【作者】 朱伟；

【导师】 孙俊英；

【作者基本信息】 苏州大学 ， 骨外科学， 2006， 硕士

【摘要】 第一部分成人骨髓间充质干细胞向成骨细胞定向诱导分化的实验研究目的:研究体外培养的成人骨髓间充质干细胞(MSCs)在特定的条件下定向诱导分化为成骨细胞,探讨其作为骨组织工程种子细胞的可行性和应用价值。方法:从骨髓血中提取MSCs,在含10%胎牛血清的高糖DMEM培养基中培养,以流式细胞仪检测MSCs的表面抗原表达。培养传代后改用含地塞米松(1 x10-8 mol/L) ,β一甘油磷酸钠(10 mmol/L )和维生素C(50 mg/L)的条件培养基培养,在相差显微镜下观察细胞形态并绘制生长曲线。免疫组化检测I型胶原(COL-I)、骨钙素(osteocalcin, OC)。NAP法进行碱性磷酸酶(ALP)染色、von Kossa法进行钙结节染色,并进行统计学分析。结果:MSCs增殖能力活跃,流式细胞仪检测MSCs显示CD105,CD44,表达阳性,CD14、CD34和CD45表达为阴性。成骨诱导培养后,可见碱性磷酸酶( ALP)染色、Ca结节染色、胶原-I (COL-I)和骨钙素(osteocalcin, OC)免疫组化染色阳性。结论:MSCs取材安全方便,易于诱导分化为成骨细胞,是研究骨组织工程的理想种子细胞。第二部分MSCs与生物活性玻璃45S5的体外生物相容的实验研究目的:研究成人MSCs与生物活性玻璃45S5在体外培养条件下的生物相容性。方法:分离培养人MSCs,置于含体积分数为10%的胎牛血清DMEM培养基中培养,传代后改用含地塞米松、β-甘油磷酸钠和维生素C的条件培养基培养,分为45S5复合细胞组和单纯细胞组,不同时间用倒置相差显微镜、透射电镜及扫描电镜观察细胞增殖和分化。细胞计数、MTT法及流式细胞仪进行细胞增殖测定,进行ALP的定量检测。并用von Kossa法行钙结节染色,结果:成人MSCs可以诱导为成骨细胞,成骨细胞体外培养时复合或不复合45S5均生长良好,表现出典型成骨细胞的形态特征和生物学特性,接种于生物活性玻璃45S5支架材料上的细胞生长良好,形成许多细小的钙结节和胶原纤维。45S5利于细胞的贴附、生长与增殖,并对细胞的功能无不良影响。结论:生物活性玻璃45S5是较理想的骨组织工程支架材料,用MSCs作为种子细胞,生物活性玻璃45S5作为支架构建组织工程更多还原

【Abstract】 Part I Induced differentiation of adult HMSCs in vitro toward osteoblastsObjective :To investigate the feasibility of inducing in vitro mesenchymal stem cells derived from adult human bone marrow differentiate into osteoblasts and potential applicability of the MSCs as the seed cells in tissue engineering. Methods: Mesenchymal stem cells were extracted from the bone marrow in blood and cultured immediately in DMEM medium containing 100 m L/L fetal bovine serum. hMSCs were analyzed by the flow cytometry to detect the surface antigens. The subcultured cells was induced to differentiate into osteoblasts with an osteoinductive medium containing dexamethasone (1x10-8 mol/L), beta-sodium glycerophosphate (10 mmol/L) and ascorbic acid (50 mg/L). Cellular morphologies were observed under phase-contrast microscope and the growth curve was drawn accordingly.. The collagen type I and osteocalcin was detected by immunohistochemistry, alkaline phosphatase in the MSCs stained by NAP, the calcified nodules were stained by von Kossa method. Results: The MSCs proliferated rapidly in in vitro culture. Flow cytometry analysis showed that in vitro expanded hMSCs expressed mesenchymal cell marker, including CD105、CD44、and didn’t express CD14、CD34、CD 45. The staining of the ALP,COL-I,osteocalcin and calcified nodules were positive after osteoinduction. Conclusions:Human bone marrow-derived MSCs can be induced to differentiate into osteoblasts through relatively simple procedures,.Therefore the MSCs is perfect seeding cells for bone tissue engineering.更多还原