【作者】 刘江华；

【导师】 廖二元；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 第一部分OPG基因敲除骨丢失鼠的骨微结构及生物力学改变目的观察护骨素（OPG）基因敲除骨丢失鼠的骨微结构、骨生物力学、骨密度、骨矿含量及骨转换生化指标的变化，探讨OPG在骨丢失中的作用。方法SPF级10周龄雌性OPG基因敲除纯合子(OPG^-/-)小鼠与野生型（WT）小鼠各10只。OPG^-/-与WT小鼠均由具有C57BL／6J×129／SV背景的OPG杂合子(OPG^+/-)小鼠相互交配产生。应用μCT测定小鼠右侧胫骨近端松质骨和皮质骨的骨微结构参数；DXA测定左侧股骨骨密度；应用三点弯曲试验检测右侧股骨的骨生物力学性能；以ELISA法检测血清骨源性碱性磷酸酶、抗酒石酸酸性磷酸酶以及NF-κB受体活化因子配基。结果与野生型小鼠比较，OPG基因敲除小鼠的胫骨松质骨体积骨密度、组织骨密度、骨体积分数、骨面积分数、骨小梁数目及骨小梁连结密度均明显下降，而结构模型指数、骨小梁厚度及骨小梁间隔明显增加；皮质骨厚度、皮质骨面积、截面总面积、截面惯性矩、皮质骨密度和皮质骨骨矿含量明显降低，内径周长与骨髓腔面积则明显增加。股骨的最大载荷、最大应力、弹性模量及刚性系数均明显下降。股骨骨密度与骨矿含量明显下降。血清骨源性碱性磷酸酶、抗酒石酸酸性磷酸酶以及核因子κB受体活化因子配基均明显升高。结论OPG基因敲除小鼠的松质骨与皮质骨出现高转换型骨丢失，伴显著的骨微结构破坏和生物力学性能下降。第二部分Zoledronic acid抗骨吸收的作用机制研究目的探讨在体内环境中二膦酸盐Zoledronic acid的抗骨吸收作用机制及其与OPG途径的关系。方法SPF级6周龄雌性OPG基因敲除小鼠纯合子(OPG^-／-)20只随机分为Zoledronic acid（Zol）组和溶媒对照组，另10只野生型小鼠（WT）为正常对照组。OPG^-／-与WT小鼠均由具有C57BL／6J×129／SV背景的OPG杂合子(OPG^+／-)小鼠相互交配产生。Zol组给予Zoledronic acid 150μg／kg，皮下注射，每周2次，相应的溶媒对照组每周2次皮下注射等容量溶媒（无菌蒸馏水），干预时间6周。应用μCT测定小鼠右侧胫骨近端松质骨和皮质的骨微结构参数；DXA测定左侧股骨总体骨密度与骨矿含量；三点弯曲试验检测右侧股骨的骨生物力学性能。ELISA法检测血清骨源性碱性磷酸酶、抗酒石酸酸性磷酸酶以及NF-κB受体活化因子配基。结果与H₂O溶媒对照组比较，Zoledronic acid组的股骨总体骨密度与骨矿含量均明显增加，达到野生型小鼠水平。股骨的生物力学参数最大载荷、最大应力、弹性模量及刚性系数明显升高。胫骨松质骨微结构参数体积骨密度、组织骨密度、骨体积分数、骨面积分数、骨小梁数目、骨小梁连接密度和骨小梁厚度均较溶媒对照组明显升高，其中，组织骨密度、骨小梁连接密度、骨体积分数和骨小梁厚度还显著高于野生型小鼠；而结构模型指数与骨小梁间隔则明显降低。胫骨皮质微结构参数皮质骨厚度、截面惯性矩、皮质骨面积、截面总面积、皮质骨密度和皮质骨矿含量均明显增高，其中，皮质骨厚度、皮质骨密度和皮质骨矿含量还显著高于野生型小鼠；而内径周长与骨髓腔面积则明显下降。血清抗酒石酸酸性磷酸酶明显下降，血清NF-κB受体活化因子配基明显上升，而血清骨源性碱性磷酸酶无明显变化。结论在小鼠体内OPG基因功能缺失状况下，Zoledronic acid仍具有良好的拮抗骨丢失作用。提示Zoledronic acid在体内的抗骨吸收作用并非依赖OPG途径，对破骨细胞的直接抑制可能是其抗骨吸收的主要途径。第三部分Genistein调节骨代谢的作用机制研究目的探讨植物雌激素Genistein在体内对骨代谢的作用与OPG途径之间的关系。方法SPF级6周龄雌性OPG基因敲除纯合子(OPG^-／-)小鼠40只随机分为Genistein（Gen）组、17β-Estradiol组（E₂）组、DMSO对照组、Zoledronic acid（Zol）组及H₂O对照组，8只野生型（WT）小鼠为正常对照组。OPG^-／-与WT小鼠均由具有C57BL／6J×129／SV背景的OPG杂合子(OPG^+／-)小鼠相互交配产生。Gen组给予Genistein 0.8mg／只／天，皮下注射。E₂组给予17β-Estradiol（0.03μg／只／天），皮下注射。相应的溶媒对照DMSO组每天皮下注射等容量的DMSO与聚乙二醇300混合物。Zol组给予Zoledronic acid 150μg／kg，皮下注射，每周2次，相应的溶媒对照H₂O组每周2次皮下注射等容量的无菌蒸馏水。干预时间为6周。应用μCT测定小鼠右侧胫骨近端松质骨和皮质的骨微结构参数；DXA测定左侧股骨总体骨密度与骨矿含量；三点弯曲试验检测右侧股骨的骨生物力学性能；ELISA法检测血清骨源性碱性磷酸酶、抗酒石酸酸性磷酸酶和NF—κB受体活化因子配基。结果与DMSO溶媒对照组比较，Genistein组与17β-Estradiol组股骨总体骨密度与骨矿含量无增加。股骨生物力学参数最大载荷、最大应力、弹性模量及刚性系数均无明显变化。胫骨松质微结构参数骨体积骨密度、组织骨密度、骨体积分数、骨面积分数、结构模型指数、骨小梁数目、骨小梁联接密度、骨小梁间隔及骨小梁厚度，和皮质的骨微结构参数皮质骨厚度、截面惯性矩、内径周长、外径周长、骨髓腔面积、皮质骨面积、截面总面积、皮质骨密度及皮质骨矿含量亦无改善。血清骨源性碱性磷酸酶、抗酒石酸酸性磷酸酶以及NF-κB受体活化因子配基水平无变化。Zoledronic acid则能显著改善OPG基因敲除小鼠的骨密度、骨生物力学性能及骨微结构，部分指标达到甚至超过野生型小鼠水平。结论在小鼠体内OPG基因功能缺失的状况下，Genistein与17β-Estradiol调节骨代谢的作用完全消失，提示Genistein与17β-Estradiol对骨代谢的作用完全依赖于OPG途径。第四部分OPG转基因小鼠模型的建立目的护骨素OPG是调节骨代谢重要的细胞因子之一，为开展其在整体水平上的功能研究，建立OPG转基因小鼠模型。方法构建带有人类OPG基因启动子的真核表达载体pCI-hOPGp-m OPG，以显微注射法转入C57BL／6J×CBA小鼠受精卵雄核中，导入假孕母鼠输卵管后培养子代小鼠，PCR鉴定子代小鼠基因型。结果构建的表达载体pCI-hOPGp-mOPG质粒经酶切与测序鉴定序列正确。在69只原代小鼠中，有7只转基因阳性的Founder小鼠。在F₁代120只小鼠中，有4系共15只转基因阳性小鼠。结论初步获得4个稳定表达OPG的转基因小鼠系，为深入研究OPG在体内的生物学功能与基因调控创造了条件。更多还原

【Abstract】 Part one Changes of bone mineral density, microarchitecture andbiomechanical property in OPG - knockout miceObjective To study the changes of bone mineral density （BMD）,microarchitecture and biomechanical property in osteoprotegerin（OPG）-knockout mice (OPG^-/-).Methods 10-week-old female OPG^-/- mice （n=10） and wild-type mice（n=10） were involved in the study. The trabecular and cortical bonemicroarchitecture was assessed by micro-CT in the right proximal tibia.BMD of the left femur was detected by DXA. The biomechanicalproperty of the right femur was determined by a three-point bending test.Serum biochemical markers of bone turnover such as bone alkalinephosphatase（B-ALP）,tartrate-resistant acid phosphatase-5b （TRACP-5b）,and receptor activator of nuclear factorκB ligand （RANKL） weredetermined by ELISA.Results Compared with wild-type mice, the OPG^-/- mice showed adecreased volumetric BMD （vBMD）,tissue BMD（tBMD）, bone volumefraction （BVF）, bone surface fraction（BSF）, trabecular number （Tb.N）,and trabecular connectivity density （Conn.D）, with an increased structuremodel index（SMI）, trabecular thickness（Tb.Th） and trabecular separation（Tb.Sp）. The cortical bone microarchitecture parameters such as corticalthickness （Ct.Th）, cortical area （Ct.Ar）, total area （Tt.Ar）, Moment ofInertia （Mm）, cortical BMD （Ct.BMD） and cortical bone mineral containt（Ct.BMC） were also decreased, with an increased inner perimeter（In.Pm） and marrow area （Ma.Ar）. Three-point bending test revealed adecrease in ultimate load, ultimate stress, stiff index and elastic modulus.The BMD was reduced by DXA determination, while serum B-ALP,TRACP-5b, and RANKL were all elevated at the same time.Conclusions The turnover rate of OPG knockout mice is high withdramatic disturbances in bone microarchitecture and deterioration in bonebiomechanical property. Part two In vivo studies of zoledronic acid on the mechanism ofanti-bone resorptionobjective To study the in vivo anti-bone resorption mechanism ofzoledronic acid.Methods Six-week-old female OPG^-/- mice （n=20） and wild-type mice（n=10） were involved in the study. OPG^-/- mice were randomly dividedinto two groups（10 for each group）, and treated with zoledronic acid at adose of 0μg/kg （H₂O group） and 150μg/kg （Zol group） subcutaneouslytwice per week. The mice were killed 6 weeks after intervention.Trabecular and cortical bone microarchitecture in the right proximal tibiawas assessed by micro-CT and the bone mineral density （BMD） of theleft femur was measured by DXA. The biomechanical property of theright femur was determined by a three-point bending test. Serum bonealkaline phosphatase （B-ALP）, tartrate-resistant acid phosphatase-Sb（TRACP-Sb） and receptor activator of nuclear factorκB ligand （RANKL）were determined by ELISA.Results Compared with the mice in H₂O group, the zoledronicacid-treated mice showed an increased total BMD and BMC, almostequal to the levels of wild-type mice.μCT analysis revealed an increasein vBMD, tBMD, BVF, BSF, Tb.Th,Tb.N and Conn.D, with a decreasein SMI and Tb.Sp; the cortical bone microarchitechitecture parameterssuch as Ct.Th, Mm, Ct.Ar, Tt.Ar, Ct.BMD and Ct.BMC were allincreased, with an decreased In.Pm and Ma.Ar. Three-point bending testshowed an increase in ultimate load, ultimate stress, elastic modulus andstress index. A decrease in serum TRACP-Sb and an increase in serumRANKL were revealed by ELISA analysis.Conclusions Zoledronic acid could effectively prevent bone lose in mice lacking osteoprotegerin, suggesting that the in vivo anti-boneresorption effect of zoledronic acid is triggered via an OPG-independentpathway, possibly by direct suppression of the activity of osteoclasts.Part three Studies of genistein effects on bone metabolism in OPG^-/-miceObjective To study genistein in vivo effects on bone metabolism inOPG^-/- miceMethods Following heterozygotes(OPG^+/-) matings, thehomozygotes(OPG^-/-)and wild type（WT）with a mixed C57BL/6J×129/SVbackground were obtained. 6-week-old female OPG^-/- mice （n=40） andwild-type mice （n=8） were involved in the study. OPG^-/- mice wererandomly divided into 5 groups （n=8 for each group）: 1) Genistein-treatedmice: treated with genistein at a maximal dose（0.8mg/day）subcutaneously（Gen）; 2) 17β-Estradiol-treated mice: treated with17β-Estradiol at a maximal dose（0.034μg/day） subcutaneously（E₂）; 3)DMSO-treated mice: treated with a mixture of dimethylsulfoxide（DMSO） and polyethylenglycol-300 subcutaneously; 4) Zoledronicacid-treated mice: treated with zoledronic acid at a dose （150μg/kg）twice per week subcutaneously （Zol）; 5) H₂O-treated mice: treated withsterilized water twice per week subcutaneously. The mice were killed 6weeks after intervention. Trabecular and cortical bone microarchitecturewas assessed by micro-CT in the right proximal tibia. The bone mineraldensity （BMD） of the left femur was measured using DXA. Thebiomechanical property of the right femur was determined by athree-point bending test. Serum bone alkaline phosphatase （B-ALP）,tartrate-resistant acid phosphatase-5b （TRACP-Sb） and receptor activator of nuclear factorκB ligand （RANKL） were determined by ELISA.Results DXA analysis revealed that the total BMD of femur was notsignificantly changed in Gen, E₂, H₂O, and DMSO groups. Three-pointbending test showed no significant differences in biomechanicalparameters including ultimate load, ultimate stress,stiff index, and elasticmodulus, and micro-CT analysis showed that trabecular bonemicroarchitectural parameters （vBMD、tBMD、BVF、BSF、SMI、Tb.N、Conn.D、Tb.Sp and Tb.Th） and cortical bone microarchitecturalparameters（Ct.Th、Mm、In.Pm、Ot.Pm、Ma.Ar、Ct.Ar、Tt.Ar、Ct.BMDand Ct.BMC） were not different either in these groups. Genistein and17β-Estradiol did not modify either serum TRACP-5b or B-ALP orRANKL levels. However, in addition to increases of bone mass,zoledronic acid could effectively improve biomechanical property andprevented the architectural bone from deteriorating in OPG^-/- mice.Conclusions Lack of osteoprotegerin in mice, the effects of genisteinas well as 17β-Estradiol on bone metabolism in vivo disappeared,suggesting that their activity on bone metabolism should be totallyOPG--dependent.Part four Estabolishment of osteoprotegerin transgenic mice.Objective osteoprotegerin（OPG） may play a key role in the regulationof bone metabolism. In order to evaluate its effects in vivo, wedeveloped an OPG transgenic mouse model.Method Expression plasmid pCI-hOPGp-mOPG containing humanOPG promoter was successfully reconstructed, and micro-injected intofertilized zygotes from C57BL/6J×CBA mice to prepare trangenic mice.Progeny were screened for the presence of the transgene by PCR. Resuts Out of the 69 founder generation, 7 founder mice wereidentified by PCR dectetion of OPG. Out of the 120 F₁ generation, 15OPG transgenic mice were obtained.Conclusion Four OPG transgenic mice lines have been developed,which could be valuable for studying the biological significance and generegulation of OPG in vivo.更多还原