课题背景：间充质干细胞主要来源于骨髓,但由于骨髓源性间充质干细胞存在高度病毒污染的可能，且随着年龄增长其细胞数量、扩增分化能力均出现明显下降趋势，故寻找一种能替代骨髓间充质干细胞，并可弥补其缺陷的间充质干细胞来源逐渐受到关注。

偏倚或不足：实验从人脐带血中分离培养出间充质干细胞，在条件培养液诱导下短期内可获得软骨细胞，如能进一步定量检测诱导分化后的软骨细胞纯度及活性将更加完善。

术语解析：间充质干细胞来源于发育早期中胚层的多能干细胞，具有高度自我更新能力和多向分化潜能，广泛存在于全身多种组织中，可在体外培养扩增，并能在特定条件下分化为神经细胞、成骨细胞、软骨细胞、肌肉细胞、脂肪细胞等，在细胞替代治疗及组织工程方面有极大的应用价值。

摘要
目的：关节软骨创伤难以自行修复，传统疗法因修复的组织以纤维软骨为主，缺少正常透明软骨的力学性能及耐用性。种子细胞是组织工程化软骨形成的首要条件，为此拟建立一套较为简便、有效和实用的脐血间充质干细胞体外分离培养体系，探讨其向软骨细胞分化的可行性。
方法：实验于2005-07/2006-09在右江民族医学院组织学与胚胎学教研室完成。①细胞来源：无妊娠并发症足月分娩后的胎盘脐静脉血，均征得供者同意，实验经医院医学伦理委员会批准。②实验方法：穿刺抽取15 mL脐带血，密度梯度法分离吸取分层界面的白色环形絮状物，其中富含单个核细胞，离心后沉积细胞用含15％胎牛血清、100 U/mL青霉素、100 U/mL链霉素的DMEM高糖培养基悬浮，调整细胞密度为1×109 L-1接种于25 T培养瓶内，放置在37 ℃、体积分数为0.05的CO2饱和湿度培养箱中培养，72 h后更换培养基，去除红细胞及其他未贴壁细胞，至70％~80％融合时传代。③实验评估：取传至第2代的脐带血间充质干细胞进行增殖能力检测；加入含终浓度为100 μg/L胰岛素样生长因子1，15％胎牛血清的DMEM软骨细胞诱导培养基，倒置显微镜下连续观察细胞生长情况，并行苏木精-伊红染色，应用扫描电子显微镜对诱导后细胞进行鉴定。
结果：①细胞形态学观察：来源于脐血的单个核细胞接种24 h少量贴壁，近似圆形或短梭形；体外培养3 d后可见分散的间充质干细胞小集落，集落细胞为长梭形，呈放射状生长；2周后细胞集落彼此融合，呈旋涡状或菊花状生长。传代后3周细胞基本融合成层。②细胞增殖能力检测：脐带血间充质干细胞传代后8~12 h贴壁，7~13 d细胞处于对数生长期，16~19 d进入平台期，扩增速度快于原代培养，倍增时间为8.5 d。③脐带血间充质干细胞向软骨细胞诱导分化结果：胰岛素样生长因子1诱导12 d，脐带血间充质干细胞苏木精-伊红染色可见分泌少量细胞外基质。扫描电子显微镜下呈现软骨细胞特点，多边形，外围被分泌细胞外基质所围绕，呈蜘蛛网样。
结论：从人脐带血中成功分离获取间充质干细胞，在体外经胰岛素样生长因子1条件培养液诱导短期内可获得软骨细胞。
关键词：脐血；间充质干细胞；软骨细胞；细胞分化；胰岛素样生长因子1

解继胜，黄海玲，唐毓金，陈维平，罗小琼.人脐带血间充质干细胞分离培养及向软骨细胞的分化[J].中国组织工程研究与临床康复，2008，12(16):3065-3068 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3065(ps).pdf]

Isolation, culture and differentiation of human umbilical cord blood-derived mesenchymal stem cells towards chondrocytes

Abstract

AIM：Self-repair is difficult for injured articular cartilage. Traditional therapy to repair cartilage mainly involves fibrocartilage, which lacks of mechanical properties and durability in normal hyaline cartilage. Seed cell is a primary condition for the formation of tissue-engineered cartilage, therefore, we established a more simple, effective and practical isolation and culture system for umbilical cord blood-derived mesenchymal stem cells (MSCs), and investigated the feasibility of chondrocyte differentiation.
METHODS: The experiment was performed at Department of Histology and Embryology of Youjiang Medical College for Nationalities between July 2005 and September 2006. ①Fetal blood was obtained from placenta of full-term delivery with no pregnant complications. The informed consent was obtained from the donors, and the protocol was approved by Hospital Medical Ethics Committee. ②15 mL umbilical cord blood was extracted by puncturing, stratified surface white ring-like floccus containing abundant mononuclear cells was isolated using density gradient method and centrifuged. Depositing cells were incubated in high-sugar DMEM culture medium containing 15% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL Streptomycin, then cells were seeded in a 25 T culture bottle after cell density was adjusted to 1×109 L-1, and incubated at 37 ℃, 0.05 volume fraction CO2 with saturated humidity. After 72 hours, the culture medium was replaced, and red blood cells and other nonadherent cells were removed. Cells began to passage at 70%-80% confluence. ③The 2nd passage of umbilical cord blood-derived MSCs were harvested for multiplication ability examination. DMEM culture medium consisting of insulin-like growth factor 1 (final concentration, 100 μg/L), and 15% FBS was added to induce chondrocyte differentiation, and cell growth was observed under an inverted microscope. Cells were HE stained and cells after induction were observed using scanning electronic microscope.
RESULTS: ①Few umbilical cord blood-derived mononuclear cells attached after 24 hours of incubation, presenting round or short spindle-shaped; After 3 days of culture in vitro, dispersed small colonies of MSCs were observed, presenting long spindle-shaped and radiated growth; Two weeks later, the cell colonies fused, and grew in whirlpool or chrysanthemum tendency. Cells were nearly confluent three weeks after culture. ②Umbilical cord blood-derived MSCs attached 8-12 hours after passage, entered log phase growth from 7 to 13 days and platform phase from 16 to 19 days, presenting a rapider proliferation than primary culture. Cell proliferation doubled on day 8.5. ③After induced with insulin-like growth factor 1 for 12 days, the MSCs secreted little extracellular matrix by HE staining. Under the scanning electronic microscope, cells presented characteristics of chondrocytes, polygon-shaped, and surrounded by extracellular matrix.
CONCULUSION: MSCs are isolated from human umbilical cord blood, and induced to differentiate into chondrocytes by insulin-like growth factor 1 in vitro.

Xie JS, Huang HL, Tang YJ, Chen WP, Luo XQ.Isolation, culture and differentiation of human umbilical cord blood-derived mesenchymal stem cells towards chondrocytes.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3065-3068
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3065(ps).pdf]