不同浓度1,25-(OH)_2维生素D_3对体外培养恒河猴骨髓基质干细胞的影响

目的 观察1,25-(OH)_2维生素D_3[1,25-(OH)_2D_3]对体外培养的恒河猴骨髓基质干细胞(bone marrow stromal stem cells,BMSC)分泌骨保护素和NF-κB受体活化因子配体(RANKL)的影响.方法 设置了10~(-12)、10~(-10)、10~(-8)moL/L 3种浓度的1,25-(OH)_2D_3试验组和一个空白对照组.在体外用含这3种浓度1,25-(OH)_2D_3的培养基和空白培养基连续培养恒河猴BMSC.通过四唑盐(MTT)比色法观察细胞增殖.通过酶联免疫吸附测试法(ELISA)检测碱性磷酸酶、骨钙素、骨保护素、RANKL的含量.通过茜红素染色检测矿化结节.结果 不同浓度的1,25-(OH)_2D_3对恒河猴BMSC增殖的影响均表现为少于7 d促进增殖,超过7 d抑制增殖,但没有明显的剂量依赖性(均P<0.05).1,25-(OH)_2D_3促进恒河猴BMSC向成骨细胞方向分化,各浓度组成骨分化指标碱性磷酸酶(ng/ml,7 d:31.40±1.25,26.50±0.50,28.47±0.25对13.48±0.26;10 d:33.37±0.68,35.30±1.57,33.27±0.67对17.14±0.55;13 d:35.37±0.12,30.47±0.25,30.27±1.25对16.55±1.13;均P<0.05)和骨钙素(ng/ml,7 d:4.47±0.29,4.00±0.60,3.73±0.78对1.63±0.55;10 d:5.63±0.57,5.17±0.15,4.30±0.10对2.17±0.15;13 d:7.03±0.15,5.53±0.25,5.27±0.31对2.23±0.55;均P<0.05)均较对照组明显升高,但各浓度组均未见典型矿化结节的形成.1,25-(OH)_2D_3促进恒河猴BMSC分化出的成骨细胞分泌骨保护素(pg/ml,7 d:72.57±0.67,68.00±1.75,64.23±0.87对30.13±1.72;10 d:62.03±1.62,51.80±1.30,28.93±0.95对18.13±1.40;13 d:65.13±0.71,62.43±2.11,44.93±1.63对36.70±0.95;均P<0.05)和RANKL(pg/ml,7 d:74.33±0.61,82.37±2.15,85.23±0.45对70.83±1.71;10 d:83.30±0.46,86.70±0.56,88.23±0.91对74.20±1.83;13 d:81.70±1.81,81.07±0.95,84.70±1.41对72.73±0.97;均P<0.05),RANKL的分泌量呈现先升高后下降的趋势,而骨保护素的分泌量则呈现出相反的变化,两者均表现出剂量依赖性.结论 1,25-(OH)_2D_3促进恒河猴BMSC向成骨细胞方向分化,并促进其分泌骨保护素和RANKL.

Effects of 1,25-(OH)_2-vitamin D_3 on proliferation, differentiation, and secretion of osteoprotegerin and RANKL in cultured marrow mesenchymal cells of rhesus monkey

Objective To study the effects of 1,25-(OH)_2-vitamin D_3[1,25-(OH)_2D_3] on proliferation,differentiation, and secretion of osteoprotegerin and RANK ligand (RANKL)in cultured marrow mesenchymal cells of rhesus monkey (RhBMSCs). Methods Three different concentrations of 1,25-(OH)_2D_3 (10~(-12) ,10~(-10), and 10~(-8)mol/L)were added to the cultured RhBMSCs in vitro. MTT was used to observe cell proliferation and ELISA technique was used to measure the concentration of alkaline phosphatase (ALP), osteocalcin, osteoprotegerin, and RANKL. Mineralization nodus was identified via alizarin red staining. Results Under different concentration of 1,25-(OH)_2D_3, RhBMSCs proliferation were promoted within 7 days but were suppressed beyond 7 days. No significant dose-dependent manner was found. Differentiation of RhBMSCs into osteoblast was promoted by 1,25-(OH)_2D_3. The levels of ALP and osteocalcin in groups with various concentrations of 1,25-(OH)_2D_3 were higher than those of control group [ALP(ng/ml) ,7 d:31.40±1.25,26.50±0.50,28.47± 0.25 vs 13.48±0.26;10 d:33.37±0.68,35.30±1.57,33.27±0.67 vs 17.14±0.55;13 d:35.37±0.12,30.47± 0. 25 , 30. 27±1.25 vs 16.55 ± 1.13 ; osteocalcin (ng/ml), 7 d:4.47±0. 29,4.00 ±0. 60,3.73±0.78 vs 1.63± 0.55;10 d:5.63±0.57,5.17±0.15,4.30±0. 10 vs 2.17±0. 15;13 d:7.03±0.15,5.53±0.25,5.27±0.31 vs 2.23±0. 55 ; all P < 0. 05], but no typical mineralization nodus was found in either group. Secretions of osteoprotegerin and RANKL from RhBMSCs were stimulated by 1,25-(OH)_2D_3 [osteoprotegerin (pg/ml), 7 d: 72.57±0.67,68.00±1.75,64.23±0.87 vs 30. 13±1.72; 10 d:62.03±1.62,51.80±1.30,28.93±0.95 vs 18.13±1.40;13 d:65.13±0.71,62.43±2.11,44.93+1.63 vs 36.70±0.95 ;RANKL(pg/ml) ,7 d:74.33+0.61, 82.37±2.15,85.23±0.45 vs 70.83±1.71 ;10 d:83.30±0.46,86.70±0.56,88.23±0.91 vs 74.20±1.83;13 d: 81.70±1.81,81.07±0.95,84.70±1.41 vs 72.73±0.97 ;all P<0.05]. The secretion of RANKL was increased at first and then decreased, whereas the secretion of osteoprotegerin had the opposite tendency. The secretions of RANKL and osteoprotegerin were both in a dose-dependent manner. Conclusions 1,25-(OH)_2D_3 promotes differentiation of RhBMSCs into osteoblasts,resulting in increased secretions of osteoprotegerin and RANKL