瘦素对hBMSCs成骨分化的作用及机制研究

EFFECT AND MECHANISM OF LEPTIN ON OSTEOBLASTIC DIFFERENTIATION OF hBMSCs

目的探讨瘦素(leptin,LEP)对hBMSCs成骨分化的作用及机制。方法采用全骨髓法培养hBMSCs,取第3代hBMSCs分为A、B、C、D、E、F组,待细胞贴壁后各组分别加入400、200、100、50 ng/mL LEP、100 ng/mL BMP和普通培养基2 mL进行培养。于培养后7 d行ALP染色、21 d行矿化结节染色,倒置相差显微镜观察染色结果;并于培养后7、14、21 d,检测各组ALP活性、骨钙素(osteocalcin,OCN)水平,筛选LEP的最佳诱导浓度;B、E、F组细胞培养7 d后,采用RT-PCR检测成骨相关基因:核心结合因子(core-binding factorα1,Cbfα1)、ALP、ColⅠ、OCN mRNA的表达。结果诱导培养7 d,ALP染色结果显示,A、B、C、D、E组细胞胞浆均呈蓝色阳性着色,F组呈弱阳性;诱导培养21 d,矿化结节染色结果显示,A、B、C、D、E组细胞染色呈阳性,F组呈阴性。B组培养后7、14、21 d,ALP、OCN活性低于E组,但显著高于其余各组,差异均有统计学意义(P<0.05),200 ng/mL LEP为最佳浓度。B、E、F组细胞培养7 d,F组Cbfα1、ALP、ColⅠ、OCN mRNA均不表达;B、E组各产物mRNA均有表达,但B组低于E组。结论LEP可促进hBMSCs成骨分化,其机制可能为LEP促进了成骨相关基因的表达。

Objective To investigate the effect and mechanism of leptin （LEP） on the osteoblastic differentiation of hBMSCs in vitro. Methods Whole bone marrow culture method was applied to culture hMBSCs and hBMSCs at passage 3 were divided into groups A, B, C, D, E and F, and when cell attachment was evident, 400, 200, 100 and 50 ng/mL LEP, 100 ng/mL BMP and common nutrient medium were added into each group, respectively. ALP staining and mineralized nodules staining were conducted at 7 and 21 days after culture, respectively. And inverted phase contrast microscope observation was performed. ALP activity and osteocalcin （OCN） level of hBMSCs in each group was detected at 7, 14 and 21 days after culture to select the best induced concentration of LEP on osteoblastic differentiation. For groups of B, E and F at 7 days after culture, RT-PCR was adopted to detect the expression of such osteogenesis-related genes as core-binding factor α 1 （Cbfα1）, ALP, Col I and OCN mRNA. Results At 7 days after induced culture, the ALP staining result showed that the endochylema in groups A, B, C, D and E were stained blue and the endochylema in the group F was slightly positive. At 21 days after induced culture, the mineralized nodules staining showed that cells in groups A, B, C, D and E were stained positively and cells in group F were negative. At 7, 14 and 21 days after culture, ALP and OCN activities in group B were less than that of group E （P 〈 0.05）, but significant higher than that of groups A, C, D and F （P 〈 0.05）, the optimal concentration of LEP was 200 ng/mL. At 7 days after culture, group F witnessed no expression of Cbfα1, ALP, Col I and OCN mRNA, while groups B and E witnessed expressions of all those indexes, but the expressions in group B were less than those of group E. Conclusion LEP can stimulate osteoblastic differentiation of hBMSCs in vitro, and the possible mechanism is its role of promoting the expression of osteoblastic related genes.