课题背景：国家自然科学基金及江西省自然科学基金资助项目。课题名称分别为“骨髓基质细胞促进脑缺血后内源性神经干细胞增殖分化的调控机制”与“骨髓基质细胞促进中风后内源性神经干细胞增殖分化机制”。现阶段研究成果表明，骨髓干细胞能促进神经干细胞的增殖及向神经元的分化，骨髓干细胞移植能促进缺血性脑血管疾病缺血区血管新生,进而促进神经功能的恢复。

相关链接：内皮祖细胞是一种能定向分化为成熟内皮细胞的成体干细胞,具有很高的增殖潜能。内皮祖细胞不仅参与人胚胎血管生成,同时也参与出生后血管新生和机体损伤后的修复过程。因此, 内皮祖细胞为组织缺血性疾病提供了一条新的并且极有临床应用潜力的治疗途径，在血管组织工程、冠状动脉粥样硬化性心脏病、缺血性脑血管疾病、肿瘤、创伤愈合等过程具有广阔的临床应用前景。临床上通过内皮祖细胞移植治疗缺血性疾病已经获得成功,虽然有学者对此提出了"移植是轻率的、冒险的"疑问,但内皮祖细胞的研究正越来越多地受到人们的关注。

同行评价：间充质干细胞来源于发育早期中胚层的多能干细胞，具有高度自我更新能力和多向分化潜能，广泛存在于全身多种组织中，可在体外培养扩增，并能在特定条件下分化为神经细胞、成骨细胞、软骨细胞、肌肉细胞、脂肪细胞等，在细胞替代治疗及组织工程方面有极大的应用价值。

摘要
目的：目前内皮祖细胞主要从骨髓、脐带血和外周血获取，但其分离培养方法尚不成熟，外周血来源因损伤小、易获取而被普遍使用，但得到的内皮祖细胞纯度较低。实验拟探讨大鼠骨髓来源内皮祖细胞体外培养、诱导扩增和生物学特性鉴定方法。
方法：实验于2006-08/2007-08 在南昌大学第一附属医院泌外研究所完成。①实验材料：2周龄SD大鼠由南昌大学医学院动科部提供，雌雄不拘,清洁级，实验过程中对动物处置符合动物伦理学标准。②实验方法：应用梯度离心法采集大鼠骨髓单个核细胞，贴壁筛选法分离内皮祖细胞,置于添加了血管内皮细胞生长因子、人碱性成纤维细胞生长因子、白血病抑制因子、表皮生长因子的M199培养液中扩增培养,对培养2周的细胞采用免疫荧光染色鉴定细胞标志物血管性血友病因子、血管内皮生长因子受体-2、CD34、CD133， 采用RT-PCR法检测内皮细胞特异性分子标志血管性血友病因子、FLK-1、Tie-2、CD34、CD133、eNOS基因表达。
结果：①培养4 d,光电显微镜下可见细胞集落形成,梭形贴壁细胞从集落中央以放射状向外周生长。培养7~10 d,细胞集落相互连接,呈典型的"鹅卵石"样外观。2周左右可见细胞排列成条索状结构并可呈"微血管样"排列生长。②贴壁细胞免疫荧光染色鉴定细胞标志物血管性血友病因子、FLK-1、CD34、CD133阳性，RT-PCR法检测内皮细胞特异性分子标志血管性血友病因子、FLK-1、Tie-2、CD34、CD133、eNOS基因阳性表达。
结论：采用梯度离心法从骨髓中分离单个核细胞，经诱导培养可实现内皮祖细胞的分离培养和体外扩增。
关键词：内皮祖细胞；骨髓源性；体外培养

邓志锋,鲁友明, 汪泱,娄远蕾, 郭菲, 谢安.大鼠骨髓源性血管内皮祖细胞的体外培养与鉴定[J].中国组织工程研究与临床康复，2008，12(16):3069-3073 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3069(ps).pdf]

In vitro culture and identification of endothelial progenitor cells from rat bone marrow

Abstract

AIM：Current isolation and culture methods of endothelial progenitor cells (EPCs) from bone marrow, cord blood and peripheral blood are not mature. Peripheral blood is a common source for EPCs but the obtained cell purity is very low. This study investigated methods of isolating, culturing, differentiation and identification of EPCs from rat bone marrow.　
METHODS: The experiment was performed at Research Institute of Urinary Surgery, First Hospital of Nanchang University from August 2006 to August 2007. ①The mononuclear cells were collected from 2-week-old SD rat bone marrow (Nanchang University Medical College, clean grade) by Ficoll density gradient centrifugation. EPCs were isolated with adherence screening method and cultured in M199 culture medium with the supplement of human basic fibroblast growth factor, leukaemia inhibitory factor, and epidermal growth factor. After 2 weeks of culture, immunofluorescence was used to identify cell markers such as von Willebrand disease factor, vascular endothelial growth factor receptor-2, CD34, and CD133; RT-PCR was employed to identify cell specific molecular markers including von Willebrand disease factor, FLK-1, Tie-2, CD34, CD133, and eNOS gene expression.
RESULTS: ①After four days of culture, cell colony was observed using light electron microscopy; adherent cells showed fusiform and radiated from the centre to the outside of the colony. While cultured for 7 days to 10 days, cell colonies were interconnected with the shape of cobblestone. After 2 weeks of culture, cells arranged in strands and capillary vessel network. ②Adherent cells were identified positive for von Willebrand disease factor, FLK-1, CD34, and CD133 by immunofluorescence staining, and positive for von Willebrand disease factor, FLK-1, Tie-2, CD34, CD133, and eNOS gene expression by RT-PCR.
CONCLUSION: EPCs are isolated from bone marrow-derived mononuclear cells, and amplified in vitro.

Deng ZF, Lu YM, Wang Y, Lou YL, Guo F, Xie A.In vitro culture and identification of endothelial progenitor cells from rat bone marrow.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3069-3073(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3069(ps).pdf]