课题背景：课题由国家高技术研究发展计划（“八六三”计划）（2006AA03Z0448）资助。冠心病、外周动脉闭塞性疾病等较多需要小口径血管（直径< 6 mm）行动脉移植手术。目前临床上已用的小口径血管移植物包括自体大隐静脉或乳内动脉、涤纶和膨体聚四氟乙烯人工血管等各有缺陷。因此，人们迫切需要研制新的小口径动脉替代物。

临床应用性：实验采用原位插管、0.1%胶原酶灌注消化法获取犬浅静脉内皮细胞，经形态学、第Ⅷ因子相关抗原免疫组织化学染色法和透射电镜检测对获得细胞进行鉴定证实为内皮细胞来源，有效提高了血管内皮细胞培养成功率。

同行评价：课题选题较好，结合临床需要，对自体血管内皮细胞培养技术加以改进，为进一步的深化研究打下了基础，对组织工程血管进一步基础和临床应用研究有重大意义。

摘要
背景：自体血管内皮细胞数量有限且体外长期培养传代后存在功能老化，是目前制约组织工程血管发展的问题。
目的：实验拟验证通过体外分离和大规模培养方式为组织工程动脉支架体外内皮化提供血管内皮细胞的可行性。
设计、时间及地点：重复测量设计，实验于2007-03/12在复旦大学附属中山医院中心实验室完成。
材料：健康杂种犬6只，雌雄不拘，体质量35~40 kg，用于获取血管内皮细胞。
方法：将犬麻醉后前肢隐静脉原位插管，采用Ⅰ型胶原酶消化法获取血管内皮细胞，体外培养、传代并大规模扩增。采用微格法每日计数培养的细胞总数。
主要观察指标：倒置显微镜观察细胞生长情况，透射电镜和细胞免疫化学进行细胞鉴定，检测原代和传代细胞的条件培养液中6-酮-前列腺素F1α和Ⅷ因子相关抗原的含量。
结果：倒置显微镜下观察静脉内皮细胞呈典型的“纺锤样”梭形细胞，单层细胞贴壁生长至融合时呈铺路石样排列。透射电镜下见内皮细胞胞浆中特征性Weibel-Palade小体。细胞免疫化学显示血管内皮细胞Ⅷ因子相关抗原抗体染色阳性。原代及传代细胞培养上清液中6-酮-前列腺素F1α和Ⅷ因子相关抗原的含量差异无显著性意义（P > 0.05）。
结论：实验成功培养、传代并大规模扩增静脉血管内皮细胞。所培养的静脉内皮细胞可以为组织工程动脉支架体外内皮化提供足够数量和功能良好的细胞。
关键词：组织工程；犬；内皮细胞；体外培养；原位获取

施德兵，符伟国，何红兵，刘震杰，张祥满，史振宇.实验犬浅静脉内皮细胞原位获取及体外培养[J].中国组织工程研究与临床康复，2008，12(20):3831-3836 [www.zglckf.com/zglckf/ejournal/upfiles/08-20/20k-3831(ps).pdf]

In situ harvest and in vitro culture of endothelial cells derived from adult canine superficial vein

Abstract

BACKGROUND：Current issues in vascular tissue engineering included limited sources and aging of autuolgous vascular endothelial cells after long-term subculture in vitro.
OBJECTIVE：To investigate the feasibility of providing vascular endothelial cells for the confluent in vitro lining of the scaffolds of tissue-engineered artery by in vitro isolation and large-scale culture.
DESIGN, TIME AND SETTING: The repeated measurement design experiment was performed at the Central Laboratory of Zhongshan Hospital Affiliated to Fudan University from March to December 2007.
MATERIALS: Six healthy hybrid canines of both genders and weighing 35-40 kg were selected to isolate canine vascular endothelial cells.
METHODS：Enzymatic endothelial cell detachment was achieved by the in situ cannulation with type Ⅰ collagenase to short saphenous vein segments of experimental canine after anesthesia. The vascular endothelial cells were cultured in vitro and mass cultured. A microgrid technique was used to enable the daily in situ quantification of available endothelial cells.
MAIN OUTCOME MEASURES: Vascular endothelial cells were identified by the morphologic characteristics observed under an inverted microscope, a transmission electron microscope and by immunohistochemical staining. The both 6-keto-PGF1α and factor Ⅷ contents in the supernatant of primary and subcultured passages were analyzed.
RESULTS: Vascular endothelial cells presented typical “spindle-shaped”, elongated, polygonal or round appearance and formed monolayer that arrayed in “cobblestone-like” morphology under the inverted microscope. Endothelial cell Weibel-Palade (“W-P”) body was sometimes observed under the transmission electron microscope. Immunohistochemical analysis indicated that these cells were positively stained for factor Ⅷ. The 6-keto-PGF1αand factor Ⅷ contents in the supernatant of primary and subcultured passages did not have significant difference (P > 0.05).
CONCLUSION：A mass of endothelial cells we cultured may be used as a source for providing well functional and a sufficient number of cells for the endothelialization of the scaffolds of tissue-engineered artery.

Shi DB, Fu WG, He HB, Liu ZJ, Zhang XM, Shi ZY. In situ harvest and in vitro culture of endothelial cells derived from adult canine superficial vein.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(20):3831-3836(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-20/20k-3831(ps).pdf]