改良的心肌微血管内皮细胞分离培养方法

目的:建立一种细胞获得率高并且操作简单的心肌微血管内皮细胞体外培养体系。方法:无菌条件下分离左心室,0.1%胶原酶消化6min后,加0.1%胰蛋白酶继续消化4min,用100μm滤网过滤消化液,离心收集滤液中血管段进行培养。用内皮细胞的重要标记物VIII因子相关抗原鉴定细胞,并通过3H-TdR掺入法观察培养细胞对碱性成纤维细胞生长因子(bFGF)的反应性。结果:细胞的获得率为1.2×106细胞/g心肌组织,显微镜观察培养细胞具有内皮细胞生长特性:单层“卵石样”排列特征;VIII因子免疫细胞化学染色呈阳性;证实获得的是内皮细胞。bFGF刺激后3H-TdR掺入率与对照组比较差异显著(P<0.05)。结论:本研究利用改良的酶消化法,缩短了消化时间,提高了细胞获得率,同时培养的内皮细胞对生长因子具有良好反应性,表明该方法是一种比较理想的体外心肌微血管内皮细胞培养体系。

An Improved Method for Isolation of Microvascular Endothelial Cells from Myocardial Tissues

Objective: To establish a convenient in vitro culture model for obtaining more cardiac microvascular endothelial cells (CMEC).Method: Left ventricles were isolated under the sterile condition.The tissue was then digested for 6 min in 0.1 collagenase,followed by 0.1 trypsin for another 4 min.The digested solution was filtered through 100 μm nylon mesh.Microvessles from the filtrate were collected by centrifugation,and cultured in M199 medium.CMEC were identified using an important marker,factor VIII antigen.In addition,the responsiveness of endothelial cells to basic fibroblast growth factor (bFGF) was observed by measuring ~ 3 H thymidine incorporation.Results: The final field of CMEC was 1.2×10~ 6 cells g tissue).The cells grew out as typical “cobblestone” under an inverted microscope.The cells were positive for factor VIII antigen.Additionally,~ 3 H thymidine incorporation of the cells treated by bFGF was significantly elevated,compared with control group(P<0.05).Conclusion: Using the improved enzyme digestion method,there was an increase in the final yield of cells and a reduction in the digestion time.These results indicated that this method was a better isolation and culture system of CMEC.