成人大隐静脉内皮细胞的培养

doi:国家高技术研究发展计划；国家自然科学基金

成人大隐静脉内皮细胞的培养

崔彬，刘迎龙^*，谢宁，于存涛，陈庆良

中国医学科学院中国协和医科大学阜外心血管病医院心血管病研究所先心病研究室，北京100037

收稿日期:2003-01-23 修回日期:1900-01-01 出版日期:2004-03-28 发布日期:2004-03-28
通讯作者: 刘迎龙

Cultivation of adult human endothelium from great saphenous vein

CUI Bin, LIU Ying-long^*,XIE Ning,YU Cun-tao,CHEN Qing-liang

Department of Congenital Heart Defect, Cardiovascular Instituteand Fuwai Hospital, PUMC & CAMS, Beijing 100037,China

Received:2003-01-23 Revised:1900-01-01 Online:2004-03-28 Published:2004-03-28
Contact: LIU Ying-long

摘要/Abstract

摘要： 目的:探讨培养成人大隐静脉内皮细胞的有效方法。方法: 采用胶原酶Ⅱ(0.2%)消化法收集大隐静脉内皮细胞, 原代培养高密度种植(1.5×10⁴/cm²),传代培养低密度种植(0.8×10⁴/cm²),自体血清(10%~20%)培养，胰酶(0.25%)-EDTA(1 mmol•L^-1)消化,1∶3传代。结果：原代培养细胞，12～24 h贴壁70%~80%，2~3 d开始生长、延伸，（12.0±2.3）d细胞90%汇合,原代培养获取细胞数7.0×10⁴/cm²。传代培养细胞，0.5～2.0 h贴壁80%~90%，3～5 h开始生长，（7.0±1.1）d长满瓶壁。传代细胞较原代细胞易于贴壁，生长旺盛。形态学可见细胞呈扁平状单层排列，梭形或多角形，典型的呈“铺路石”状改变；CD₃₁相关抗原免疫荧光染色标记可见内皮细胞呈特异性黄绿色荧光；超微结构观察可见细胞表面有微绒毛,细胞浆内特异性的长杆状Weibel-Palade（WP）小体。结论：该方法确实、有效，为组织工程内皮细胞的研究和应用提供了新的途径。

关键词: 内皮血管, 细胞培养

Abstract: Objective To investigate the suitable method to cultivate the adult human endothelium from the great saphenous vein. Methods Endothelial cells (EC) from small discarded segments of human great saphenous vein of patients undergoing coronary artery bypass surgery were digested with 2% collagenase Ⅱ in medium 199,seeded into culture flask with high density about (1.5×10⁴/cm²),grew in medium containing the 10%~20% human serum collected from the same patient. After reaching confluence, the EC were detached with a 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA) solution (1 mmol•L^-1),and seeded into culture flask with low density about (0.8×10⁴/cm²),　subcultivated as described for primary cultures　(split ratio 1∶3). Results Primary endothelial cells were confluenced about (12.0±2.3)d and 7.0×10⁴/cm² EC or more could be achieved,the growth of passage cells were more exuberant and confluenced about (7.0 ±1.1) d. The EC retained spindle-shaped or cobblestone appearance,Weibel-Palade (WP) could be found by the transmission electron microscopy, and CD31 antigens were positive in the EC by the immunofluorescent histochemistry. Conclusion The results suggest that it is a useful method for study on cultured endothelial cells and tissue engineering.

Key words: endothelium, cell culture

中图分类号:

Q813.11

崔彬，刘迎龙，谢宁，于存涛，陈庆良. 成人大隐静脉内皮细胞的培养[J]. J4, 2004, 30(2): 221-223.

CUI Bin, LIU Ying-long,XIE Ning,YU Cun-tao,CHEN Qing-liang. Cultivation of adult human endothelium from great saphenous vein[J]. J4, 2004, 30(2): 221-223.

[1]	乔莎, 黄映辉, 王世宝, 孙延霞, 卢振霞. 不同培养方法对乳腺癌MDA-MB-231细胞DNA甲基化状态的影响[J]. 吉林大学学报(医学版), 2016, 42(02): 271-276.
[2]	那键,马超,霍维玲,秦瑞云,许永,王涛,吴晓东,谷贵山. 雌性乳鼠颅骨来源的成骨细胞培养方法的建立及其评价[J]. 吉林大学学报(医学版), 2013, 39(1): 170-173.
[3]	王鹏\|许洁\|李昕\|于顺\|何欣. α-突触核蛋白功能片段对原代培养大鼠神经元突起的促生长作用[J]. J4, 2012, 38(6): 1101-1105.
[4]	赵斌\|申玉芹\|林崇韬\|林泓兵. 小型猪牙周膜细胞原代培养及鉴定[J]. J4, 2011, 37(3): 475-478.
[5]	刘学文, 胥向红, 郎森阳, 蔡爱民. p38MAPK蛋白在体外培养神经元中的表达及W-7的干预作用[J]. J4, 2011, 37(1): 47-50.
[6]	闫嘉晴, 林崇韬, 孙营营. 不同年龄供体人牙周膜细胞原代培养成功率的比较[J]. J4, 2010, 36(1): 95-98.
[7]	董智勇, 李玉林, 徐红, 张丽红. 毛囊来源的表皮干细胞在不同培养条件下细胞生长情况的比较及效果评价[J]. J4, 2009, 35(5): 880-883.
[8]	崔俐, 张巨, 林卫红, 马涤辉, 齐亚丽, 付海龙, 孙强, 常明, 吕晓红. 谷氨酸对体外培养胎鼠神经干细胞的损伤作用[J]. J4, 2009, 35(3): 451-455.
[9]	刘超，苗雷英，孙宏晨，乔春燕，刘金钟，柯小亮. 大鼠颌下腺细胞培养方法建立及生物学特性研究[J]. J4, 2009, 35(2): 218-221.
[10]	张殿君，刘一，付长峰，宿晓云，王芳，李相军. 新生大鼠脊髓源性神经干细胞的分离培养及分化鉴定[J]. J4, 2009, 35(2): 230-232.
[11]	朱勇，陈良万，林若柏，韩子阳，康明强. SD大鼠骨髓间充质干细胞体外培养、表型鉴定及标记[J]. J4, 2009, 35(2): 326-329.
[12]	兰珊珊，赵自然，李玉新，鲍永利，王淼，张舵. 人脂肪干细胞的分离、体外培养及鉴定[J]. J4, 2008, 34(6): 1079-1082.
[13]	赵岩，李才，林风武，田琳，李相军，石艳，苗春生. 尾加压素Ⅱ对体外高糖培养大鼠肾小球系膜细胞增殖的影响及其意义[J]. J4, 2008, 34(6): 931-934.
[14]	何津,王英,张晓霞，李玉林 . 人胚胎生殖腺嵴和肠背系膜来源的成纤维样细胞作为饲养层体外培养人胚胎生殖细胞方法的建立[J]. J4, 2008, 34(5): 894-898.
[15]	贾晓晶，张志良，曲雅勤，翟成伟，杨太权，Wolf-Dieter Rausch,龚守良. 帕金森病体外实验细胞模型的建立[J]. J4, 2008, 34(5): 778-781.