系统性红斑狼疮模型鼠骨 BMP/Smads信号通路状态的研究

目的 :探讨狼疮鼠(MRL/lpr)骨BMP/Smads信号通路表达情况。方法 :分离小鼠股骨制备组织切片,常规苏木素-伊红(HE)染色,观察骨质情况;免疫组化观察骨组织中骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)表达;密度梯度离心法和贴壁筛选法分离培养骨髓间充质干细胞(bone marrow masenchymal stem cells,BMMSCs),细胞爬片后免疫荧光法观察BMP-2、p-Smad1/5/8蛋白表达情况;BMP-2诱导BMMSCs向成骨细胞分化,RT-PCR法检测BMMSCs ALP、Runx2基因m RNA水平,ALP染色鉴定早期成骨情况。结果:狼疮鼠骨皮质较正常鼠减少,皮质骨占骨体积比例较正常鼠减低(P<0.01);狼疮鼠股骨BMP-2表达与正常鼠无明显差别(P>0.05);细胞免疫荧光显示狼疮鼠BMMSCs BMP-2、p-Smad1/5/8表达减弱(P<0.05);狼疮鼠BMMSCs BMP-2刺激7 d后ALP活性较正常鼠减低,BMP-2刺激3 d后ALP m RNA水平较正常鼠减弱(P<0.01),Runx2 m RNA水平较正常鼠无明显差别(P>0.05)。结论 :狼疮鼠骨BMP/Smads信号通路处于抑制状态。

Status of BMP/Smads signaling on the bone from mouse model of systemic lupus erythematosus

Objective:To investigate the status of BMP/Smads signaling pathway on the bone of lupus mice(MRL/lpr). Methods:The femurs of the mice were isolated,and the bone structure was detected by hematoxylin-eosn(HE)staining. The protein expression levels of bone morphogenetic protein-2 (BMP-2)were measured by immunohistochemistry. Bone marrow mesenchymal stem cells(BMMSCs)were isolated by the density gradient centrifugation and the adherence screening methods. After BMMSCs growing on coverslips,the protein of BMP-2 and PSmad1/5/8 was valued by immumofluorescence method. The mRNA expressions of ALP,Runx2 of BMMSCs were measured by Real-time PCR,and the Alkaline phosphatase (ALP)staining was performed for measuring the early osteogenic differentiation induced by BMP-2. Results:Compared with the controlled mice,the cortex of MRL/lpr mice were reduced (P < 0.01). There was no difference in the BMP-2 expression on the bone of the two groups by immunohistochemistry detection(P > 0.05). The expressions of BMP-2 and PSmad1/5/8 in BMMSCs of lupus mice were lower than those of the control group(P < 0.05). After 7 days of BMP-2 stimulation,ALP activities of BMMSCs from lupus mice were decreased compared with the control group. The mRNA levels of ALP in lupus mice were lower than that of C3He/HeJ mice (P < 0.01),while there were no differences in the Runx2 mRNA levels between the two groups after BMP-2 stimulation(P > 0.05). Conclusion:BMP/Smads signaling was inhibited in the bone of lupus mouse.