猪主动脉瓣内皮细胞的分离与鉴定

Isolation and identification of endothelial cells from porcine aortic valve

目的:探讨胶原酶消化法分离猪主动脉瓣内皮细胞的可靠性及可重复性。方法:通过胶原酶消化法从新鲜猪主动脉瓣膜表面分离并培养主动脉瓣内皮细胞,通过免疫荧光染色法和流式细胞仪对获得的瓣膜内皮细胞(VEC)的表型加以鉴定。结果:通过胶原酶消化法可成功分离VEC,光镜下VEC呈铺路石样分布,为典型的内皮细胞形态。免疫荧光染色示VEC的CD31表达为阳性,波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA)表达为阴性,分离获得的细胞中VEC阳性率高,间质细胞污染率较低;流式细胞仪示分离获得的细胞Vimentin表达阴性(3.06%),CD31表达阳性(99.01%),再次验证分离获得的VEC纯度高。结论:胶原酶消化法可从猪主动脉瓣膜分离获得纯度较高的VEC,该方法简单,可重复性好。

Objective:To investigate the reliability and reproducibility of isolating endothelial cells from porcine aortic valve by collagenase digestion.Methods:The aortic valve endothelial cells(VECs)were isolated and cultured from fresh porcine aortic valve surface by collagenase digestion.Immunofluorescence staining and flow cytometry were used to identify the phenotype of VECs.Results:The cells isolated presented typical morphology of endothelial cells,arranging as paving stones under light microscopy.VEC could be successfully isolated by collagenase digestion.Immunofluorescence staining showed that CD31 was positive,while Vimentin andα-SMA were negative in VECs,indicating that positive rate of VECs in cells isolated was high and contamination rate by interstitial cells was low.It was verified by flow cytometry,which showed that Vimentin positive rate was 3.06%and CD31 positive rate was 99.01%in cells isolated.Conclusions:Collagenase digestion can be used to isolate aortic high-pure VECs from porcine aortic valve.This method is simple and reproducible.