比较不同传代人骨髓间充质干细胞的生物学特性:推荐第3～6代细胞可用于临床

Biological characteristics of human bone marrow-derived mesenchymal stem cells at different passages:Third to sixth passages are recommended for clinical application

背景:人骨髓内间充质干细胞含量稀少,且随着年龄增加或体质衰弱细胞数量会逐渐减少,因此进行大量扩增非常必要,但目前关于扩增后各代骨髓间充质干细胞的生物学特性是否相同仍无准确定论。目的:分析比较不同传代人骨髓间充质干细胞的生物学特性,着眼于推荐其应用于临床组织工程的实际需求。设计、时间及地点:细胞学体外观察,于2008-03/10在北京中医药大学附属东直门医院骨科和中心实验室完成。材料:骨髓间充质干细胞来源于非造血系统疾病的患者骨髓,由北京中医药大学附属东直门医院骨科提供。方法:从患者髂后上棘抽取骨髓,Percoll法分离培养骨髓间充质干细胞。当细胞融合至90%以上时胰酶消化传代,定期在倒置显微镜下观察各代细胞形态变化,取第2代细胞用于指标检测。主要观察指标:细胞形态及免疫表型,MTT检测细胞活性,流式细胞仪分析细胞分裂及凋亡坏死比例。结果:传代培养的骨髓间充质干细胞形态均一,呈梭形、纺锤形,克隆样生长,培养至9代后生长减慢,并出现胞浆空泡化、胞体变大等老化现象。第2代细胞表面标志CD44,CD106,CD105均呈阳性表达,而CD34,CD45则呈阴性。骨髓间充质干细胞增殖到第9代MTT值达到最高,从第10代开始MTT值下降。细胞增殖到11代,分裂期细胞数量仍呈上升趋势,但从第6代开始凋亡细胞数量明显增加,第8代以后凋亡细胞数量达到60%以上。结论:通过对各代骨髓间充质干细胞生物学特性分析,推荐使用第3～6代骨髓间充质干细胞用于临床治疗使用。

BACKGROUND：Mesenchymal stem cells are few in human bone marrow,and their number will decrease with aging or body weakening,so a large amount of amplification is necessary.However,the biological characteristics of human mesenchymal stem cells of each passage remain poorly understood.OBJECTIVE：To analyze and compare the biological characteristics of each passage of bone marrow-derived mesenchymal stem cells （MSCs） so as to provide a basis for clinical demands of tissue engineering.DESIGN,TIME AND SETTING：Cytological observation in vitro.The experiment was performed at the Department of Orthopedics and Central Laboratory,Dongzhimen Hospital,Beijing University of Chinese Medicine from March to October 2008.MATERIALS：From bone marrow of patients with non-hematopoietic disease,MSCs were provided by Department of Orthopedics,Dongzhimen Hospital,Beijing University of Chinese Medicine.METHODS：Bone marrow was collected form posterior superior iliac spine of patient,MSCs were isolated and cultured by Percoll method.When the cells were confluent at 90%,they were trypsinized and observed by inverted miscroscopy.The second passage of cells were collected for index detection.MAIN OUTCOME MEASURES：Cell morphological characteristics and immunophenotype;cell activity was detected by MTT;cell division and apoptosis in the proportion of necrosis were analyzed by flow cytometry analysis.RESULTS：The passaged MSCs exhibited uniform appearance in fusiform shape,and their growth was slowed down after 9 passages,exhibiting cytoplasm vacuolization and body enlarging.The second passage of MSCs was positive for CD44,CD106,and CD105,but negative for CD34 and CD45.MTT values peaked at passage 9,and gradually decreased since passage 10.At passage 11,the number of MSCs at division stage was increased,but from the sixth passage,the number of apoptotic cells increased significantly,reaching more than 60% at passage 8.CONCLUSION：According to biological characteristics analysis of MSCs at each passage,the third to the sixth passage cells are recommend for clinical therapy.