ERα基因过表达对去卵巢骨质疏松小鼠骨密度及钙磷代谢的影响

Effects of ERαgene overexpression on bone mineral density and calcium and phosphorus metabolism inovariectomized osteoporosis mice

目的:探讨雌激素受体α(ERα)基因过表达对去卵巢骨质疏松小鼠骨代谢及钙磷代谢的影响,为靶向基因治疗骨质疏松症提供实验依据。方法:选择SPF级雌性小鼠30只,随机分为假手术组、模型组和ERα过表达组,每组10只。采用双侧卵巢切除法制备绝经后骨质疏松小鼠模型,模型建立完成后,ERα过表达组通过椎体髓腔注射的方法转染携带小鼠ERα基因的重组腺病毒载体,模型组转染空载病毒,假手术组不作处理。实时荧光定量PCR(q RT-PCR)检测小鼠骨组织ERα基因表达;分别于造模后检测小鼠股骨骨密度(BMD);采用Micro-CT扫描测量骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)、骨小梁分离度(Tb.Sp)、骨体积分数(BV/TV),并进行股骨生物力学强度检测;采用全自动生化仪检测血清中钙(Ca)、磷(P)、骨钙素(BGP)、碱性磷酸酶(ALP)含量;采用免疫组化检测骨组织中基质金属蛋白酶的组织抑制剂1(TIMP-1)和单核细胞趋化蛋白-1(MCP-1)的蛋白表达水平。结果:与假手术组相比,模型组小鼠骨组织ERα基因表达水平显著降低、BMD、BV/TV、Tb.Th、最大载荷、刚性系数、Ca和P降低,而Tb.Sp、BGP和ALP显著升高(P<0.05);ERα过表达组小鼠骨组织ERα基因表达水平、BMD、BV/TV、Tb.Th、最大载荷、刚性系数、Ca和P较模型组小鼠显著上升;而Tb.Sp、BGP和ALP显著降低(P<0.05)。与假手术组相比,模型组小鼠骨组织中TIMP-1平均光密度值显著降低而MCP-1平均光密度值升高,而ERα过表达组TIMP-1平均光密度值显著升高而MCP-1显著降低(P<0.05)。结论:ERα基因过表达通过调节骨密度、骨参数、骨代谢、钙磷代谢指标及组织中TIMP-1和MCP-1的表达水平,改善骨质疏松。

Objective:To investigate the effects of estrogen receptorα(ERα)gene overexpression on bone metabolism and calcium and phosphorus metabolism in ovariectomized osteoporosis mice,and to provide experimental basis for targeted gene therapy of osteoporosis.Methods:Thirty SPF female mice were randomly divided into sham operation group,model group and ERαoverexpression group with 10 mice in each group.After the model was established,the ERαoverexpression group was transfected with recombinant adenovirus vector carrying mouse ERαgene by intraspinal injection.The model group was transfected with empty virus,and the sham operation group was not treated.The expression of ERαgene in bone tissue of mice was detected by quantitative Real-time PCR(q RT-PCR).Bone mineral density(BMD)of mouse femur was measured after modeling.Trabecular number(Tb.N),trabecular thickness(Tb.Th),trabecular segregation(Tb.Sp),bone volume fraction(BV/TV)and biomechanical strength of femur were measured by micro-CT scanning.Serum levels of calcium(Ca),phosphorus(P),osteocalcin(BGP)and alkaline phosphatase(ALP)were measured by automatic biochemical analyzer.The expressions of tissue inhibitor of metalloproteinases 1(TIMP-1)and monocyte chemotactic protein 1(MCP-1)in bone homogenate were detected by Immunohistochemistry.Results:Compared with sham operation group,the expression level of ERαgene in bone tissue of model group was decreased significantly,the levels of BMD,BV/TV,Tb.Th,maximum load,rigidity coefficient,Ca and P were decreased,while the levels of Tb.Sp,BGP and ALP were increased significantly(P<0.05).Compared with the sham operation group,the expression level of TIMP-1 protein in the bone tissue of the model group was significantly decreased,while that of MCP-1 protein was increased,while that of the ERαoverexpression group was increased while that of MCP-1 was decreased(P<0.05).The levels of ERαgene expression,BMD,BV/TV,TB.Th,maximum load,rigidity coefficient,Ca and P in the ERαoverexpression group were significantly higher than those in the model group,while Tb.Sp,BGP and ALP were significantly lower(P<0.05).Compared with the sham operation group,mean optical density of TIMP-1 in the bone tissue of the model group was significantly decreased,while that of MCP-1 was significantly increased,and that of the ERαoverexpression group was significantly increased while that of MCP-1 was significantly decreased(P<0.05).Conclusion:ERαgene overexpression can improve osteoporosis by regulating bone mineral density,bone parameters,bone metabolism,calcium and phosphorus metabolic indicators and the expression levels of TIMP-1 and MCP-1 in tissues.