大鼠脊髓损伤后骨髓间充质干细胞源性成骨细胞内信号传导通路的变化

摘要

Objective To investigate the changes in Wnt/β-catenin,bone morphogenic protein (BMP),estrogen receptor (ER) and insulin-like growth factor (IGF) signaling pathways in bone marrow mesenchymal stem cells (BMSCs) differentiation to the osteoblasts after spinal cord injury (SCI) and understand the mechanism of osteoporosis after SCI.Methods Forty 6-week-old male rats were divided into SCI group (n =20) and control group (n =20) according to the random number table.Rats in SCI group were submitted to laminar osteotomy at T10-12 and given lower thoracic cord sharp transection.In control group,rat lower thoracic cord was only exposed without transaction.Femoral bone marrow density (BMD) of rat right side was determined at postoperative 3 months.Femoral bone marrow was harvested from rat left side.After BMSCs osteoblast differentiation,cells were harvested and used for examining expression of genes associated with the signaling pathways in the two groups using microarray technology and real-time PCR analysis.Results BMD in SCI group was significantly lower in the ephiphyses and metaphyses[(0.176 ± 0.017)g/cm2 and (0.170 ±0.016)g/cm2] compared to that in control group [(0.257 ± 0.023) g/cm2 and (0.196 ± 0.013) g/cm2,P ＜0.05].Microarray and PCR analysis revealed Wnt/β-catenin (eg.Wnt1,Wnt3a,Wnt5a,Lrp5,Ctnnb1,Lef1 and Axin),BMP (Tgfb1 and Bmpr1),IGF -1 (eg.IGF1 R,c-fos and c-Jun),and ER (eg.Esr1) signaling pathways in osteoblasts were significantly down-regulated in SCI group compared to these in control group (P ＜ 0.05).Conclusions The Wnt/β-catenin,BMP,ER,and IGF-1 signaling pathways in osteoblasts are significantly down-regulated after SCI,resulting in profound BMD loss.This indicates that these signaling pathways are implicated in the osteoporosis after SCI.%目的 观察脊髓损伤(SCI)后骨髓间充质干细胞(BMSCs)向成骨细胞分化过程中Wnt/β-catenin、骨形态发生蛋白(BMP)、雌激素受体(ER)及胰岛素样生长因子(IGF)信号传导途径相关基因表达的变化,探讨SCI后骨质疏松的发生机制. 方法 40只雄性6周龄SD大鼠按随机数字表法分为SCI组(20只)与对照组(20只).SCI组在T1o ～ 12节段行椎板切除暴露脊髓,用解剖刀锐性横断下胸髓;对照组仅暴露下胸髓而不横断.术后3个月,两组大鼠右股骨先测定其骨密度(BMD).术后3个月,取两组大鼠左侧股骨骨髓,培养BMSCs并成骨分化后,收集细胞做基因微点阵分析4种信号传导通路相关基因表达,进一步应用实时荧光定量-PCR分析细胞内相关基因表达差异. 结果 SCI组股骨远段双能X线吸收测量法测定的BMD[(0.176士0.017)g/cm2]显著小于对照组[(0.257 ±0.023)g/cm2] (P ＜0.05);SCI组股骨干BMD[(0.170±0.016)g/cm2]也显著小于对照组[(0.196±0.013)g/cm2] (P＜0.05).基因微点阵及实时荧光定量-PCR分析结果均证实SCI组BMSCs源性成骨细胞内Wnt/β-catenin(如Wnt1、Wnt3a、Wnt5a、Lrp5、Ctnnb1、Axin、Lef1)、BMP(如Tgfb1、Bmpr1)、IGF-1(如IGF1R、c-fos、c-Jun)和ER(如Esr1)信号传导途径等相关基因表达均明显低于对照组. 结论 SCI后成骨细胞内Wnt/β-catenin、BMP、ER和IGF-1信号传导途径均显著下调,导致BMD明显降低;提示4种信号传导通路均参与了SCI后骨质疏松的发生.