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背景:骨髓间质干细胞作为种子细胞具有巨大的分化潜力和优势,但对于再生障碍性贫血或骨髓源性肿瘤等疾病的应用受限。现已发现肌源性干细胞具备与其相似的优越性,已引起相关领域研究者的注意。目的:观察兔肌源性干细胞的生物学特性,并对其进行表型分析。设计、时间和地点:细胞体外观察,于2005-08/2006-03在中山大学附属第二医院医研中心完成。材料:清洁级1.5月龄新西兰大白兔1只,增殖培养基为DMEM-LG+体积分数0.1胎牛血清+100g/L马血清,融合培养基为DMEM-LG+2%胎牛血清。方法:兔麻醉后取大腿肌肉,采用差速贴壁Preplate技术分离培养肌源性干细胞,以Ⅺ胶原酶、dispase蛋白酶及胰蛋白酶分步消化,沉淀用增殖培养基重悬,接种在胶原包被的培养瓶中,该培养瓶为PP1。PP1于37℃、含体积分数为0.05的CO2培养箱中静置过夜,随后将悬液转移到另一个胶原包被的培养瓶,此为PP2。随后同法建立PP3,PP4,PP5,PP6。将PP6接种到6孔板进行融合实验,分为两组:一组使用增殖培养基培养,在汇合度超过50%后继续培养,不传代;另一组使用融合培养基进行培养,汇合度达30%时传代培养。主要观察指标:收集PP1~PP6,采用流式细胞仪、免疫细胞化学及WesternBlot法鉴定细胞表型。通过不同汇合度及不同浓度血清培养,检测PP6融合情况。结果:PP6细胞>80%为desmin+,>70%为Bcl-2+,>95%为CD45-,提示为高浓度肌源性干细胞,随着纯化步骤的进行,α-SMA表达逐渐减弱,至高度纯化的PP6时已无α-SMA表达。PP6在高汇合度(>50%)或低血清(仅含2%血清)培养时,极易融合成肌管或肌细胞链,骨骼肌肌球蛋白呈阳性表达。结论:肌源性干细胞具有高水平表达desmin,Bcl-2,极低水平表达CD45,不表达α-SMA的生物学特性,在高汇合度或低血清培养条件下能够多向分化。
BACKGROUND: Bone marrow mesenchymal stem cells have great potential and advantages as seed cells, but their application to diseases such as aplastic anemia or bone marrow-derived tumors is limited. Muscle-derived stem cells have been found to have similar advantages and have drawn the attention of researchers in related fields. Objective: To observe the biological characteristics of rabbit myogenic stem cells and analyze their phenotype. DESIGN, TIME AND SETTING: The in vitro observation of cells was performed at the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University from August 2005 to March 2006. MATERIALS: One grade of New Zealand white rabbits with a clean grade of 1.5 months old were cultured in DMEM-LG + 0.1% fetal bovine serum and 100% fetal bovine serum. The fusion medium was DMEM-LG + 2% fetal bovine serum. Methods: Thigh muscles were obtained after anesthesia in rabbits. Muscle-derived stem cells were isolated and cultured by differential preplate technique. The myogenic stem cells were digested with collagenase, dispase and trypsin. The pellet was resuspended in proliferation medium and inoculated on collagen The flasks were PP1. PP1 was allowed to stand overnight at 37 ° C in a CO2 incubator containing 0.05 volume fraction and the suspension was then transferred to another collagen-coated flask, PP2. Then with the law to establish PP3, PP4, PP5, PP6. Inoculation of PP6 into 6-well plates for fusion experiments was divided into two groups: one group was cultured in proliferation medium and continued to be cultured when the confluency exceeded 50%, and the other was not passaged; the other group was cultured with fusion medium, 30% subculture. MAIN OUTCOME MEASURES: PP1 ~ PP6 were collected and the cell phenotypes were identified by flow cytometry, immunocytochemistry and Western blotting. Through different confluence and different concentrations of serum culture, detection of PP6 fusion. Results: The expression of α-SMA in 80% of desmin +, 70% of Bcl-2 + and 95% of CD45- was observed in PP6 cells. Highly purified PP6 has no alpha-SMA expression. When cultured in high confluence (> 50%) or low serum (containing only 2% serum), PP6 readily integrates into myotubes or myocyte chains and possesses positive skeletal myosin expression. CONCLUSION: Myogenic stem cells have high level of expression of desmin, Bcl-2, low level of expression of CD45, do not express the biological characteristics of α-SMA, under high confluency or low serum culture conditions can be multi-directional differentiation.