摘要
背景:骨髓间充质干细胞易于从骨髓中分离,来源取材方便,对供体损伤小,具有强大的增殖能力及多向分化潜能,便于自体移植,如能成功诱导为软骨细胞将是软骨组织工程理想的种子细胞。目的:观察体外诱导骨髓间充质干细胞向软骨细胞的定向分化,并对分化后细胞进行鉴定。设计、时间及地点:细胞形态学、蛋白质组学的比较观察于2005-05-03/2007-12-30在沈阳医学院临床中心实验室完成。材料:骨髓间充质干细胞来源于二三个月龄新西兰健康大白兔,体质量2～2.5kg。方法:采用全骨髓法分离培养兔骨髓间充质干细胞。取生长良好的细胞第4代细胞消化传代,以1×105密度接种于24孔板中将生长状态良好的细胞在含有地塞米松、碱性成纤维细胞生长因子、维生素C及转化生长因子β的培养液中诱导分化。主要观察指标:采用免疫组化的方法进行细胞鉴定,RT-PCR法检测诱导分化细胞II型胶原的表达。结果:骨髓间充质干细胞经成骨诱导后,由长梭形变为椭圆形及肾形。免疫组织化学方法显示所培养间充质干细胞不表达CD34、CD45,但CD105显阳性。骨髓间充质干细胞在未经诱导时不表达Ⅱ型胶原蛋白,经诱导后在500～750bp之间可见明显条带。结论:骨髓间充质干细胞经含地塞米松、碱性成纤维细胞生长因子维生素C及转化生长因子β等培养液诱导后,可以在体外向软骨细胞转化,高表达Ⅱ型胶原,并经细胞形态学及蛋白质组学RNA水平鉴定验证。
BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are easily collected from bone marrow and conveniently used in autologeus transplantation, and possess the potential of strong proliferation and multiple differentiations, and have little injury to the donor. BMSCs can be an ideal seed cells in cartilaginous tissue engineering, if BMSCs can differentiate into chondrocytes. OBJECTIVE： To investigate the in vitro directional differentiation of BMSCs into chondrocytes, and identify the differentiated cells. DESIGN, TIME AND SETTING： The cell morphology and proteomics experiment was Clinical Central Laboratory, Shenyang Medical College from May 3^rd, 2005 to December 30^th, 2007. MATERIALS： BMSCs were harvested from bone marrow of New Zealand healthy rabbits aged two or three months weighing 2-2.5 kg. METHODS： BMSCs were isolated by a whole bone marrow method. At the fourth passage, BMSCs were digested and incubated in a medium containing dexamethasone, basic fibroblast growth factor, vitamin C and transforming growth factor β in a 24-well plate at a density of 1 × 10^5. MAIN OUTCOME MEASURES： Cells were identified by immunohistochemistry. Expression of collagen Ⅱ was detected by reverse transcription-polymerase chain reaction. RESULTS： BMSCs changed from spindle into ellipse and reniform after osteogenic induction. Immunohistochemistry showed that BMSCs were negative for CD34 and CD45, but positive for CD105. BMSCs did not express collagen Ⅱ, but significant strap was detected at 500-750 bp after induction. CONCLUSION： Cell morphology and proteomics RNA verified that BMSCs can differentiate into chondrocytes in vitro, highly express collagen Ⅱ after the induction in a medium containing dexamethasone, basic fibroblast growth factor, vitamin C and transforming growth factor β.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第38期7495-7498,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
辽宁省科技厅资助项目(2005225017)~~
