藻酸盐凝胶三维培养条件下体外定向诱导骨髓间充质干细胞成软骨  

Directional induction of bone marrow mesenchymal stem cells into chondroblast in three-dimensional culture using algingate microspheres

作　　者：朱丽明[1] 崔颖[2] 

机构地区：[1]辽宁医学院,辽宁省锦州市121001 [2]辽宁医学院附属第一医院耳鼻喉科,辽宁省锦州市121001

出　　处：《中国组织工程研究与临床康复》2009年第21期4041-4044,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：辽宁省自然科学基金资助项目(20062198)~~

摘　　要：背景:目前有关诱导骨髓间充质干细胞向软骨细胞分化的研究不少,但选用何种生长因子调节以及如何保证其持续高效刺激尚处于探索阶段。目的:实验以软骨源性形态发生蛋白1(cartilage-derived morphogenetic protein1,CDMP1)和转化生长因子β1(transforming growth factor-β1,TGF-β1)作为主要诱导因子,通过三维培养微环境诱导,观察独立及联合应用时,促进骨髓间充质干细胞向软骨细胞表型分化的能力。设计、时间及地点:体外观察实验,于2007-04/2008-04在辽宁医学院附属医院耳鼻喉实验室完成。材料:选择辽宁医学院附属第一医院自然流产或咪嗦流产的12～20周胚胎,胚胎的使用经孕妇同意。实验经医院医学伦理委员会批准。方法:无菌条件下取出胚胎股骨,用密度梯度离心法和贴壁法分离纯化人胚骨髓间充质干细胞,体外培养传代。采用特定的诱导培养液使骨髓间充质干细胞向软骨分化,按培养基内添加生长因子的不同分成4组:①联合诱导组:无血清的软骨诱导液+CDMP1(300μg/L)+TGF-β1(10μg/L)。②TGF-β1诱导组:无血清的软骨诱导液+TGF-β1(10μg/L)。③CDMP1诱导组:无血清的软骨诱导液+CDMP1(300μg/L)。④对照组:无血清的软骨诱导液。先进行单层细胞培养10d,再置于藻酸盐凝胶中继续培养11d。主要观察指标:①于培养21d后倒置显微镜观察细胞在凝胶球中的生长和增殖情况。②二甲基亚甲基蓝比色法测定各组标本的铵聚糖含量。③免疫组织化学染色检测Ⅱ型胶原的表达。结果:第21天后,倒置显微镜观察藻酸盐凝胶呈现透明胶冻状,有黏性。加入不同生长因子的藻酸盐凝胶,细胞呈散在分布于凝胶介质中,呈现"悬浮"状态。第21天时联合诱导组铵聚糖含量、Ⅱ型胶原表达阳性率高于其他3组,差异有显著性意义(P<0.05);TGF-β1诱导组、CDMP1诱导组铵聚糖含量、Ⅱ型胶原表达阳性率高于对照组,差异有显�BACKGROUND： There are plenty of studies regarding induction of bone mesenchymal stem cells （BMSCs） differentiate into chondrecytes; however, it is need to be explored how to use growth factor in regulating and stimulating cells. OBJECTIVE： To explore the effect of combined or alone use of the growth factor on ability of BMSCs differentiate into chondrocyte phenotype in vitro under three-dimensional tissue environment, the cartilage-derived morphogenetic protein 1 （CDMP1） and tansforming growth factor-β 1 （TGF-β 1 ） are served as inducing factor. DESIGN, TIME AND SETTING： The in vitro observation was completed in the Department of Otorhinolaryngology, First Affiliated Hospital of Liaoning Medical University from April 2007 to April 2008. MATERIALS： Human embryo spontaneous abortion at 12-20 weeks of gestation was collected with signed agreements of the pregnant women, and the experiment was approved by the hospital Ethics Committee. METHODS： BMSCs were isolated from femora of the human fetuses and purified by density gradient centrifugation and adhesion method in vitro. Different growth factors were added to induce BMSCs cell differentiation into chondrocytes. （1）Combination group： induction medium＋CDMP1 （300 μ g/L） ＋TGF-β1 （10 μ g/L）; （2）TGF-β 1 group： induction medium＋ TGF-β 1 （10 μ g/L） （3）CDMP1 group： induction medium＋ CDMP1 （300 μg/L）; （4）Control group： The monolayer cells were cultured for 10 days followed by additional 11 days culture in alginate microspheres. MAIN OUTCOME MEASURES： The growth and proliferation of BMSCs were observed by inverted microscope at 21 days after culture. Contents of glycosaminoglycan （GAG） in each group were measured by methylene blue colorimetric assay. In addition, the expression of type I｜ collagen was determined by immunohistochemical staining. RESULTS： The inverted microscopy demonstrated that alginate was transparent colloid with viscidity at 21 days after culture, which presented i

关 键 词：藻酸盐凝胶 骨髓间充质干细胞 生长因子 细胞诱导 

分 类 号：R318[医药卫生—生物医学工程]