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目的 研究大鼠骨髓基质细胞 (r MSCs)的生长特点和在诱导条件下的成骨特性。方法 使用密度梯度法分离成年大鼠骨髓基质细胞进行培养 ,保留贴壁细胞传代 ,观察、测试在培养液中添加成骨诱导剂地塞米松 (Dex) 10 - 8mol/ L、β-甘油磷酸钠 (β- GP) 10 mm ol/ L,抗坏血酸 (AA) 5 0 μg/ ml条件下骨髓基质细胞的生长变化和成骨分化。结果 形态学观察表明 ,r MSCs贴壁细胞呈集落生长 ,有成纤维细胞样外观。利用 MTT法和流式细胞术 (FCM)测定增殖的结果表明 ,传代次数增加 ,r MSCs的增殖活性升高。成骨诱导剂促进骨髓基质细胞的增殖 ,而且对多次传代细胞促增殖作用明显。成骨诱导剂促进骨髓基质细胞成骨 ,在诱导 3周时即出现明显的钙化结节。组织化学染色结果显示 ,非诱导条件下 MSCs的碱性磷酸酶 (AL P)水平会随传代而升高。传 3代 r MSCs的 AL P的表达较弱 ,诱导一周后 AL P的表达明显升高 ,超过未加诱导剂的传一代大鼠成骨细胞 (OB)的 AL P表达。结论 本实验表明所培养的 r MSCs仍处于低分化水平 ,具有骨祖细胞的特性。
Objective To study the growth characteristics and osteogenic properties of rat bone marrow stromal cells (r MSCs) under induction conditions. Methods Adult rat bone marrow stromal cells were isolated and cultured using density gradient method. The adherent cells were preserved for passage. The osteoblasts were treated with dexamethasone (10 - 8mol / L), sodium β - glycerophosphate (β-GP) 10 mm ol / L, ascorbic acid (AA) 50 μg / ml in bone marrow stromal cells growth and osteogenic differentiation. Results Morphological observation showed that the adherent cells of r MSCs showed colony growth with fibroblast-like appearance. The results of proliferation assay by MTT assay and flow cytometry (FCM) showed that the proliferation of r MSCs increased after the passage of time. Osteogenic inducers promote the proliferation of bone marrow stromal cells, but also promote the proliferation of multiple subcultures. Osteogenic inducers promote bone marrow stromal cells to osteoblast, and obvious calcified nodules appeared at 3 weeks after induction. Histochemical staining results showed that the ALP level of MSCs increased with the passage of time under non-induced conditions. The expression of AL P in the third generation of r MSCs was weak, and the expression of AL P in the first week after induction was significantly higher than that in the primary cultured rat osteoblasts without any inducer. Conclusion This experiment shows that the cultured r MSCs are still at a low level of differentiation and have the characteristics of osteoprogenitors.