摘要
背景:软骨组织工程是骨科研究的热点,在种子细胞的研究上由于软骨细胞存在老化现象更多的研究集中在干细胞向软骨细胞分化上。分化条件经历单细胞因子、联合细胞因子及贯续细胞因子的应用。目的:观察改良诱导方案诱导SD大鼠脂肪源性干细胞定向分化为软骨细胞的能力。设计、时间及地点:观察性实验,于2008-06/12在遵义医学院珠海校区中心实验室完成。材料:SPF级健康SD大鼠5只,鼠龄18～22d,体质量约25g,用于脂肪干细胞的培养基扩增。方法:在无菌条件下从SD大鼠腹股沟区取皮下脂肪组织约3g,Ⅰ型胶原酶消化后,1000r/min离心10min,待细胞长满瓶底80%时用2.5g/L胰蛋白酶消化,按1:2或1:3比例传代培养。加入软骨诱导培养基14d后阿新蓝染色及Ⅱ型胶原免疫组织化学检测脂肪干细胞向软骨细胞的诱导分化。主要观察指标:流式细胞仪检测大鼠脂肪干细胞表面CD44、CD45和CD49d的表达。结果:SD大鼠脂肪组织分离培养出的细胞经流式细胞仪检测CD44、CD49d表达阳性,CD45表达阴性。加入软骨诱导培养基定向诱导72h后,细胞生长缓慢,逐渐变成圆形,并渐渐有软骨结节形成;诱导分化2周后,阿新蓝染色及Ⅱ型胶原免疫组织化学检测阳性。结论:SD大鼠脂肪组织能够分离培养出脂肪干细胞,生物学特性与骨髓间充质干细胞相似,能向软骨细胞分化。
BACKGROUND： Cartilage tissue engineering is a hotspot of orthopedic research, specially in the study of seed cells, and more research focus on the differentiation of stem cells into the cartilage cells because of aging chondrocytes. The differential conditions experienced the application of single cytokine, unite cytokines and sequential cytokines. OBJECTIVE： To observe the possibility of differentiation of the Sprague Dawley rat adipose-derived stem cells （ADSCs） into chondrocytes. DESIGN, TIME AND SETTING： The observational experiment was performed at the Central Laboratory of Zhuhai Campus, Zunyi Medical College between June and December 2008.
MATERIALS： Five SPF-grade, healthy Sprague Dawley rats aged 18 22 days and weighing about 25 g were applied in the amplification of the culture medium of ADSCs.
METHODS： Subcutaneous adipose tissue of about 3 g was obtained from lilac region of rats under the aseptic condition and was digested with I -collagenase, then centrifuged for 10 minutes at 1 000 r/min. When cells covered 80% of the bottom, they were treated with 2.5 g/L trypsin digestion and were subcultured according to the ratio of 1：2 or 1：3. Following 14 days treatment of chondrocyte inductor, the differentiation of ADSCs into chondrocytes was measured using Alcian blue staining and type Ⅱ collagen immunohistochemistry.
MAIN OUTCOME MEASURES： The expression of CD44, CD45, and CD49d were detected using flow cytometry.
RESULTS： ADSCs isolated from the Sprague Dawley rat inguinal fat pads were positive for CD44, CD49d, but negative for CD45 using flow cytometry. ADSCs grew slowly and become round and formed cartilage nodules gradually by adding chondrogenic medium 72 hours after induction orientation. Alcian blue staining and type ~I-collagen immunohistochemistry exhibited a positive reaction 14 days after induction orientation.
CONCLUSION： ADSCs can be isolated from Sprague Dawley rat adipose tissue. Their biological characteristics are similar with bone marrow mesenchymal stem cells and have the potential to differentiate into chondrocytes.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第36期7052-7056,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
贵州省科技厅资助项目(黔科通NY字[2006]62-2)
珠海市医学重点专科建设基金资助(珠卫[2008]80)~~
