兔肌源性干细胞的生物学特性及表型分析

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＋100gm/L血清，融合培养基为DMEM-LC＋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Western Blot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时己无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。

Biological characteristics and phenotype of rabbit muscle-derived stem cells

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) have a remarkable differentiation potential and superiority as a type of seed cells,but their application is limited in the presence of certain diseases,such as aplastic anemia and myelogenous neoplasm.The present studies have found that seed cells called muscle-derived stem cells (MDSCs) have brought more and more attention,because of their capability of stir-renewal and multi-diffcrentiation like B MSCs.OBJECTIVE: To explore the biological characterization of the muscle-derived stem cells (MDSCs) from rabbits,and analyze the phenotype.DESIGN,TIME AND SETTING: Cell in vitro observation experiment was performed at the Medical Research Center of Second Affiliated Hospital of Sun Yat-sen University from August 2005 to March 2006.MATERIALS: A New Zealand rabbit (1.5 months old,clean grade) was enrolled for the preparation of Muscle-derived stem cells.Growth medium was DMED-LG added with 10% fetal bovine serum and 10% horse serum and fusion medium was DMEM-LG added with 2% fetal bovine serum.METHODS: The muscle mass was removed from the anesthetized rabbit to isolate MDSCs.These cells were dissociated using three enzymes (collagenase XI,dispase and trypsin) respectively.Sediment was resuspended.Then preplate technique was used.The muscle cell extract was plated on a collagen-coated culture flask with growth medium.The flask was called PP1.PPI was kept overnight in a 37 ℃ incubator containing 5% CO2,After that,the suspension was transferred to another collagen-coated culture flask,which was called PP2.PP3,PP4,PP5 and PP6 were constructed later following the same procedures.The cells adhered in PP6 were collected,plated in 6-well plates,and divided into 2 groups.Growth medium was used in one group,in which the cells were kept growing at a degree of confluence beyond 50%,and fusion medium was used in the other one,in which the cells were passaged with a degree up to 30%.MAIN OUTCOME MEASURES: The cells from PP1 to PP6 were collected,and the characterization was identified preliminary by Flow cytomctry,Immunocytochemistry and Western Blotting.The fusion of cells in PP6 was detected at different confluence degrees and concentration of medium.RESULTS: The cells in PP6 showed ＞ 80% desmin+,＞ 70% Bcl-2+,＞ 95% CD45,which indicated that MDSCs were in a high concentration.The expressions of α-SMA in the cells were decreasing with the Preplate technique used and the cells in PP6 almost had no α -SMA expression.When passaged at a high confluence (＞ 50%) or cultured with low concentrations of serum (2% serum),the cells in PP6 had a strong tendency of fusing into myotubes or cell chains and were skeletal myosin+.CONCLUSION: MDSCs,which are capable of multi-differentiation under a high fusion or low serum conditions,express dcsmin and Bcl-2 highly,but extreruelv little CD45 and no α -SMA.