Objective: To separate peripheral blood mesenchymal stem cells (PBMSC) and peripheral blood endothelial progenitor cells (PBEPC) from peripheral blood, and investigate the biological characteristics of composite cell sheets of PBMSC and PBEPC.
Methods: The peripheral blood of healthy adult New Zealand white rabbits was extracted and PBMSC and PBEPC were separated by density gradient centrifugation. Morphological observation and identification of PBMSC and PBEPC were performed. The 3rd generation of PBMSC and PBEPC were used to construct a composite cell sheet at a ratio of 1∶1, and the 3rd generation of PBMSC was used to construct a single cell sheet as control. The distributions of cells in two kinds of cell sheets were observed by HE staining. In addition, the expression of alkaline phosphatase (ALP), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) in the supernatants of cell sheets were observed by ELISA at 1, 5, and 10 days after osteogenic induction.
Results: The morphology of PBMSC was spindle-shaped or polygonal, and PBMSC had good abilities of osteogenic and adipogenic differentiation. The morphology of PBEPC was paved stone-like, and the tube-forming test of PBEPC was positive. Two kinds of cell sheets were white translucent. The results of HE staining showed that the composite cell sheet had more cell layers and higher cell density than the single cell sheet. The expressions of ALP, OCN, and VEGF in the supernatant of the two groups of cell sheets increased with the time of induction. The expression of OCN in the group of composite cell sheet was significantly higher than that in the group of single cell sheet on the 5th and 10th day, ALP on the 10th day was significantly higher than that in the group of single cell sheet, VEGF expression on the 1st, 5th, and 10th day was significantly higher than that in the group of single cell sheet, all showing significant differences ( P<0.05), and there was no significant difference between the two groups at other time points ( P>0.05).
Conclusion: PBMSC have stable differentiation ability, and they have good application prospects because of their minimally invasive access. Composite cell membranes constructed by co-culture of two kinds of cells and induction of membrane formation provides a new idea and exploration for tissue defect repair.
目的: 从外周血中分离获取外周血来源间充质干细胞（peripheral blood mesenchymal stem cells，PBMSC）和外周血内皮祖细胞（peripheral blood endothelial progenitor cells，PBEPC），构建复合细胞膜片并初步研究其生物学特性。.
方法: 抽取健康成年新西兰大白兔外周血，采用密度梯度离心法分离获取 PBMSC 和 PBEPC，分别行形态学观察及鉴定。取第 3 代 PBMSC 和 PBEPC 以 1∶1 比例进行成膜诱导形成复合细胞膜片，取单纯第 3 代 PBMSC 同法制备单一细胞膜片。取两种细胞膜片行 HE 染色，观察细胞膜片内细胞分布情况；分别对两种细胞膜片行成骨诱导，收集诱导 1、5、10 d 的上清液，利用 ELISA 法检测两种细胞膜片上清液内 ALP、骨钙素（osteocalcin，OCN）和 VEGF 的表达情况。.
结果: PBMSC 细胞形态单一，呈纺锤形或多角形，具备良好的成骨、成脂分化能力；PBEPC 细胞形态单一，呈铺路石样，成管试验阳性。两种细胞膜片均呈白色半透明膜状物。HE 染色示，与单一细胞膜片组相比，复合细胞膜片组膜片更厚，具备更多细胞层数及更高的细胞密度。ELISA 法检测示，随诱导时间延长，两组细胞膜片上清液中 ALP、OCN 和 VEGF 表达量均有所增加。复合细胞膜片组诱导培养 5、10 d OCN 表达量显著高于单一细胞膜片组，10 d ALP 表达量显著高于单一细胞膜片组，1、5、10 d VEGF 表达量均显著高于单一细胞膜片组，差异均有统计学意义（ P<0.05）；其余各时间点两组间各蛋白表达量比较差异均无统计学意义（ P>0.05）。.
结论: PBMSC 具备稳定的 MSCs 分化能力，因其取材微创具备良好的应用前景。通过与 PBEPC 共培养、成膜诱导等手段构建复合细胞膜片，为组织缺损的修复提供了一种新的思路与探索。.
Keywords: Peripheral blood mesenchymal stem cells; cell sheets; endothelial progenitor cells; tissue engineering; tissue repair.