骨髓间充质干细胞移植对去巢骨质疏松大鼠骨代谢及RANKL/OPG/TRAF6信号通路的影响

Effects of bone marrow mesenchymal stem cell transplantation on bone metabolism and RANKL/OPG/TRAF6 signaling pathway in osteoporotic rats

目的探究骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSC)移植对去卵巢骨质疏松大鼠骨代谢的影响及其作用机制。方法将7周龄40只清洁级SD雌性大鼠按随机数字表法分为4组:假手术组、模型组、移植组、阳性对照组,除假手术组外其余组均进行双侧卵巢切除术构建骨质疏松大鼠模型。模型构建2个月后,移植组大鼠尾静脉注射80µl/kg含有BMSC的PBS溶液,假手术组和模型组尾静脉注射等量的PBS溶液,阳性对照组大鼠每天灌胃给与仙灵骨葆0.5 g/100 g。治疗14 d后收集各组大鼠血清和股骨。苏木精-伊红染色(HE)观察股骨组织病理学变化;Micro-CT法测量骨密度(bone mineral density,BMD)与骨参数;酶联免疫吸附测定(ELISA)试剂盒检测骨钙素、骨保护素、碱性磷酸酶、胰岛素样生长因子1表达水平;分光光度计法测定血清钙、磷、镁水平;蛋白免疫印迹(Western blot)检测各组大鼠股骨中核因子κb配体受体激活剂(receptor activator of nuclear factorκb ligand,RANKL)、骨保护素(osteoprotegerin,OPG)、肿瘤坏死因子受体相关蛋白6(tumor necrosis factor receptor-associated protein 6,TRAF6)和核因子kappa B亚基1(nuclear factor kappa B subunit 1,NF-kB1)蛋白水平表达。结果与假手术组比较,模型组大鼠BMD降低(0.28±0.01)g/cm^(3),骨体积分数(bone volume fraction,BV/TV)降低(19.73±2.02)%,骨小梁厚度(trabecular thickness,Tb.Th)降低(0.082±0.008)mm,骨小梁数量(trabecular number,Tb.N)降低(1.60±0.17)mm^(-1),骨小梁分离度(trabecular separation/spacing,Tb.Sp)升高(0.273±0.024)mm,骨保护素降低(489.49±55.29)ng/L,碱性磷酸酶降低(229.13±15.05)U/L,胰岛素样生长因子1降低(236.64±14.32)µg/L,骨钙素升高(1.866±0.109)µg/L,钙水平升高(11.98±1.09)mg/dl,磷水平升高(6.85±0.68)mg/dl,镁水平降低(0.62±0.04)mg/dl,RANKL相对表达水平升高(1.05±0.09),OPG相对表达水平降低(0.58±0.08),RANKL相对表达水平升高(0.74±0.10),NF-kB1相对表达水平升高(1.01±0.11)(P<0.05);移植组大鼠BMD降低(0.38±0.04)g/cm^(3),BV/TV降低(26.73±2.74)%,Tb.Th降低(0.094±0.006)mm,Tb.N降低(2.67±0.09)mm^(-1),Tb.Sp升高(0.241±0.026)mm,骨保护素基本不变(720.09±67.41)ng/L,碱性磷酸酶基本不变(269.48±14.15)U/L,胰岛素样生长因子1降低(335.95±24.13)µg/L,骨钙素升高(1.392±0.153)µg/L,钙水平升高(7.12±0.53)mg/dl,磷水平升高(4.54±0.32)mg/dl,镁水平基本不变(0.87±0.08)mg/dl,RANKL相对表达水平升高(0.59±0.05),OPG相对表达水平降低(0.97±0.10),RANKL相对表达水平升高(0.45±0.06),NF-kB1相对表达水平升高(0.72±0.06)(P<0.05);阳性对照组大鼠BMD降低(0.36±0.05)g/cm^(3),BV/TV基本不变(28.72±3.20)%,Tb.Th基本不变(0.096±0.011)mm,Tb.N基本不变(2.85±0.24)mm^(-1),Tb.Sp基本不变(0.241±0.027)mm,骨保护素降低(716.78±36.90)ng/L,碱性磷酸酶基本不变(270.65±18.59)U/L,胰岛素样生长因子1降低(336.94±17.50)µg/L,骨钙素升高(1.377±0.101)µg/L,钙水平升高(7.13±0.80)mg/dl,磷水平升高(4.58±0.71)mg/dl,镁水平基本不变(0.89±0.04)mg/dl,RANKL相对表达水平升高(0.55±0.08),OPG相对表达水平降低(0.98±0.13),RANKL相对表达水平基本不变(0.40±0.05),NF-kB1相对表达水平升高(0.65±0.09)(P<0.05)。结论BMSC移植可通过调控骨代谢、血清钙、磷、镁水平改善去卵巢大鼠骨质疏松症,这可能与RANKL/OPG/TRAF6通路有关。

Objective To investigate the effect of bone marrow derived mesenchymal stem cells(BMSC)transplantation on bone metabolism and its mechanism in ovariectomized osteoporosis rats.Methods Forty clean SD female rats aged 7 weeks were divided into 4 groups according to the random number table method:sham operation group,model group,the transplantation group,positive control group,in addition to control the rest of the group were performed bilateral oophorectomy build osteoporosis rats model,after 2 months of model establishment,rats in transplantation group were injected with 80µl/kg PBS solution containing bone marrow mesenchymal stem cells through tail vein,rats in sham operation group and model group were injected with the same amount of PBS solution through tail vein,and rats in positive control group were given Xianlinggubao(0.5 g/100 g)by gavage every day.Serum and femur were collected 14 days after treatment.Hematoxylin and eosin staining(HE)was used to observe the histopathological changes of femur.Micro-CT was used to measure bone mineral density and bone parameters.The expression levels of osteocalcin,osteoprotegerin,alkaline phosphatase and insulin-like growth factor 1 were detected by enzyme-linked immunosorbent assay(ELISA)kit.The serum levels of calcium,phosphorus and magnesium were measured by spectrophotometer.The protein expressions of RANKL,OPG,TRAF6 and NF-KB1 in femur of each group were detected by Western blot.Results Compared with the sham operation group,the bone mineral density(BMD)of the model group was decreased by(0.28±0.01)g/cm^(3),bone volume fraction(BMD)was decreased by(0.28±0.01)g/cm^(3).BV/TV)decreased by(19.73±2.02)%,trabecular thickness(Tb.Th)decreased by(0.082±0.008)mm,trabecular number(Tb.N)decreased by(1.60±0.17)mm^(-1)and trabecular separation/spacing(Tb.Sp)increased(0.273±0.024)mm,osteoprotegerin(489.49±55.29)ng/L,alkaline phosphatase(229.13±15.05)U/L,insulin-like growth factor-1(236.64±14.32)µg/L,and osteocalcin were decreased(1.866±0.109)µg/L,calcium(11.98±1.09)mg/dl,phosphorus(6.85±0.68)mg/dl,and magnesium decreased(0.62±0.04)mg/dl),the relative expression level of RANKL increased(1.05±0.09),the relative expression level of OPG decreased(0.58±0.08),the relative expression level of RANKL increased(0.74±0.10),and the relative expression level of NF-kB1 increased(1.01±0.11)(P<0.05);bone mineral density,bone mineral density,bone mineral density BMD(0.38±0.04 g/cm^(3),BV/TV(26.73±2.74)%,Tb.Th(0.094±0.006)mm,Tb.N(2.67±0.09)mm^(-1)and Tb.Sp were decreased(0.241±0.026)mm),osteoprotegerin(720.09±67.41)ng/L,alkaline phosphatase(269.48±14.15)U/L,insulin-like growth factor 1(IGF-1)decreased(335.95±24.13)µg/L,and osteocalcin increased(1.392±0.153)µg/L,calcium(7.12±0.53)mg/dl,phosphorus(4.54±0.32)mg/dl,magnesium(0.87±0.08)mg/dl.RANKL relative expression level increased(0.59±0.05),OPG relative expression level decreased(0.97±0.10),RANKL relative expression level increased(0.45±0.06),NF-kB1 relative expression level increased(0.72±0.06)(P<0.05);bone mineral density,bone mineral density,bone mineral density BMD(0.36±0.05)g/cm^(3),BV/TV(28.72±3.20)%,Tb.Th(0.096±0.011)mm,Tb.N(2.85±0.24)mm^(-1)Tb.Sp was basically unchanged(0.241±0.027)mm,osteoprotegerin was decreased(716.78±36.90)ng/L,alkaline phosphatase was basically unchanged(270.65±18.59)U/L,and insulin-like growth factor 1 was decreased(336.94±17.50)μg/L,osteocalcin(1.377±0.101)µg/L,calcium(7.13±0.80)mg/dl,phosphorus(4.58±0.71)mg/dl,and magnesium(0.89±0.04)remained unchanged mg/dl,the relative expression level of RANKL increased(0.55±0.08),the relative expression level of OPG decreased(0.98±0.13),the relative expression level of RANKL was basically unchanged(0.40±0.05),and the relative expression level of NF-kB1 increased(0.65±0.09)(P<0.05).Conclusion Bone marrow mesenchymal stem cell transplantation can improve osteoporosis in ovariectomized rats by regulating bone metabolism and serum levels of calcium,phosphorus and magnesium,which may be related to RANKL/OPG/TRAF6 pathway.