中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (14): 2607-2611.doi: 10.3969/j.issn.1673-8225.2010.14.028

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

自体富血小板血浆对人骨髓来源内皮祖细胞功能活性的影响

赵忠海，李洪秋，阿  良，陈  宾，汪少伯   

  1. 沈阳医学院奉天医院骨科，辽宁省沈阳市  110024
  • 出版日期:2010-04-02 发布日期:2010-04-02
  • 作者简介: 赵忠海，男，1969年生，辽宁省葫芦岛市人，汉族，1992年锦州医学院毕业，副主任医师，主要从事骨外科工作，特别是创伤外科的临床实践。 zzh-hy@126.com
  • 基金资助:

    沈阳医学院课题(20063021)，课题名称：富血小板血浆对骨折修复及血管内皮影响的基础研究。

Effects of autologous platelet-rich plasma on function and activity of human bone marrow-derived endothelial progenitor cells  

Zhao Zhong-hai, Li Hong-qiu, A Liang, Chen Bin, Wang Shao-bo   

  1. Department of Orthopaedics, Fengtian Hospital, Shenyang Medical College, Shenyang   110024, Liaoning Province, China
  • Online:2010-04-02 Published:2010-04-02
  • About author:Zhao Zhong-hai, Associate chief physician, Department of Orthopaedics, Fengtian Hospital, Shenyang Medical College, Shenyang 110024, Liaoning Province, China zzh-hy@126.com
  • Supported by:

    the Project of Shenyang Medical College , No. 20063021*.

PDF

433

被引次数

5

摘要：

背景：富血小板血浆是经过特殊方法提取的血小板含量丰富的血浆，相较普通血清含有更丰富的细胞因子，如血小板衍生因子、转化生长因子β、血管内皮生长因子等。
目的：观察富血小板血浆对人骨髓血来源的内皮祖细胞生物学特性的影响，拟探讨富血小板血浆在骨创伤修复血管化中的作用。
方法：抽取16名健康志愿者自体外周静脉血获取富血小板血浆。采用密度梯度离心法获取骨髓血内皮祖细胞，将培养8 d的内皮祖细胞随机分为对照组和富血小板血浆组，胎牛血清组采用培养基为体积分数为10%胎牛血清配比高糖DMEM培养基加青霉素、链霉素各100 U/mL；无血清对照组采用高糖DMEM培养基加青霉素、链霉素各100 U/mL。相差显微镜观察细胞生长情况，激光共聚焦显微镜鉴定，AC133和vWF双染阳性细胞为正在分化的内皮祖细胞，MTT法检测细胞培养6，12，48 h增殖情况，Transwell小室检测迁移，Matrigel管腔形成实验检测管腔形成能力。
结果与结论： ①各组细胞均在接种后12~24 h开始贴壁，并由圆形逐步变化为梭型、多角形、不规则形状等。②富血小板血浆作用于内皮祖细胞6 h后，A490值明显高于对照组(P < 0.05)；12 h时其促进作用更强(P < 0.01)。在0~48 h，随时间延长，富血小板血浆促进内皮祖细胞增殖的作用相应增强。③富血小板血浆可提高内皮祖细胞的迁移能力，6 h开始明显增强(P < 0.05)，于12 h达高峰(P < 0.01)，48 h有所下降，但仍明显高于对照组(P < 0.01)。④富血小板血浆显著增强内皮祖细胞体外管腔形成能力，在作用时间为48 h最为显著，并且形成的管腔样结构也较对照组复杂。

关键词: 富血小板血浆, 内皮祖细胞, 增殖, 分化, 骨缺损

Abstract:

BACKGROUND: Platelet-rich plasma is the plasma containing rich platelet extracted by special method. Compared with common serum, platelet-rich plasma contains richer cytokines, such as platelet derived growth factor, transforming growth factor beta and vascular endothelial growth factor.
OBJECTIVE: To observe effects of platelet-rich plasma on biological characteristics of human endothelial progenitor cells (EPCs), and to discuss the repair function of platelet-rich plasma on bone defects.
METHODS: Platelet-rich plasma was collected from autologous peripheral vein blood of 16 healthy volunteers. EPCs were harvested from bone marrow blood by density gradient centrifugation. EPCs following 8 days of culture were randomly assigned to control group, platelet-rich plasma group, fetal bovine serum group and serum-free control group. In the fetal bovine serum group, cells were incubated in high-glucose DMEM containing 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. In the serum-free control group, cells were incubated in high-glucose DMEM containing 100 U/mL penicillin and 100 U/mL streptomycin. EPC growth was observed under a phase contrast microscope and a laser confocal microscope. AC133- and vWF- positive cells (double staining) were differentiated EPCs. EPC proliferation, migration and tubule formation capacity were respectively measured by the MTT assay, Transwell chamber assay and Matrigel tubule assay at 6, 12, 48 hours following culture.
RESULTS AND CONCLUSION:  ① Cells from each group began to adhere to the wall, and changed from round to spindle, polygonal and irregular shapes at 12-24 hours following incubation. ② A490 value in EPCs was significantly greater following 6 hours of treatment with platelet-rich plasma compared with the control group (P < 0.05), and the promotion effect became stronger at 12 hours (P < 0.01). At 0-48 hours, with prolonged time, the promotion effect of platelet-rich plasma on EPC proliferation enhanced. ③ Platelet-rich plasma could elevate EPC migration, and the effect became significantly enhanced at 6 hours (P < 0.05), peaked at 12 hours (P < 0.01), decreased at 48 hours, and still significantly greater than the control group (P < 0.01). ④ Platelet-rich plasma obviously enhanced tubule formation capacity in EPCs in vitro, especially at 48 hours, and the tubule-like structure is more complicated compared with the control group.

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