【摘要】 背景:脂肪间充质干细胞分泌各种骨重塑需要的细胞因子和生长因子,被认为是骨再生的优良候选细胞。骨形态发生蛋白2与脂肪间充质干细胞对骨再生有协同作用,并能显著增强脂肪间充质干细胞的成骨分化作用。目的:探讨脂肪间充质干细胞成骨分化条件培养基与骨形态发生蛋白2联合应用对大鼠绝经后骨质疏松症的影响。方法:8-10月龄雌性SD大鼠75只,随机取60只采用卵巢切除方法建立绝经后骨质疏松症大鼠模型,余15只进行假手术,未切除卵巢。将60只造模成功大鼠随机分为4组:骨质疏松症组、条件培养基组、骨形态发生蛋白2组、联合治疗组,通过尾静脉分别注射DMEM培养基、脂肪间充质干细胞成骨分化条件培养基、骨形态发生蛋白2、脂肪间充质干细胞成骨分化条件培养基联合骨形态发生蛋白2。治疗12周,取各组大鼠股骨和血清,组织学观察骨小梁数量和结构以及骨小梁间距,ELISA检测血清P1NP、ALP、TRAP、OPG、RANKL水平,Western blot、Realtime PCR检测RANKL、OPG蛋白和m RNA水平,细胞因子芯片分析脂肪间充质干细胞成骨分化条件培养基中细胞因子水平。结果与结论:(1)与假手术组相比,骨质疏松症组大鼠的骨小梁间距扩大,骨小梁数量明显减少,骨小梁失去正常结构并且不连续。与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组大鼠骨小梁间距扩大较少,骨小梁结构更完整、更连续;(2)与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组大鼠血清P1NP和ALP水平显著升高(P <0.05),血清TRAP水平显著降低(P <0.05);(3)与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组RANKL/OPG比值显著降低(P <0.01),从而促进更多的骨形成;(4)脂肪间充质干细胞成骨分化条件培养基含有多种与骨形成密不可分的细胞因子,包括骨形态发生蛋白4和7、白血病抑制因子、脑源性神经营养因子、骨保护素、胰岛素样生长因子1等;(5)结果表明,脂肪间充质干细胞成骨分化条件培养基与骨形态发生蛋白2联合应用可减轻卵巢切除大鼠骨质疏松,可能成为治疗绝经后骨质疏松症的新方案。更多还原

【Abstract】 BACKGROUND: Adipose-derived mesenchymal stem cells(ADSCs) secrete various cytokines and growth factors required for bone remodeling, which are considered to be excellent candidate cells for bone regeneration. Bone morphogenetic protein 2(BMP2) and ADSCs have a synergistic effect on bone regeneration and can significantly enhance the osteogenic differentiation of ADSCs. OBJECTIVE: To explore the efficacy of conditioned medium of ADSCs combined with BMP2 on postmenopausal osteoporosis in a rat model. METHODS: Healthy and female Sprague-Dawley rats aged 8-10 months(n=75) were obtained from the Animal Center of Shenyang Medical College. Ovariectomy was performed to induce postmenopausal osteoporosis in 60 rats, and the remaining 15 rats underwent surgeries without removal of the ovaries(sham group). Ovariectomized rats were randomized into four groups: osteoporosis group, conditioned medium group, BMP2 group, and conditioned medium+BMP2 group, followed by injection of DMEM medium, ADSCs conditioned medium, BMP2, and ADSCs conditioned medium+BMP2 via the tail vein, respectively. After 12 weeks of treatment, femurs and serum samples of each group were taken. The number and structure of trabecular bone and trabecular spacing were detected histologically. Serum P1 NP, ALP, TRAP, OPG, RANKL levels were detected by ELISA. Western blot and real-time PCR were used to detect RANKL and OPG at protein and mRNA levels. Cytokine chip analysis of cytokines in ADSCs conditioned medium was performed. RESULTS AND CONCLUSION: Compared with the sham group, increased trabecular spacing, fewer trabeculae and marked trabecular disconnection were observed in osteoporosis rats. The trabeculae in conditioned medium+BMP2 group appeared to be more complete and continuous with less widened spacing compared with the osteoporosis, conditioned medium and BMP2 groups. Serum levels of P1 NP and ALP were dramatically higher, while TRAP level group was decreased significantly in the conditioned medium+BMP2 group compared with the other groups(both P < 0.05). In the conditioned medium+BMP2 group, RANKL/OPG ratio was reduced significantly compared with the other groups(P < 0.01), further promoting bone formation. ADSCs conditioned medium contained a variety of cytokines that were essential for bone formation and remodeling, including bone morphogenetic proteins 4 and 7, leukemia inhibitory factor, brain-derived neurotrophic factor, osteoprotegerin, and insulin-like growth factor 1. These results demonstrate that ADSCs conditioned medium combined with BMP2 can mitigate osteoporosis induced by ovariectomy and it may be an attractive strategy to treat postmenopausal osteoporosis.更多还原