雷帕霉素对铁蓄积高转换骨质疏松小鼠骨生成的影响

Effect of rapamycin on osteogenesis in high⁃turnover osteoporosis mice with iron accumulation

目的 研究雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)抑制剂雷帕霉素(rapamycin, Rapa)对铁蓄积高转换骨质疏松骨生成的影响。方法 将16只8周龄野生(wide type, Wt)雌性小鼠和48只8周龄铁调素敲除(△Hep)转基因雌性小鼠予以卵巢切除(OVX),分为四组(Wt+OVX组、△Hep+OVX组、△Hep+OVX+羧甲基纤维素(carboxymethylcellulose, CMC)组和△Hep+OVX+Rapa组)。Rapa为腹腔注射,3mg/(kg·d),持续2个月。CMC为Rapa溶剂,注射剂量及持续时间同Rapa。末次注射结束后4组小鼠处死取血清和骨组织标本。用股骨微型计算机断层扫描(micro computed tomography, micro-CT)检测股骨骨量及骨密度(bone mineral density, BMD)等参数,扫描电镜、HE染色检测骨量及骨小梁结构,生物力学实验检测股骨弯曲弹性模量、弯曲能量、最大弯曲应力、弯曲刚性系数,血清成骨指标如碱性磷酸酶(alkaline phosphatase, ALP)、Ⅰ型前胶原N端前肽(procollagenⅠN-terminal propeptide, P1NP)、骨钙素(osteocalcin, OCN)等采用ELISA检测,成骨基因如Runt相关转录因子2(runt-related transcription factor 2,Runx2)、成骨相关转录因子SP7(transcription factor SP7,SP7)、ALP等表达采用RT-PCR检测。结果 股骨micro-CT显示,与Wt+OVX组相比,△Hep+OVX组和△Hep+OVX+CMC组BMD、骨体积分数(bone volume to total volume, BV/TV)、骨小梁数量(trabecular number, TB.N)、骨小梁厚度(trabecular thickness, TB.Th)、连接密度(connection density, ConnD)明显减少,骨小梁分离度(trabecular separation, TB.Sp)、结构模型指数(structure model index, SMI)明显增加;而△Hep+OVX+Rapa组较△Hep+OVX组和△Hep+OVX+CMC组BMD、BV/TV、TB.N、TB.Th、ConnD增加,TB.Sp、SMI减少(P<0.05)。股骨电镜、胫骨HE染色显示,与Wt+OVX组相比,△Hep+OVX组和△Hep+OVX+CMC组骨量及骨小梁明显减少,使用雷帕霉素后骨量及骨小梁明显改善(P<0.05)。生物力学显示,与Wt+OVX组相比,△Hep+OVX组和△Hep+OVX+CMC组弯曲弹性模量、弯曲能量、最大弯曲应力、弯曲刚性系数明显降低,注射雷帕霉素后明显恢复(P<0.05)。与Wt+OVX组相比,△Hep+OVX组和△Hep+OVX+CMC组血清成骨指标(ALP、P1NP、OCN)、骨组织成骨基因(Runx2、SP7、ALP)表达明显减少,雷帕霉素干预后明显增加(P<0.05)。结论 雷帕霉素可以提高铁蓄积高转换骨质疏松小鼠的骨量及成骨活性,具有改善骨生成作用。

Objective To study the osteogenic effect of rapamycin,an inhibitor of mTOR,on high-turnover oste-oporosis with iron accumulation.Methods Sixteen 8-week-old wild(Wt)female mice and forty-eight 8-week-old(△Hep)transgenic female mice were ovariectomized(OVX).They were divided into four groups(Wt+OVX group,△Hep+OVX group,△Hep+OVX+Carboxymethylcellulose(CMC)group,and△Hep+OVX+Rapa group).Rapa with a dose of 3 mg/(kg·d)was injected intraperitoneally for 2 months.CMC was the solvent of Rapa,and the dose and duration of in-jection were the same as△Hep+OVX+Rapa group.After the last injection,four groups of mice were executed to collect ser-um and bone tissue samples.Bone mass,bone mineral density(BMD)and other indexes of femur were measured by micro-CT.Bone mass and trabecular structure were measured by scanning electron microscopy(SEM)and HE staining.The femo-ral bending modulus of elasticity,bending energy,maximum bending stress,and bending rigidity coefficient were measured by biomechanical experiments.The expression of serum osteogenic-indexes[alkaline phosphatase(ALP),procollagenⅠN-terminal propeptide(P1NP),osteocalcin(OCN)]were detected by ELISA,and the expression of osteogenic genes[runt-related transcription factor 2(Runx2),transcription factor SP7(SP7),ALP]in bone were detected by RT-PCR.Results Micro-CT of femer showed that the BMD,bone volume to total volume(BV/TV),trabecular number(TB N),trabecular thickness(TB Th)and connection density(ConnD)significantly decreased but trabecular sepa-ration(TB Sp),structure model index(SMI)significantly increased in△Hep+OVX group and△Hep+OVX+CMC group by comparing with Wt+OVX group.The BMD,BV/TV,TB N,TB Th,and ConnD significantly increased but TB Sp,SMI significantly decreased in△Hep+OVX+Rapa group by comparing with△Hep+OVX group and△Hep+OVX+CMC group(P<0.05).Compared with Wt+OVX group,femoral scanning electron microscopy and tibial HE staining showed that the bone mass and trabecular bone in△Hep+OVX group and△Hep+OVX+CMC group decreased significantly,but they were improved significantly after rapamycin treatment(P<0.05).Compared with Wt+OVX group,the femoral bending modulus of elasticity,bending energy,maximum bending stress,and bending rigidity coef-ficient in△Hep+OVX group and△Hep+OVX+CMC group decreased significantly,and recovered significantly after ra-pamycin injection(P<0.05).Compared with△Wt+OVX group,the expression of serum osteogenic-indexes(ALP,P1NP,OCN)and bony osteogenic-genes(Runx2,SP7,ALP)in△Hep+OVX group and△Hep+OVX+CMC group decreased significantly,but increased significantly after rapamycin intervention(P<0.05).Conclusion Rapamycin,which has positive effect on osteogenesis,can improve bone mass and osteoblastic activity in high-turnover osteoporosis with iron accumulation.