表皮生长因子对外周血间充质干细胞原代克隆形成的影响及其传代后多向分化潜能

Effects of epidermal growth factor on primary clone formation rate of peripheral blood mesenchymal stem cells and its multi-directional differentiation potential after passage

背景:目前已有很多实验证明外周血中存在间充质干细胞,但由于所用分离筛选方法、培养条件的不同导致了结果的多样性,且实验可重复性较差。目的:观察表皮生长因子对体外分离培养的原代小鼠外周血间充质干细胞克隆形成的影响,以及外周血间充质干细胞传代后的多向分化潜能。设计、时间及地点:细胞学体外观察,于2007-10/2008-09在南京医科大学骨与干细胞研究中心完成。材料:雄性成年C57BL/6J小鼠16只,购于南京医科大学骨与干细胞研究中心实验动物中心。重组人表皮生长因子为Sigma公司产品。方法:无菌条件下从小鼠心脏采血,裂解红细胞后应用贴壁法分离外周血间充质干细胞,通过传代进行纯化和扩增。将原代细胞分2组进行体外培养,实验组向α-MEM培养液中加入10μg/L表皮生长因子,对照组给予不含表皮生长因子的α-MEM培养液,16d后行亚甲基蓝染色,计算克隆面积。取第3代外周血间充质干细胞,以1×104/cm2密度接种后,分别向成骨和脂肪方向诱导分化。主要观察指标:外周血间充质干细胞的生长特性,外周血间充质干细胞体外诱导成骨、成脂能力。结果:倒置相差显微镜下,外周血间充质干细胞贴壁生长,形态类似于骨髓间充质干细胞,呈成纤维细胞样;但其形成克隆的时间较骨髓间充质干细胞有所推迟,且原代细胞克隆形成率低于骨髓间充质干细胞;亚甲基蓝染色结果示实验组外周血间充质干细胞克隆形成率明显高于对照组(P<0.05)。外周血间充质干细胞成骨诱导后,碱性磷酸酶染色、总胶原染色及茜素红染色均呈强阳性;成脂诱导后油红染色呈阳性,有一定数量的脂滴形成。结论:外周血中存在少量的间充质干细胞,能自我增殖,表皮生长因子可有效促进原代外周血间充质干细胞克隆的形成;体外分离培养的外周血间充质干细胞具备成骨和成脂分化特性。

BACKGROUND： There is mesenchymal stem cells （MSCs） in peripheral blood, but study results are various due to various isolation method and culture condition. The study has bad repeatability. OBJECTIVE： To observe the effects of epidermal growth factor on forming efficiency of colony fibroblastic units of primary cultured mice in vitro, and the multi-directional differentiational potential of peripheral blood MSCs after passage. DESIGN, TIME AND SETTING： The cytology in vitro study was conducted at the Research Center of Bone and Stem cells, Nanjing Medical University from October 2007 to September 2008. MATERIALS： A total of 16 male adult C57BL/6J mice were purchased from Animal Experimental Center, Research Center of Bone and Stem cells, Nanjing Medical University. Recombinant human epidermal growth factor （Sigma, USA） was used in this study. METHODS： The blood samples were harvested in aseptic condition from the hearts of mice. After erythrocyte lysis, peripheral blood MSCs were harvested in vitro by adherence method, purified and amplified by passage. Primary cultured cells were assigned into 2 groups for in vitro culture. Cells in the experimental group were incubated in α -MEM medium containing 10μg/L epidermal growth factor. Cells in the control group were incubated in α -MEM medium without epidermal growth factor. Cells were stained with methylene blue after 16 days culture to calculate clone area. At the third passage, peripheral blood MSCs were incubated at 1×10^4/cm^2 and differentiated into osteoblasts and adipocytes. MAIN OUTCOME MEASURES： Growth characteristics, osteoblast and adipogenic induction in vitro of peripheral blood MSCs were measured. RESULTS： Under an inverted phase contrast microscope, peripheral blood MSCs were adherent and presented fibroblast-like cells. Clone time was quite late in peripheral blood MSCs compared with bone marrow MSCs. Cloning efficiency of primary cells were lower than bone marrow MSCs. Methylene blue staining results suggested that cloning efficiency of peripheral blood MSCs was significantly higher in the experimental group than in the control group （P 〈 0.05）. After osteoplastic induction, peripheral blood MSCs had strongly positive reactions for alkaline phosphatase staining, total collagen staining, alizarin red staining. After adipogenic induction, peripheral blood MSCs had a positive reaction for oil red O staining, with the presence of some lipid droplets. CONCLUSION： Peripheral blood MSCs circulate in low number, can proliferate. Epidermal growth factor can improve clone formation of peripheral blood MSCs. In vitro cultured peripheral blood MSCs have the differentiational properties of osteoblasts and adipocytes.