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背景:利用骨髓间充质干细胞的多方向分化潜能,选择合适的生物材料和适当的生长因子联合应用,可达到修复组织缺损的目的。目的:观察兔骨髓间充质干细胞的生长特点及其在诱导条件下的成骨能力。设计:单一样本实验。单位:右江民族医学院组织胚胎学教研室、右江民族医学院附属医院。材料:实验于2004-06在右江民族医学院组织胚胎学教研室完成。新西兰大白兔,雄性,体质量2.0～2.5kg。方法:抽取兔骨髓组织,梯度离心后,保留贴壁细胞传代,稳定传代后改用诱导成骨培养液(50mL/L胎牛血清的Dulbecco改良培养基含10mmol/Lβ-甘油磷酸钠,10nmol/L地塞米松,50mg/L维生素C)进行培养,隔日换培养液1次。通过倒置显微镜观察、碱性磷酸酶染色、骨连接素、骨桥素免疫细胞化学染色等方法观测骨髓间充质干细胞的成骨特性。主要观察指标:①骨髓间充质干细胞的形态学观察。②骨髓间充质干细胞的成骨特性。结果:①骨髓间充质干细胞的形态学观察:原代兔骨髓间充质干细胞七八天即可长满,并可稳定传代,传代细胞五六天即可传代。②骨髓间充质干细胞的成骨特性:碱性磷酸酶免疫组化染色可见胞浆中出现大量棕褐色颗粒,对照染色细胞胞浆未见着色;骨连接素、骨桥素免疫组织化学检测可见胞核染成淡蓝色,胞浆内出现大量的棕黄色颗粒,呈明显强阳性,对照染色中胞浆内没有出现黄色颗粒。结论:兔骨髓间充质干细胞用地塞米松、维生素C和β-甘油磷酸钠培养液联合诱导培养后,符合成骨细胞的形态特征和生物学特性,具有成骨活性,可在短期内为体外构建组织工程化骨提供自体种子细胞。
BACKGROUND: The purpose of repairing tissue defects is to utilize the potential of multidirectional differentiation of bone marrow-derived mesenchymal stem cells and select the suitable biomaterials for combination with appropriate growth factors. OBJECTIVE: To observe the growth characteristics of rabbit bone marrow mesenchymal stem cells and their osteogenic ability under induction. Design: Single sample experiment. Unit: Youjiang Medical College for Histology Department of Embryology, Youjiang Medical College Affiliated Hospital. MATERIALS: Experiments were performed at the Department of Embryology, Youjiang Medical College for Nationalities in June 2004. New Zealand white rabbits, male, body mass 2.0 ~ 2.5kg. Methods: Rabbit bone marrow tissue was collected and the adherent cells were preserved after gradient centrifugation. Stably passaged, the osteoblast culture medium (50mL / L fetal bovine serum supplemented with 10mmol / L sodium β-glycerophosphate and 10nmol / L dexamethasone, 50mg / L vitamin C) for culture, alternate culture medium every other day. The osteogenic properties of bone marrow mesenchymal stem cells were observed by inverted microscope, alkaline phosphatase staining, osteonectin and osteopontin immunocytochemical staining. MAIN OUTCOME MEASURES: ① Morphological observation of bone marrow mesenchymal stem cells. ② Bone marrow mesenchymal stem cells osteogenic properties. Results: (1) Morphological observation of bone marrow mesenchymal stem cells: Primary rabbit bone marrow mesenchymal stem cells can grow over seven or eight days, and can be passaged stably. The passage cells can be passaged for five or six days. Osteoblasts of bone marrow mesenchymal stem cells: Alkaline phosphatase immunohistochemical staining showed a large number of cytoplasm brown particles, the control staining cytoplasm no staining; osteonectin, osteopontin immunohistochemistry can be seen Nuclei stained light blue, a large number of brown granules appeared in the cytoplasm, was strongly positive, no yellow granules cytoplasm control staining. CONCLUSION: Rabbit bone marrow mesenchymal stem cells cultured with dexamethasone, vitamin C and β-glycerophosphate can induce morphological and biological characteristics of osteoblasts with osteogenic activity and can be used in vitro for a short period of time Build tissue-engineered bone to provide autologous seed cells.