【作者】 吴星火；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分联合应用小剂量脂多糖和大剂量甲强龙诱导兔骨坏死的实验研究目的激素性骨坏死发生发展的病理生理机制目前还不明确，合适的实验模型是骨坏死治疗研究的基础。本实验研究设计一新型兔骨坏死模型制作方案，采用低剂量的脂多糖和高剂量的甲强龙诱导骨坏死。材料与方法38只大白兔分为实验组和对照组，实验兔连续两次静脉注射脂多糖（LPS，10μg／kg），然后连续三次肌注甲强龙（MPS,20 mg／kg）（L-LPS×2+H-MPS×3）。组织学评估双侧股骨和肱骨近端和远端部分的病理改变。其它评估指标包括不同评估时间点的MRI扫描、骨内压检测、血清纤溶酶原活性剂／抑制剂比值、血清胆固醇、甘油三酯和低密度脂蛋白胆固醇和高密度脂蛋白胆固醇的比值（LDL／HDL ratio）。结果注射MPS后6刷，MRI扫描发现73．3％（22／30）兔出现骨坏死影响学改变，T₁WI表现为不规则低信号，T₂WI表现为不规则低信号或高信号；组织学证实，90％（27／30）兔子出现骨坏死，呈特征性骨坏死的病理学改变。其它结果包括骨髓脂肪细胞增大、骨内压增高、血液凝固异常和高脂血症。结论总之，此改良新型骨坏死诱导模型可有效地阐明激素性骨坏死发生发展的病理生理变化，以利于骨坏死的预防和治疗研究。第二部分粒细胞集落刺激因子（G-CSF）和干细胞因子（SCF）单独及联合应用对间充质干细胞生物学行为的影响目的本实验主要研究粒细胞集落刺激因子（G-CSF）和干细胞因子（SCF）单独及联合应用对骨髓间充质干细胞（MSCs）的粘附、迁移、增殖和成骨能力的影响。材料与方法分离、培养兔骨髓MSCs，采用流式细胞技术分析培养至第三代MSCs的谱系和表面标志表型，行细胞粘附特性测定、Transwell迁移实验、细胞增殖实验、成纤维细胞集落形成实验（CFU-F）和ALP活性测定。结果流式检测细胞标记物示，培养至第3代的细胞表型为CD29⁺／CD45^-，CD105⁺／CD34^-,CD90⁺／HLADR^-。免疫细胞化学染色示抗波形蛋白抗体阳性。因此，据该群细胞的免疫表型可以确定该群细胞为MSCs。G-CSF或SCF诱导后骨髓基质干细胞的黏附性增强，其中G-CSF的效果优于SCF；联合应用G-CSF／SCF可显著增强骨髓基质干细胞对FN的黏附。SCF对MSCs迁移作用的潜能和效果类似G-CSF。联合应用G-CSF和SCF可导致更强烈的细胞迁移效应。G-CSF／SCF对MSCs的增殖有明显的促进作用（*P＜0．05，**P＜0．01），联合应用G-CSF／SCF的细胞生长状态好于两者的单用，可有效的促进细胞增殖：与MSCs形成CFU-F的数目成正性剂量效应关系。G-CSF与SCF均抑制骨髓基质干细胞的成骨分化（*P＜0．05，**P＜0．01），各组各检测时间点ALP的活性均较对照组明显降低。结论骨髓基质干细胞有诱人的应用前景，本研究发现G-CSF／SCF可抑制MSCs的分化，促进其增殖再生，其协同作用可导致重要的生物学效应。第三部分联合应用粒细胞集落刺激因子和干细胞生长因子（G-CSF／SCF）治疗兔激素性骨坏死的实验研究目的大量研究证实骨髓衍生干细胞（Bone marrow-derived stem cell，BMSC）可用于治疗塌陷前期股骨头坏死。本研究，我们通过建立兔骨坏死模型，评估G-CSF／SCF动员自体BMSCs修复激素性骨坏死的潜能。材料与方法我们采用低剂量的脂多糖和甲强龙（L-LPS×2+H-MPS×3）建立兔骨坏死模型，分为治疗组和对照组。治疗组兔皮下注射G-CSF 100μg／kg和SCF 25μg／kg，连续干预5天；对照组给予等量的生理盐水。抽取血液，采用酶联免疫吸附测定法（ELISA）检测血清骨钙素的水平；采用磁共振（MRI）行影像学检查。采集实验动物双侧股骨和肱骨，处理成石蜡切片和硬组织切片，以进行免疫组织化学、组织学和组织形态学检测分析。结果应用G-CSF／SCF动员后，白细胞的平均数目和单核细胞的相对数目明显增多。G-CSF／SCF治疗组兔血清OCN蛋白表达明显增高。MRI检查发现，通过G-CSF／SCF干预后，骨坏死区和修复区呈现出活性修复界面。定量分析发现，治疗组的新生血管和血管密度分别是对照组的3．3倍和2．6倍。组织学和组织形态学证实，G-SCF／SCF组治疗4周后，新生骨容量明显高于对照组。结论联合应用G-CSF／SCF动员BMSC至骨坏死区，可促进早期骨坏死区骨的生长与修复，是治疗骨坏死值得探索的新策略。更多还原

【Abstract】 PartⅠExperimental osteonecrosis induced by a combination of low-doselipopolysaccharide and high-dose methylprednisolone in rabbitsObjective The pathogenetic mechanisms involved in steroid-inducedosteonecrosis are poorly understood. Appropriate experimental models of thehuman disease are indispensable to the understanding of successful treatmentmodalities for osteonecrosis. In the present experiment we devised a novelrabbit model of steroid-induced osteonecrosis by use of LPS and MPS toinvestigate the development of osteonecrosis.Materials and Methods 38 rabbits were assigned into the treatment groupcontrol group. In treatment group, two injections of 10μg/kg body weight ofLPS were given intravenously, and then three injections of 20 mg/kg bodyweight of MPS were given intramuscularly, at a time interval of 24 h（L-LPS× 2 + H-MPS×3）. Tissue assessments were performed on proximal third anddistal condyles of femora and humeri obtained 6 weeks after theadministration of LPS and MPS. MRI of these regions and intraosseouspressure of proximal femur were obtained at 0 and 6 weeks. Otherassessments included serum plasminogen activator/inhibitor ratio, cholesterollevel, LDL/HDL ratio, and triglyceride levels at various time points.Results 6 weeks after last injection of MPS ,MRI findings showed irregularlow signal on T₁-weighted images and irregular low or high signal onT₂-weighted image in 73.3%（22/30） rabbits. Histologically, 90% of the rabbits（27/30） in the treatment group developed ON 6 weeks after last injection ofMPS. Other results included bone marrow fat cell enlargement , a rise inintraosseous pressure, coagulation abnormalities and hyperlipidaemia.Conclusions On the whole, this is a novel modified animal model of steroidassociated osteonecrosis and it would be useful for elucidating thepathogenesis of steroid associated osteonecrosis and developing preventiveand therapeutic strategies. PartⅡEffects of granulocyte colony-stimulating factor and stem cell factor,alone and in combination, on the biological behaviours of bonemarrow mesenchymal stem cellsObjective The effects of granulocyte colony-stimulating factor （G-CSF） andstem cell factor （SCF） on the proliferation and osteogenic differentiationcapacity of bone marrow mesenchymal stem cells （MSCs） were studied in theexperiment.Materials and Methods Bone marrow MSCs were collected from rabbitssuccessfully, and treated with various concentrations of G-CSF, SCF or acombination of the two. Flow cytometric analyse, MTT test, CFU-F assay,and alkaline phosphatase （ALP） activity measurement were employed.Results: The results of flow cytometry showed that immunophenotype of thecells were CD29⁺/CD45^-,CD105⁺/CD34^-,CD90⁺/HLADR^- MSCs wereshown to constitutively express low levels of c-kit which could be enhancedby SCF. G-CSF and SCF had an obvious facilitative effect on the proliferationof MSCs in a dose-dependent fashion. In addition, G-CSF and SCF would beeffective in reversibly preventing their differentiation, as showed by thedecrease of ALP activity, leading to self-renewal rather than differentiative cell divisions. The effects of G-CSF were superior to SCF. And cells in thegroup treated with combination of G-CSF and SCF showed more powerfuleffects than the groups treated with G-CS, SCF, or none of the two.Conclusion: On the whole, these studies demonstrated that MSCs responsedto G-CSF, SCF, and to G-CSF plus SCF in a manner that suppresseddifferentiation, and promotes proliferation and self-renewal, and support theview that these factors could act synergistically.PartⅢA Combination of Granulocyte Colony- Stimulating Factor and Stem CellFactor Ameliorates Steroid-associated Osteonecrosis in RabbitsObjective Bone marrow-derived stem cell （BMSC） has been highlighted forthe treatment of osteonecrosis before collapse of the femoral head. In the study,the potential of G-CSF/SCF- mobilized BMSCs to repair steroid-associatedosteonecrosis were assessed in rabbits.Materials and Methods Osteonecrosis was induced by low-dose lipopolysaccharide and subsequent pulsed high-dose methylprednisolone.Rabbits in the treated group were subjected to subcutaneous injections ofG-CSF at a dose of 100μg/kg and SCF at a dose of 25μg/kg per day for 5days; and rabbits in the control group were given saline. Blood samples werecollected and serum osteocalcin was detected by ELISA. Radiological analysiswas performed by Magnetic Resonance Imaging. Then bilateral femora andhumeri were harvested and processed to paraffin sections and hard tissuesections for immunohistochemical, histologic and histomorphometric analysis.Results The mean number of leukocytes and the relative numbers ofmononuclear cell significantly increased after mobilization. All rabbitsdisplayed a marked increase in OCN protein expression in response toG-CSF/SCF. MRI scans showed reactive interface between the necrotic andreparative zones after G-CSF/SCF administration. Quantitative analysisshowed that new vessel and vessel density in the treatment group was 3.3-foldand 2.6-fold grater than the control group, respectively. The histologic andhistomorphometric analysis revealed that the new bone volume wassignificantly higher in the G-SCF/SCF group than in control group at 4 weeks.Conclusion G-CSF/SCF -induced mobilization of BMSC in the necrotic focimay represent a promising strategy for promoting functional bone repair of theearly-stage osteonecrosis.更多还原