摘要
[目的]探讨软骨细胞外基质微环境对源于人脐带Wharton胶的间充质干细胞增殖的影响,观察该微环境促进干细胞向软骨细胞方向分化的潜能。[方法]①取3%(w/v)猪关节软骨细胞外基质和2.4%(w/v)海藻酸盐为原料,按体积比1∶1的比例充分混合,将其用带16 G针头的1 ml注射器匀速滴入氯化钙溶液制作软骨细胞外基质/海藻酸盐混合凝胶微球,通过扫描电镜观察该微球内部结构。②将人脐带Wharton胶中间质干细胞(WJMSCs)混于软骨细胞外基质/海藻酸混合液,滴入氯化钙溶液形成复合干细胞的混合凝胶微球进行培养,设为实验组;同理WJMSCs混合于单纯藻酸盐凝胶并形成微球作为对照组。③通过扫描电镜和FDA-PI染色观察上述两组微球内细胞的生长粘附情况,体外培养4周,经组织学和DNA测定观察干细胞的表型及增殖情况。[结果]①构建的软骨细胞外基质/海藻酸盐混合凝胶微球呈半透明乳白色,内部具有较多不规则的孔隙结构,具有三维立体培养细胞的环境特点。②比较实验组和对照组不同细胞培养时间段细胞增殖量,发现实验组细胞增殖率高于对照组,第28 d时两组细胞DNA含量的差异有统计学意义,(P﹤0.05)。③实验组中细胞呈圆形或椭圆形,形成明显的软骨陷窝样的结构,有大量细胞外基质分泌,对照组中细胞呈圆形,陷窝样的结构不明显。④组织学染色观察发现:实验组中培养的干细胞明显有向软骨细胞方向分化的趋势。[结论]软骨细胞外基质微环境不仅利于人脐带Wharton胶中间质干细胞的增殖,而且具有将WJMSCs向软骨细胞方向诱导分化的潜能。
[Objective] To investigate the effects of the microenvironment of the cartilage extracellular matrix （ECM） on the proliferation of Wharton＇s jelly - derived mesenchymal stem cells （WJMSCs） from the umbilical cord and determine if this microenvironment can promote the differentiation of stem cells toward chondrocytes. [ Methods ] （ 1 ） The mixed - gel micro- spheres used were composed of a 3% （w/v） acellular matrix made up of cartilage suspension and 2.4% alginate solution and were dropped into a calcium chloride solution by using a syringe. We observed the features of the 3 - dimensional （3 - D） mixed gel microspheres by scanning electronmicroscopy. （2） WJMSCs from the human umbilical cord were mixed with the ECM - alg- inate gel microspheres, and this mixture was used as the experimental set, and stem cells with simple alginate gel microspheres were used as the control set. （3） The adhesion and activity of the cells were observed by scanning electronmicroscopy and fluo- rescein diacetate/propidium iodide （ FDA - PI） staining, and the differentiation and proliferation of the cells were detected by histological staining and DNA analysis after 4 weeks of culture in vitro. [ Results ] （ 1 ） The mixed - gel microspheres com- posed of alginate and cartilage ECM were translucent and ivory in color, with more interior irregular gap structure ; they provided a3 - D environment for the cultured cells. （2） The cell count in the experimental set was higher than that in the control set ;both these counts increased with increase in the culture period. Significant differences were observed between the DNA content for the 2 sets at 4 weeks （P 〈 0. 05） . （3） The cells in the experi- mental set were round or oval, formed an obvious cartilage lacunar structure, and secreted a large quantity of ECM, and the cells in the control set were round and formed a dimple -like structure that was not obvious. （4） Histological staining indicated that thestem cells in the experimental set
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2013年第11期1127-1132,共6页
Orthopedic Journal of China
基金
国家自然科学基金资助项目(编号:30973047,81000810,81071458)
863计划项目(编号:2102AA020502)
关键词
脐带Wharton胶间充质干细胞
软骨细胞外基质
藻酸盐
软骨组织工程
umbilical cord Wharton＇ s jelly derived mesenchymal stem cells, cartilage extracellular matrix, algi-nate, cartilage tissue engineering
