血管内皮细胞生长因子对人脐静脉间充质干细胞生长增殖的影响

Vascular endothelial cell growth factor effects on growth and proliferation of human umbilical vein mesenchymal stem cells

背景:在体外构建组织工程材料的过程中,快速获得足够数量且纯度高的种子细胞极为重要,但目前人脐静脉间充质干细胞原代培养的增殖率仍较低。目的:观察血管内皮细胞生长因子对人脐静脉间充质干细胞原代生长、增殖的影响。设计、时间及地点:细胞学体外观察,于2008-03/07在辽宁医学院解剖学实验室完成。材料:正常健康产妇顺产或剖宫产的新生儿脐带20条,由锦州市凌河区妇幼保健院及辽宁医学院附属第一医院提供。方法:采用胶原酶消化法分离人脐静脉间充质干细胞,以1×108L-1的密度接种于24孔培养板,设立2组,实验组加入150g/L血管内皮细胞生长因子,对照组不加入任何细胞因子。主要观察指标:观察细胞形态变化,MTT法检测细胞生长曲线,免疫细胞化学法鉴定细胞CD166的表达。结果:接种后6h细胞开始贴壁,48h后细胞完全贴壁生长,出现呈椭圆形、多角形的内皮细胞,以及呈梭形的成纤维样细胞,有的形成漩涡状生长集落;原代培养1周后细胞以梭形为主;传代后24h细胞基本完成贴壁,48h细胞开始分裂增殖,5～7d可见多核的成纤维样细胞贴壁,呈长杆状、三角形、梭形等。与对照组比较,实验组细胞生长状态较好,增殖较快,单位时间内获得的细胞数量明显增加(t=2.235,P<0.05),CD166阳性细胞率显著升高(t=1.638,P<0.01)。结论:血管内皮细胞生长因子可促进人脐静脉间充质干细胞贴壁,且更有利于间充质干细胞的增殖和纯化。

BACKGROUND： During in vitro construction of tissue-engineered materials, it is important to rapidly obtain enough seed cells with high purity. However, proliferation rate of primary culture of human umbilical vein mesenchymal stem cells （hUV-MSCs） is low. OBJECTIVE： To investigate the influence of vascular endothelial cell growth factor （VEGF） on the primary culture and proliferation of hUV-MSCs. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the Laboratory of Department of Anatomy, Liaoning Medical University from March to July 2008. MATERIALS： A total of 20 neonatal umbilical cord following spontaneous delivery or caesarean delivery in normal healthy partudents, which were supplied by Maternal and Child Health Faculty, Linghe, Jinzhou and First Hospital Affiliated to Liaoning Medical University. METHODS： The hUV-MSCs were isolated by the collagenase digestion method. At 1 ×10^8/L, hUV-MSCs were incubated in a 24-well culture plate, and then divided into two groups. These in the experimental group were treated with 150 g/L VEGF, whereas those in the control group were left intact. MAIN OUTCOME MEASURES： Cell morphology; Cell growth curve using MTT assay; CD166 expression in cells was identified using immunocytochemical method. RESULTS： At 6 hours, cells adhered, and at 48 hours, cells completely adhered. Oval or polygonal endothelial cells and spindle fibroblasts were found, and some formed whirlpool-shaped colonies. Following 1 week primary culture, a majority of cells were spindle. After 24 hours passage, cells adhered. Till 48 hours, ceils began division growth. At 5 -7 days, long, triangle and spindle-shaped fibroblasts with multiple nuclei were presented. Compared with the control group, cells in the experimental group showed good growth and rapid proliferation. Cell count significantly increased in unit time （t=2.235, P 〈 0.05）. The rate of CD166-positive cells significantly increased （t=1.638, P 〈 0.01 ）. CONCLUSION： VEGF can promote hUV-MSCs adherence and accelerate the proliferation and purity of MSCs.