摘要
目的:观察在有成骨诱导剂存在的条件下,大鼠骨髓基质干细胞向成骨细胞转化的能力。方法:实验于2005-07/12在锦州医学院中心实验室完成。选取清洁级2月龄SD大鼠6只,无菌条件下取双侧股骨,制备单细胞悬液。采用贴壁培养与传代结合方法分离纯化大鼠骨髓基质干细胞,将2代细胞置于含有1×10-7mol/L地塞米松、10mol/Lβ-甘油磷酸钠、50mg/L抗坏血酸成骨诱导剂的培养基中,培养21～30d。应用倒置显微镜观察骨髓基质干细胞与诱导后细胞形态,描绘生长曲线,流式细胞仪检测细胞周期,并用碱性磷酸酶染色和VON-KOSSA法检测成骨能力。结果:6只大鼠均进入结果分析。①骨髓基质干细胞形态学观察结果:在成骨诱导剂里细胞增殖变缓慢,呈长梭形、成纤维细胞样或不规则形。②细胞生长曲线:1～2d为潜伏期,细胞增殖不明显;3d后细胞增殖明显加快,进入对数生长期;6d后增殖变慢为平台期。经计算细胞群体倍增时间为38h。③细胞增殖周期检测结果:G0/G1期为(82.12±4.60)%,S期为(14.35±2.32)%,G2/M期为(0.87±0.30)%。④成骨能力检测结果:细胞碱性磷酸酶染色阳性率为86%,VON-KOSSA染色提示有钙结节形成。结论:大鼠骨髓基质干细胞在有成骨诱导剂存在的情况下成骨能力较高。
AIM： To study the osteogenic ability of bone marrow stromal stem cells （BMSCs） with the existing bone-forming agents in rats. METHODS： The experiment was done at the Central Laboratory of Jinzhou Medical College between July and December in 2005. Six clean degree SD rats of 2 months old were selected to take out the thigh bone of both sides under the aseptic conditions for preparing the single cell suspension. The BMSCs were purified by the coalesce culture of adhesion and passage, then the cells of passage 2 were cultured in the L-DMEM medium including 1× 10^-1 mol/L dexamethasone, 50 mg/L ascorbic acid-2- phosphate and 10 mol/L β-glycerolphosphate for 21-30 days. The BMSCs and induced cells were observed by invert microscope. The growth curve was drawn and the cell cycles were tested by flowing cytometry. The ability of osteogenic differentiation was evaluated by alkaline phosphatase （ALP） staining and Von-Kossa method.
RESULTS： The data of six rats were all analyzed.①The morphologic results of BMSCs： In osteogenic induced medium, the cells grew slowly and displayed spindle shape, fibroblast-like or irregular shape.②The cell growth curve： Within 1-2 days that was latency period, it was no obvious cell proliferation; Three days later, the cells grew fast obviously and came into logarithmic phase; After 6 days, the cells grew slowly and this phase was called plain phase. The cell double time was 38 hours by calculating.③The result of cell cycle： G0/G1 was （82.12±4.60）%, S was （14.35±2.32）%, G2/M was （0.87±0.30）%. ④The result of osteogenesis： The positive rate was 86.0% by ALP staining and Von-Kossa staining revealed there were calcium nodules appeared. CONCLUSION： The osteogenic ability of rat BMSCs is quite high on the condition of existence of bone-forming agents.
出处
《中国临床康复》
CSCD
北大核心
2006年第37期11-13,i0001,共4页
Chinese Journal of Clinical Rehabilitation
