摘要
背景:常规诱导方法诱导的细胞生长缓慢,数量少,使用生长因子可加快诱导速度,但生长因子价格昂贵限制了它的使用。目的:验证未加生长因子的诱导液体外诱导人骨髓间充质干细胞向成骨细胞分化的效率。设计、时间及地点:对比细胞学观察,于2007-07/2008-02在青岛大学医学院附属医院中心实验室完成。材料:骨髓来源自体干细胞移植治疗糖尿病患者,均征得患者同意。方法:采用密度梯度法分离培养骨髓间充质干细胞,应用0.1μmol/L地塞米松、10mmol/Lβ-甘油磷酸钠、50μmol/L维生素C对第3代骨髓间充质干细胞进行诱导,同时设立对照组,不加入诱导剂。主要观察指标:相差显微镜观察骨髓间充质干细胞的形态变化,诱导后经碱性磷酸酶染色和钙结节染色观察细胞形态变化。结果:第3代骨髓间充质干细胞在相差显微镜下呈均匀分布生长,形态为长梭形。经成骨诱导7d后培养细胞逐渐汇合呈铺路石状,随着诱导时间的延长,局部细胞呈重叠生长,间充质逐渐堆积、间充质中矿盐沉积,形成多个结节,并逐渐融合。培养14d可形成倒置显微镜下可见的褐色点状矿化结节中心;培养21d形成小片状矿化结节,von Kossa法测定可见钙结节呈黑色,碱性磷酸酶活性测定为阳性。结论:骨髓间充质干细胞易诱导分化为成骨细胞,且增殖快,成骨能力强。
BACKGROUND： Cells grew slowly in a low number after conventional induction. Growth factor can accelerate induction speed, but growth factor is expensive. OBJECTIVE： To verify the efficiency of differentiation of human bone marrow mesenchymal stem cells （BMSCs） into osteoblasts in vitro in a inductor without growth factor. DESIGN, TIME AND SETTING： The control cytology observation experiment was conducted from July 2007 to February 2008 at the Center Laboratory of Hospital Affiliated to Medical College of Qingdao University. MATERIALS： Bone marrow was collected from diabetic patients undergoing autologous stem cell transplantation. Informed consent was obtained from patients. METHODS： Using density gradient method, BMSCs were separate and primary cultured. At the third passage, BMSCs were incubated in 0.1 μ mol/L dexamethasone, 10 mmol/L β -glycerine sodium, and 50 μ mol/L vitamin C. BMSCs in the control group were left intact. MAIN OUTCOME MEASURES： Morphology of BMSCs was observed under a phase contrast microscope. Morphological changes were measured by alkaline phosphatase staining and calcified nodules staining after induction. RESULTS： Under the phase contrast microscope, the third passage of BMSCs was uniform, spindle. At 7 days, cells were confluence. Some cells were overlapped over time. Mesenchymal tissues gradually piled up, with mineralization, multiple nodes. At 14 days, brown mineralized nodes were found under an inverted microscope. At 21 days, mineralization in fragmentis were formed. Von Kossa method demonstrated black calcified nodules and positive alkaline phosphatase. CONCLUSION： BMSCs can be induced into ostecblasts easily, with high and rapid proliferation.
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7483-7486,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
