课题背景：课题为上海市科委基金资助重点项目(04JC14012)。外周血中的内皮前体细胞经过简单的静脉穿刺即可获得，在体外可以迅速分化为成熟的内皮细胞，增殖能力旺盛，且可有效减少血小板的黏附和血栓形成。人们发现内皮前体细胞虽然有多种优势，但单纯内皮前体细胞构成血管材料也存在问题：如应用至体内生理功能低下；血管材料缺乏弹性；在流体力学环境下，黏附能力下降等。本课题将内皮前体细胞与平滑肌共同培养，为克服这些不足作相应探索。

应用要点：①实验证实了内皮前体细胞在Ⅰ型胶原凝胶上与平滑肌细胞一样，具有很强的增殖能力，同时在平滑肌细胞帮助下可形成血管样结构。②并进一步证明了两种种子细胞共同培养的适宜比例。

相关链接：组织工程血管是利用血管壁的正常细胞和生物可降解材料来制备、重建和再生血管替代材料。多年前，就有研究者尝试用成熟内皮细胞和平滑肌细胞这两种血管壁的主要组成细胞联合培养构建组织工程血管。近年来，随着干细胞生物学的发展，开始有学者尝试将成体干细胞，如内皮前体细胞和间充质干细胞等作为种子细胞应用于组织工程血管构建研究。

摘要
目的：

由内皮前体细胞分化的内皮细胞与成熟平滑肌细胞联合培养可以为组织工程血管的形成提供更接近天然的条件。实验拟进一步证明两种细胞联合培养可促进血管内皮细胞生长，以及两种细胞的最适比例。
方法：实验于2006-01/2007-05在复旦大学上海医学院分子生物学实验室完成。①实验材料：新鲜脐带取自国际第一妇婴保健医院，产妇知情同意。②实验方法及评估：从人脐动脉中用组织块培养法分离并原代培养血管平滑肌细胞，免疫荧光方法鉴定其平滑肌肌动蛋白表达情况；采用密度梯度离心法分离脐血中的内皮前体细胞，经体外定向诱导分化和免疫荧光方法鉴定其表型；无菌条件下制作Ⅰ型胶原凝胶，在其上以3∶1~4∶1的比例混匀联合培养内皮前体细胞与平滑肌细胞，并观察两种细胞联合培养时的形态，分析两种细胞最合适的种植比例；免疫荧光方法观察在Ⅰ型胶原凝胶联合培养中CD31、vWF的表达以及内皮细胞的生长情况。
结果：①原代培养的平滑肌细胞呈典型“波峰谷”形态，荧光染色平滑肌肌动蛋白呈阳性。②脐血来源的内皮前体细胞定向诱导分化为内皮细胞后，呈现出“铺路石”样形态，表达CD31、vWF，结合荆豆凝集素，提示具备成熟内皮细胞特性。③在Ⅰ型胶原凝胶上两种细胞增殖力均旺盛，与平滑肌细胞以3∶1~4∶1的比例种植在Ⅰ型胶原凝胶培养一段时间后，内皮前体细胞可从平滑肌细胞处得到支持，形成血管样网络结构。④共培养1周后，CD31与vWF阳性细胞即内皮前体细胞分化而来的内皮细胞互相连接，形成环形结构。
结论：内皮前体细胞和平滑肌细胞以3∶1~4∶1共培养的模式可以促进微血管样结构的形成。
关键词：内皮前体细胞；平滑肌细胞；联合培养；Ⅰ型胶原凝胶；血管组织工程

谢尚喆，刘水，方宁涛，高红阳，王松梅，潘銮凤．内皮前体细胞与平滑肌细胞联合培养构建组织工程血管：最适比例验证[J].中国组织工程研究与临床康复，2008，12(20):3826-3830 [www.zglckf.com/zglckf/ejournal/upfiles/08-20/20k-3826(ps).pdf]

Co-culture of endothelial progenitor cells and smooth muscle cells to construct tissue-engineered blood vessel: Optimum culture ratio

Abstract

AIM：Co-culture of endothelial precursor cells (EPCs)-differentiated endothelial cells and smooth muscle cells (SMCs) makes the natural tissue-engineered blood vessels possible. This study explored the promoting effect of the co-culture of endothelial cells and SMCs on vascular endothelial cells (VECs) and investigated the optimal ratio of two kinds of cells.
METHODS: The experiment was performed at the Laboratory of Molecular Biology, Shanghai Medical College of Fudan University between January 2006 and May 2007. Fresh umbilical core was provided by Shanghai First Women and Children Health Care Hospital with the informed consent from the puerperant. Human SMCs were separated from the arteries of umbilical cords. The smooth muscle phenotype was confirmed by indirect α-actin immunofluorescent staining. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, and induced to differentiate into endothelial cells, followed by assessing their phenotype by immunofluorescent staining. Type Ⅰ collagen gel was fabricated aseptically, on which EPCs and SMCs were co-cultured at different ratios (3∶1 to 4∶1). The morphology of co-cultured cells was examined. Immunofluorescent staining was applied to observe CD31 and vWF expressions and endothelial cells growth on type Ⅰ collagen gel.
RESULTS: Primarily cultured SMCs displayed specific hill and valley morphology and expressed SM α-actin confirmed by immunofluorescent staining. EPC-derived endothelial cells showed a typical cobblestone morphology, expressed vWF and CD31, and bind human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1. This suggested cell population became endothelial in nature. After seeded on type Ⅰ collagen gel, the cells grew well. At ratio of 3∶1 to 4 ∶ 1, EPCs with SMCs formed blood vessel-like network structure. vWF and CD31 immunofluorescent staining showed EPCs formed micro-vascular network only one week after co-cultured with SMCs on type Ⅰ collagen gel.
CONCLUSION: EPCs and SMCs co-culture at ratios of 3∶1 to 4 ∶1 can facilitate the formation of blood vessel-like network.

Xie SZ, Liu S, Fang NT, Gao HY, Wang SM, Pan LF. Co-culture of endothelial progenitor cells and smooth muscle cells to construct tissue-engineered blood vessel: Optimum culture ratio.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(20):3826-3830(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-20/20k-3826(ps).pdf]