pulmonary microvascular endothelial cells and to identify the cultured primary cells. Methods the peripheral lung tissue of SD rats was used to carry out primary culture of rat lung microvascular endothelial cells. The primary culture technology of pulmonary microvascular endothelial cells was improved from animal selection, sampling, perfusion, explant, culture medium and additives. From the cell morphological features, immunohistochemical detection of vascular endothelial cell surface markers (expression of CD31 antigen and factor VIII related antigen), fluorescein isothiocyanate labeled lectin and pulmonary microvascular endothelial cell specific binding and identification of cultured pulmonary microvascular endothelial cells were observed by fluorescence microscope. The cell purity was detected by flow cytometry, and the growth curve of the cell was measured by thiazolium. Results the primary culture of pulmonary microvascular endothelial cells was polygonal, and was monolayer, which was typical cobblestone or paving stone growth. After culture, it could be changed into spindle like growth or aggregation growth. Immunohistochemistry showed that the expression of CD31 and von Willebrand factor antigen; FITC labeled lectin binding test positive. The cell growth was good and the purity of the cells was 93.2%. Conclusion the improved primary culture method is simple and reliable, and the obtained pulmonary microvascular endothelial cells have less pollution, high purity and good state, which maintain the structure and characteristics of endothelial cells.
【作者单位】：中山大学附属第一医院重症医学科SICU;
【基金】：国家自然科学基金(81071536),国家自然科学基金青年基金(81201452) 广东省教育厅育苗基金(2012LYM_0006)
【分类号】：R329.2
【正文快照】： 血管内皮细胞不仅具有屏障功能,同时还具有调节血管张力,调节抗凝、凝血和纤溶,表达黏附分子,参与血管活性物质的代谢等多种功能,被认为是一个“代谢和内分泌器官”[1]。肺的血管内皮细胞尤其是肺微血管内皮细胞(pulmonary microvascularendothelial cells,PMVECs)广泛涉及多

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