河北医科大学学报 ›› 2021, Vol. 42 ›› Issue (1): 7-11.doi: 10.3969/j.issn.1007-3205.2021.01.002

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胶原酶消化刮取再消化法分离培养犬静脉内皮细胞的实验研究

  

  1. 1.江苏大学附属医院胸心外科，江苏 镇江 212001；2.江南大学附属医院胸心外科，江苏 无锡 214062；3.上海交通大学医学院附属第九人民医院胸心外科，上海 200011
  • 出版日期:2021-01-25 发布日期:2021-02-04
  • 作者简介:李刚（1985-），男，安徽马鞍山人，江苏大学附属医院主治医师，医学硕士，从事组织工程小口径血管构建研究。
  • 基金资助:
    江苏省自然科学基金（BK20171149）；上海市卫生和计划生育委员会科研课题（201740129）

Experimental study on isolation and culture of canine vascular endothelial cells by collagenase digestion scrape and repeat digestion techniques

  1. 1.Department of Thoracic and Cardiac Surgery, the Affiliated Hospital of Jiangsu University, Jiangsu 
    Province, Zhenjiang 212001, China; 2.Department of Thoracic and Cardiac Surgery, the Affiliated 
    Hospital of Jiangnan University, Jiangsu Province, Wuxi 214062, China; 3.Department of 
    Thoracic and Cardiac Surgery, Shanghai Ninth People′s Hospital of Shanghai 
    Jiaotong University, School of Medicine, Shanghai 200011,China
  • Online:2021-01-25 Published:2021-02-04

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摘要： 目的  探讨犬大隐静脉内皮细胞（endothelial cells，ECs）体外分离培养的新方法。
方法  通过将内膜翻转后结扎两端，0.1%胶原酶消化后刮下内皮层，用胶原酶再消化法获取内皮细胞，用低糖DMEM培养基再加入适量内皮细胞生长因子培养，以0.25％胰蛋白酶进行消化传代培养。用免疫荧光法、免疫组织化学法及光镜、扫描电镜法鉴定所得细胞系。
结果  本方法分离的细胞在体外培养2周后光镜下胞体呈单层铺路石状排列，免疫荧光和免疫组织化学检测Ve-Cadherin阳性，扫描电镜观察见细胞呈梭形以及细胞表面较多微绒毛。本方法与传统的Veronica Tisato分离法比较，细胞存活率显著提高［（75.39±12.61)% vs （47.28±9.59）%，P＜0.01］。
结论  采用胶原酶消化刮取再消化法能可靠地获取纯化的犬大隐静脉ECs，获取的ECs在体外可以连续大量增殖传代。


关键词: 大隐静脉, 内皮细胞, 胶原酶类, Ve-Cadherin抗原

Abstract: Objective  To explore a novel culture method of vascular endothelial cells(ECs) in vitro from canine saphenous vein. 
Methods  Vascular inner membrane was inverted, and both ends of the vein were ligated, followed by digestion with 0.1% collagenase and mechanical scrape. Endothelial cells in canine saphenous vein were obtained by collagenase redigestion, and then cultured with low glucose DMEM medium. Appropriate amount of endothelial cell growth factor was then added for culture. Cells were cultured using 0.25% trypsin and then subcultured. The cultured cell lines were identified by immunofluorescence, immunohistochemistry, light and scanning electron microscopy. 
Results  The monolayer cells were formed at 14 days after culture in vitro. The ECs arrayed like pitching stone under light microscopy. By transmission electron microscopy, ECs were found spindle shaped and there were many microvilli on the cell surface; Ve-Cadherin in the ECs showed positive reaction by immunofluorescence and immunohistochemistry. Compared with the traditional Veronica Tisato separation method, the cell survival rate using this method was significantly improved ［(75.39±12.61)% vs. (47.28±9.59)%, P＜0.01］. 
Conclusion  Aplication of collagenase digestion, mechanical scrape and collagenase redigestion can reliably obtain purified ECs of canine saphenous vein, which could be proliferated and successively passaged in vitro.

Key words: great saphenous vein, endothelial cells, collagenase, Ve-Cadherin antigen

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