摘要
目的:应用转化生长因子β1,体外诱导脂肪干细胞向成软骨细胞表型分化,探讨其作为组织工程化软骨种子细胞的可行性。方法:实验于2005-04/2006-06在华中科技大学同济医学院公卫实验室完成。①取大鼠腹股沟处脂肪,酶消化法分离、培养脂肪干细胞,体外传代培养。②取第3代细胞通过转化生长因子β1、地塞米松和维生素C诱导脂肪干细胞向软骨细胞分化。③诱导后14d观察细胞形态变化,进行阿辛蓝染色检测软骨基质的分泌、免疫组织化学检测细胞Ⅱ型胶原的表达,采用Western-blot和反转录-聚合酶链反应检测诱导前后成软骨相关的Sox9,蛋白聚糖与Ⅱ型胶原的表达。结果:①细胞接种的最初几日,细胞呈圆形,1周后贴壁细胞呈长梭形,体积增大;14d后贴壁生长细胞基本长满单层,中心细胞排列紧密,形态与骨髓间充质干细胞相似。诱导培养后,细胞形态逐渐由梭型向多角形、多边形转变。诱导14d后多数细胞呈平坦的多边角形状细胞;其夹杂多角突起状或多角纺锤状细胞。②诱导后阿辛蓝染色示糖胺聚糖均匀分布于基质中。③免疫组织化学染色示基质中Ⅱ型胶原表达阳性。④反转录-聚合酶链反应检测成软骨相关的Sox9、蛋白聚糖、Ⅱ型胶原mRNA表达阳性。⑤Western-blot印迹检测细胞诱导后Ⅱ型胶原蛋白表达阳性。结论:脂肪干细胞在特定培养液的诱导下可向成软骨细胞表型分化,并能分泌软骨细胞特异性基质,有望成为软骨组织工程新的细胞来源。
AIM： To investigate the feasibility of rat adipose-derived stromal cells （ADSCs） as the seed cell of tissue engineered cartilage after in vitro differentiation into chondrogenic phenotype with induction of transforming growth factor-beta （TGF-β）.
METHODS： The experiment was accomplished in the Laboratory of Pubic Health, Tongji Medical College of Huazhong University of Science and Technology between April 2005 and June 2006. ①ADSCs isolated from rat inguinal fat pads ware digested with collagenase, cultured and passaged in vitro. ②The cultured ADSCs at the 3^rd passage ware induced to differentiate into chondrocytes in induction medium containing TGF-β, desamethason and vitamin C.③Fourteen days later, matrix of cartilage cells was detected by alcian blue staining, and cartilage specific collagen Ⅱ was detected by istry. The expression of Sox9, proteoglycan and collagen g in the induced ADSCs ware also detected by Western-blot and reverse transcription-polymerase chain reaction （RT-PCR）.
RESULTS： ①At the initial days of inoculation, the cells ware round; one week later, the attached cells increased to the long fusiform; 14 days later, the attached cells ware identical with bone marrow mesenchymal cells in morphology, and covered with the whole monolayer, with the central cells arranged tightly. The induced ADSCs transformed to polygon and presented fiat after 14-day induction, including spindle-shaped or polygonal-shaped.② Histological staining of proteoglycan with alcian blue staining for cartilage specific collagen Ⅱ revealed deposition of typical cartilage extraceUular matrix. ③Immunohistochemical staining showed the positive expression of collagen Ⅱ .④ RT-PCR gene expression analysis of characteristic chondrogenic related genes, such as Sox9, proteoglycan as wall as collagen Ⅱ ware positive. ⑤Westem-blot detection confirmed positive collagen Ⅱ expression by 14 days induction.
CONCLUSION： ADSCs can differentiate to be chongrogenic phenotype when cultured in a defined medium, and secrete the specific matrix of chondrocytes. ADSCs can likely to be served as optimal autogenous cell source for cartilage tissue enginearing.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第10期1805-1807,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金项目(30471753)~~
