大鼠骨髓基质干细胞定向分化为成骨细胞及体外增殖过程中地塞米松的抑制性干预

Inhibitory effect of dexamethasone on the proliferation and differentiation of bone marrow stromal cells into osteoblasts in rats

目的:地塞米松在体外诱导骨髓基质干细胞向成骨细胞分化过程中起着关键性作用。验证骨髓基质干细胞向成骨细胞分化的能力,观察成骨细胞分化早期地塞米松对骨髓基质干细胞体外增殖的抑制效果。方法:实验于2006-09/12在南方医科大学组织工程研究中心完成。①实验方法:取5周龄雄性SD大鼠10只,经颈椎脱位法处死后取股骨,去除双侧干骺端,用DMEM高糖完全培养基冲洗骨髓腔,收集骨髓细胞,离心后按(1～2)×107L-1密度接种,加入条件培养液(DMEM高糖完全培养基,体积分数为0.1的标准胎牛血清,50mg/L维生素C,10mmol/L的B-甘油磷酸钠,100U/mL青霉素、100U/mL链霉素)进行体外培养。分别于细胞传代培养后第0,2,4天向培养基中加入1μmol/L地塞米松1mL,并设立仅加入等量培养基的空白对照组。②实验评估:以2d为间隔,倒置显微镜下观察细胞生长情况。采用CellTiter96试剂盒各组细胞增殖情况。结果:①骨髓基质干细胞向成骨细胞的分化:原代培养中贴壁细胞多呈长梭形,少数呈小圆形或三角形。原代培养六七天后进行传代,多数细胞在加入地塞米松后逐渐呈均一的长梭形,随着时间延长呈叠形多层排列,细胞外基质明显增多,并逐渐形成多个小结样结构。空白对照组细胞形态欠均一,少数细胞呈多边形或三角形,细胞外基质明显少于地寒米松组,罕见小结样结构。传代后10～12d可达80%～90%致密层,细胞生长速度较原代细胞明显增快,至第10代细胞仍未出现衰老现象。②骨髓基质干细胞的增殖检测:与空白对照组比较,细胞传代培养后第0,2,4天加入地塞米松,干预处理8,10,12d时的细胞数量均明显下降(t=5.0445～11.3795,P均<0.01)。结论:①传代的骨髓基质干细胞经地塞米松处理后,细胞形态趋于成熟,生长速度加快,可定向分化为成骨细胞。②在向成骨细胞分化早期,地塞米松能够抑制骨髓基质干细胞的体外增殖。

AIM： Dexamethasone plays important roles in the differentiation of bone marrow stromal cells （BMSCs） into osteoblasts in vitro. The article is designed to verify the potential of BMSCs differentiating into osteoblasts, and observe the inhibitive effect of dexamethasone on the proliferation of BMSCs during in early differentiation into osteoblasts. METHODS： The experiment was performed at the Research Center of Tissue Engineering, Southem Medical University from September to December 2006. （1）Totally 10 5-week-old male SD rats were selected. These rats were killed by dislocation of cervical vertebra to obtain femur and remove bilateral metaphysis, Medullary cavity was washed with DMEM high glucose complete medium, Bone marrow cells were collected, centrifugated and inoculated at （1-2）×10^7 L^-1, and then cultured in DMEM high glucose complete medium containing fetal calf serum （FCS） of 0.1 volume fraction, 50 mg/L vitamin C, 10 mmol/L B sodium glycerophosphate, 100 U/mL penicillin and 100 U/mL streptomycin in vitro. 1 μmol/L dexa＇methasone 1 mL was added in the medium immediately, at days 2 and 4 after cell serial subcultivation, respectively. The blank control group was treated with complete medium of the same volume. （2）Cell growth was observed under inverted microscope every three days. Cell proliferation in the two groups was determined by Cell Tit er 96 kit. RESULTS： （1）BMSCs differentiating into osteoblasts： Adherent cells were long fusiform mostly, and few were small round or triangle. Six or seven days after primary culture, most BMSCs showed even long fusiform after intervened with dexamethasone. With the prolongation of time, these cells with more extracellular matrix were multiplayer shape, and gradually formed many nod-like structures. BMSCs in the blank control group were not even; a few cells showed polygon or triangle; extracellular matrix was significantly less than that in the dexamethasone group and nod-like structure was rare. At days 10-12 after passage, about 80%-90% compactum appeared, and BMSCs grew rapidly as compared with the primary cells. No senilism BMSCs were found in the 10^th generation cells. （2）Proliferation of BMSCs： The BMSCs were treated with dexamethasone immediately, at days 2 and 4 after cell passage, and cell number significantly reduced at days 8, 10 and 12 as compared with the blank control group （t =5.044 5-11,379 5, P 〈 0.01 ）. CONCLUSION： （1）After intervened with dexamethasone, BMSCs become mature, grow rapidly and can orientedly differentiate into osteoblasts. （2）Dexamethasone inhibits BMSCs proliferation in early differentiation into osteoblasts.