【摘要】 目的:观察骨髓间充质干细胞在定向诱导分化条件下向成骨细胞、脂肪细胞分化的特征及相互关系。方法:实验于2004-10/2005-04在泸州医学院心肌电细胞实验室完成。日本大耳兔10只,经兔髂嵴无菌抽取骨髓,采用Percoll液进行梯度离心法和体外细胞贴壁分离培养法相结合对兔骨髓间充质干细胞进行分离、纯化,将获得的原代和传代培养的骨髓间充质干细胞,分别用含成脂诱导剂和含成骨诱导剂的改良的伊格尔培养基进行实验组(向脂肪细胞和成骨细胞的定向诱导培养,对诱导培养后的细胞进行生长形态学特征观察)照组(采用不加成脂、成骨诱导剂的伊格尔培养基),两组,对进行成脂及成骨诱导后的苏丹脂肪染色、碱性磷酸酶活性检测、胞外矿化基质测定,并进行细胞成脂和成骨率比较。结果:从10只实验动物中均成功提取到骨髓间充质干细胞,其中有2只动物的骨髓间充质干细胞在原代培养时出现污染,8只获得成功进入结果分析。①在经过21d成脂诱导培养后实验组的许多细胞胞浆内均出现较多的高折光性的脂滴,苏丹脂肪染色呈橘红色,同时Kossa法也检测出少许成骨分化,其比例为70%(28/40)干细胞可转化为含有脂滴的脂肪细胞;10%(4/40)干细胞转化为含有钙结节的成骨细胞。②经过21d向成骨诱导培养后实验组出现一些细胞基质逐渐堆积、并出现基质矿盐沉积,VonKossa法测定形成的钙结节,同时苏丹脂肪染色也检测出少许向脂肪细胞分化,其比例为40%(16/40)细胞可转化为成骨细胞,20%干(8/40)骨髓间充质干细胞可转化为脂肪细胞。③对照组用苏丹脂肪染色后显示5%(2/40)骨髓间充质干细胞分化为脂肪细胞,用VonKossa法测定矿化结节形成比例和碱性磷酸酶活性测定结果成骨率10%(4/40)。结论:在适当诱导条件下骨髓间充质干细胞可一部分转化为脂肪细胞,另一部分转化为成骨细胞,两者分化存在一定关系:分化的脂肪细胞多,则成骨细胞少;分化的成骨细胞多,则脂肪细胞少。更多还原

【Abstract】 AIM: To observe the characters and interactive correlation of adipogenic and osteogenic differentiation of mesenchymal stem cells derived from bone marrow. METHODS: The experiment was conducted in the Institute of Heart Electrophysiology, Luzhou Medical Collage from October 2004 to April 2005. By Percoll density gradient centrifugation, in vitro adherent culture, mesenchymal stem cells MSCs were isolated from rabbit bone marrow and were purified. After that, some MSCs were induced into adipotic cell in adipogenesis supplement medium, others were induced into osteogenetic cell in the osteogenesis supplement medium. The Sudan fat stain, activity of alkaline phosphatase was detected, extracellular mineralized matrix was determined, and the adipogenic and osteogenic rates were compared. RESULTS: MSCs were successfully derived from 10 rabbits, those in 2 rabbits occurred contamination at primary culture, and finally 8 rabbits were survived successfully. ① After 21 days, many lipid droplet of high refractivity occurred in many cytoplasm in the treatment group, the Sudan fat stain showed orange, and Kossa method also detected a few osteogenesis, approximately 70% 28/40 and 10% 4/40 of the MSCs were transformed into adipocytic and osteogenetic cells in medium containing adipogenesis supplement medium respectively. ② After 21 days, some cell matrix in the treatment group gradually accumulated, and matrix mineral deposition was observed, Von Kossa method detected that calcium nod formed, and the Sudan fat stain also detected that approximately 40% 16/40 and 20% 8/40 of MSCs were transformed into osteogenetic and adipocytic cells in medium containing osteogenesis supplement medium respectively. ③ Sudan fat stain showed that 5% 2/40 BMCs differentiated into adipocytic cells in the control group, the Von Kossa method detected that the proportion of mineralized nod formation and the osteogenetic rate of detected activity of alkaline phosphatase was 10% 4/40. CONCLUSION: MSCs have the capacity to undergo adipocytic and osteogenetic differentiation, what’s more, these results also demonstrate an inverse differentiation relationship between adipogenesis and osteogenesis in vitro.更多还原