【摘要】 目的探讨骨形态蛋白2(BMP-2)/Smads信号通路在低剂量X射线促成骨细胞分化及矿化中的作用。方法将成骨细胞分为照射组及对照组,照射组给予单次0.1 Gy X射线照射,对照组处于相同的环境下不进行X射线照射。照射后24、48、72 h,采用CCK-8法检测细胞增殖活性,采用对硝基苯磷酸二钠法检测细胞碱性磷酸酶(ALP)活性,采用茜素红染色剂进行细胞矿化染色、十六烷基吡啶试剂进行定量;利用RT-PCR检测BMP-2、Smad1、Smad5、Smad4、成骨相关因子2(Runx2)、锌指结构转录因子(Osterix)及骨钙素(OCN)的mRNA表达;另取成骨细胞分为0.1 Gy组、0.1 Gy拮抗剂组、control组及control拮抗剂组。0.1 Gy组、control组处理同上,两个拮抗剂组分别在0.1 Gy组及control组处理的基础上加入浓度为10μg/m L的头蛋白(NOGGIN)拮抗剂0.5 mg,照射72 h后,分别采用Western blotting及ELISA法检测上述各指标蛋白的表达,并进行各组间的比较。结果照射组与对照组各时间成骨细胞增殖活性比较差异无统计学意义(P均>0.05)。与对照组比较,照射后48 h,照射组ALP活性升高(P<0.05);照射后72 h,照射组细胞矿化定量增加(P<0.05);照射后48 h,照射组BMP-2、Smad1、Smad5、Smad4、Runx2、Osterix及OCN的mRNA表达上调(P均<0.05);照射后72 h,照射组Smad1 mRNA相对表达量高于对照组(P<0.05)。照射后72 h,0.1 Gy组BMP-2、Smad1、Smad4、Smad5、Runx2、Osterix及OCN的蛋白相对表达量高于control组(P均<0.05);0.1 Gy拮抗剂组与control拮抗剂组上述各指标蛋白表达均下降,两组间各指标蛋白表达差异无统计学意义(P均>0.05)。结论 BMP-2蛋白在低剂量X射线照射的成骨细胞中表达升高,其可通过其自分泌/旁分泌方式激活细胞Smads信号通路进而调控下游成骨基因的表达,从而促进成骨细胞分化与矿化。更多还原

【Abstract】 Objective To investigate the role of bone morphogenetic protein-2(BMP-2)/Smads signal pathway in low-dose X-irradiation promoting the osteogenic differentiation and mineralization of osteoblastic UMR-106 cells. Methods The osteoblasts were divided into the irradiation group and control group. The irradiation group was given a single 0. 1 Gy X-ray irradiation,and the control group was not subjected to X-irradiation in the same environment. At 24,48,and 72 h after irradiation,the cell proliferation and cellular alkaline phosphatase(ALP) activity were evaluated by CCK-8 assay and Disodium p-Nitrophenyl phosphate tetrahydrate(p NPP),and we used Alizarin Red Stainer for the cellular mineralization and quantitative cetyl pyridine reagent. The mRNA and protein levels of BMP-2,Smad1,Smad5,Smad4,Runt-related transcription factor 2(Runx2),Osterix,and osteocalcin(OCN) were assessed by RT-PCR,Western blotting,and ELISA. The other osteoblasts were divided into 0. 1 Gy group,0. 1 Gy antagonist group,control group and control antagonist group. The 0. 1 Gy group and control group were treated as the above. Two antagonist groups were added with 10 μg/m L of NOGGIN antagonist 0. 5 mg on the basis of the same treatment of the 0. 1 Gy group and the control group,and then the protein expression of the above indexes was detected and compared. Results There was no significant difference in osteoblast proliferation activity between the irradiation group and the control group at each time point(P > 0. 05). At 48 h,the ALP activity of the irradiation group significantly increased as compared with that of the control group(P < 0. 05). At72 h after irradiation,the quantification of cell mineralization in the irradiation group increased(P < 0. 05). At 48 h after irradiation,the mRNA expression of BMP-2,Smad1,Smad5,Smad4,Runx2,Osterix,and OCN was up-regulated in the irradiation group(all P < 0. 05). At 72 h after irradiation,the expression of BMP-2,Smad1,Smad5,Smad4,Runx2,Osterix,and OCN was up-regulated in the irradiation group(all P < 0. 05). At 72 h after irradiation,the NOGGIN antagonists inhibited the expression of these above proteins(all P < 0. 05). There was no significant difference in the expression of the above indicators between the 0. 1 Gy antagonist group and the control antagonist group(all P > 0. 05). Conclusion BMP-2 protein expression is up-regulated in the osteoblasts under low-dose X-irradiation,and the mechanism may be that the autocrine/paracrine mode of BMP-2 protein activates the Smads signaling pathway and regulates the expression of downstream osteogenic genes,and thus promotes the differentiation and mineralization of osteoblasts.更多还原