【作者】 程少容；

【导师】 王仁法；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2007， 博士

【摘要】 临床上非创伤性股骨头缺血性坏死（Avascular necrosis of femoral head, AvNFH）非常多见,致残率很高。其中,激素性AvNFH占第一位。随着现代医学影像技术的快速发展,形态与功能相结合的影像学技术将成为研究疾病的新思路。在激素诱导的股骨头缺血性坏死的发生和发展过程中研究股骨的微循环及血流灌注情况显得格外重要,MR灌注成像能评估单位体积骨组织的血流灌注,了解血流量和血流速度,从而评价骨组织的活性。因此,我们采用动态增强磁共振成像的方法,并结合血液流变学指标来评估激素性股骨头缺血性坏死（AvNFH）模型不同时期股骨头的血流灌注状态,达到从另外的角度研究该疾病的目的。此外,我们还采用免疫组织化学的方法,研究了不同时相点兔缺血坏死股骨头内血管内皮生长因子（VEGF）和骨形态发生蛋白-2（BMP-2）表达的动态变化特点,以探讨两者在激素诱导的骨坏死发生发展过程中发挥的作用;采用骨髓间充质干细胞体外培养技术,将细胞学和形态学的方法相结合,从新的角度探讨了激素性AvNFH的发病机制。本实验分为两个部分:第一部分,选28只健康新西兰大白兔,随机分二组: A组:模型组（马血清加用甲基氢化泼尼松龙）; B组:对照组。A, B两组分别于用药前和用药后2周、4周、6周、8周,以相同方法行MR常规及动态增强、普通增强MR扫描,根据股骨头的时间-信号强度曲线计算股骨头动态增强MR成像参数。并于用药前和用药后的4周、8周取静脉血进行血液流变学指标检测。动物处死后取双侧股骨头制作病理切片, HE染色光镜观察。采用免疫组化的方法测定使用激素后2周、4周、6周、8周骨组织中VEGF和BMP-2的表达及变化。上述研究显示结果为:1.病理组织学切片显示:随着用药时间的延长,骨小梁空虚骨陷窝数逐渐增多,骨细胞核固缩,甚至出现骨坏死,骨髓腔内造血组织逐渐减少而脂肪组织明显增多,脂肪细胞体积增大,数量增多;2.反映早期激素性AvNFH血流灌注状态的时间-信号强度曲线的最大斜率随着时间的延长逐渐减小（p<0.05）,相应的骨小梁空虚骨陷窝数逐渐增多（p<0.01）;3.与正常组比较,实验组全血粘度（高切、低切）、血浆粘度及红细胞压积均升高,其中低切全血粘度及红细胞压积的升高有显著性（p<0.05）;4.动态增强参数SSmax与低切全血粘度呈负相关, r=-0.65, p<0.05;5.随着激素的应用,成骨细胞、内皮细胞、骨基质中VEGF蛋白表达呈下降趋势;软骨细胞、成骨细胞及骨髓细胞间基质中BMP-2蛋白表达逐渐降低。小结:1.动态增强MRI可以评估AvNFH的血流灌注状态,能反映骨坏死的病理发展过程;2.糖皮质激素抑制了骨组织中VEGF和BMP-2的表达,从而抑制了骨内新生血管和新生骨组织的形成,使骨组织局部缺血,缺氧状态难以修复重建,这一研究结果阐明了股骨头血流减少的原因;3.VEGF、BMP-2在激素诱导的骨坏死发生发展过程中起着重要的作用。第二部分,通过贴壁法分离获取骨髓间充质干细胞（MSCs）,按加入地塞米松终浓度的不同将传三代的MSCs随机分为4组:空白对照组、10-8 mol/L处理组、10-7 mol/L处理组、10-6 mol/L处理组。倒置相差显微镜下观察细胞形态变化,细胞培养至第8d时用MTT方法检测其增殖,第21d时终止培养,对各组脂肪细胞进行油红O染色,并对其进行计数;采用方差分析和t检验对上述结果进行统计学分析。上述研究显示结果为:（1） MSCs形态学观察:传代细胞接种贴壁后多呈纺锤形,加地塞米松进行分组培养后,增殖速度减慢,于培养第10d,发现10-6 mol/L处理组细胞浆内开始出现小圆形脂滴,主要集中于细胞核周围,随着培养时间延长,其它处理组也相继出现小脂滴,且小脂滴逐渐融合成大的脂肪泡,即含甘油三脂的囊泡,细胞亦逐渐增大,由原来的梭形变成圆形和多角形。（2）地塞米松抑制MSCs增殖:10-8 mol/L处理组、10-7 mol/L处理组、10-6 mol/L处理组细胞的吸光度值依次为0.289±0.023、0.221±0.015,0.154±0.013,与对照组细胞的吸光度值（0.352±0.012）相比较,有显著性差异（P<0.05）。（3）地塞米松促进MSCs分化为脂肪细胞:对照组、10-8 mol/L处理组、10^-7 mol/L处理组、10^-6 mol/L处理组中,其脂肪细胞阳性率依次为1.8±0.7,46.2±3.2,77.1±3.4,83.6±2.8 （%）。各实验组与对照组比较,差异有显著性意义（P<0.05）。小结:地塞米松不仅能直接抑制MSCs增殖,还可诱导其向脂肪细胞分化,且分化程度和地塞米松浓度呈正比。这一现象和激素性骨坏死的病理改变甚为相似,对阐明激素性骨坏死的病理生理机制提供实验依据。总而言之,本实验从活体动物水平和离体细胞水平两方面进行研究,观察了激素性股骨头缺血性坏死动物模型和原代培养的骨髓间充质干细胞各相关指标的变化,两者既是相互独立的,又能够通过糖皮质激素紧密地联系在一起。我们用动态增强MRI的方法观察到,在激素诱导的股骨头缺血性坏死的发生和发展过程中,其血流灌注状态逐渐减低;骨组织中VEGF和BMP-2的表达减低,从而抑制了骨内新生血管和新生骨组织的形成,使骨组织局部缺血,这在一定程度上解释了股骨头血流减少的原因;此外,通过细胞学和形态学相结合的方法观察到,地塞米松可直接诱导骨髓间充质干细胞分化为脂肪细胞,而且地塞米松浓度越高,脂肪细胞分化越多,研究表明大剂量应用激素后股骨头骨髓内脂肪细胞增多,造成骨髓内压升高,导致骨微循环障碍,同时骨修复过程缓慢,这阐明了股骨头缺血性坏死的病理变化过程。更多还原

【Abstract】 The avascular necrosis of the femoral head (AvNFH), among which the most cases were induced by steroid hormone, is a common disease. It often occurs and causes disabilities in clinic. With the development of modern medical imaging technologies,the imaging technology of morphology and function will become a new way during disease research. It is important that we explore the microcirculatin and blood perfusin about femoral head. MR perfusion can evaluate the blood perfusin of bone tissue,and get the blood flow and velocity and so evaluate the viability of bone tissue. We evaluated femoral perfusion in the steroid-induced AvNFH animal model, using dynamic contrast-enhanced magnetic resonance imaging combined with hemorrheology, to explore the value of dynamic contrast-enhanced MRI. Otherwise,to explore the influence of VEGF through the observation of the expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2(BMP-2) in bone tissue during the progress of steroid induced osteonecrosis with immunohistochemistry. By the methods of cell and morphology we investigate further the pathogenetic mechanism of steroid-induced AvNFH with the cultured bone marrow mesenchymal stem cell in vitro.This study is divided into two parts:Firstly,twenty-eight white New Zealand rabbits were used and randomly divided into two groups. Group A: model group (horse serum and prednisone);Group B: control group. All rabbits were examined by non-enhanced, dynamic contrast-enhanced and conventional enhancement MRI scan in the same method and in the second week, fourth week, sixth week and eighth week after steroid treatment. Vein blood was attained for hemorrheology before treatment and in the fourth week, eighth week. After rabbits were killed, all femoral heads were made paraffin section and HE stained specimen for pathological investigation. Their specimens of femoral heads were taken for the expression and alteration of VEGF and BMP-2 in the second week, fourth week, sixth week and eighth week after steroid treatment by means of immunohistochemical staining and examined under microscope.These results were showed as follows: (1) By light microscopy, there were osteocytes pyknosis or necrosis,empty bone lacunae and fat tissue with much more adipocytes increased but hematopoietic tissue decreased with treatment of prednison. (2) The steepest slope (SSmax) of time-signal intensity curve gradually declined in the early stage decreased gradually (p<0.05). Correspondingly, the number of empty lacuna increased gradually (p<0.01). (3) Compared with control group, the index of model group include of whole blood (high and low slice), viscosity of plasma and packed cell volume elevated, but only whole blood (low slice), packed cell volume were significant (p<0.05). (4) The parameter SS of dynamic contrast-enhanced MRI correlated negatively with whole blood(low slice)(r=-0.65,P<0.05). (5) The expression of VEGF protein in the osteoblasts,vascular endothelial cells and bone matrix became lower in the femoral head of model group in the second,fourth week,but increased slightly in the sixth and eighth week. The expression of BMP-2 protein in cartilage cell,osteoblasts,matix between bone marrow cells descended during treatment with prednison.These results suggested that (1) Dynamic contrast-enhanced MRI can evaluate the state of blood perfusion of femoral head and show the pathologic changes during the progress of osteonecrosis. (2) The expression of VEGF and BMP-2 protein in bone tissue are inhibited by glucocorticoid during the steroid-induced necrosis of the femoral head,then angiogenesis and new bone tissue are restrained and so the degree of ischemia, anoxia of the local environment in bone tissue become serious,which explains the decrease in femoral blood perfusion.(3) It is indicated that VEGF and BMP-2 play an important role during the progress of osteonecrosis.Secondly,MSCs were separated and cultured in 6-well plates according to the different biological characteristics between MSCs and hematopoietic stem cells. And then the third-MSCs were planted in 6-well plates, which were divided into four groups by random: empty control, group treated with 10-8 mol/L Dex, group treated with 10-7 mol/L Dex, group treated with 10-6 mol/L Dex. The cells morphology was detected by using inverted phase-contrast microscope, whose proliferation was observed by using MTT assay. After four groups of MSCs were cultured for 21 days, they were stained with oil red O. Adipocytes was investigated and counted under a light microscope. These results were showed as follows: (1) Investigation of morphology of MSC:MSCs were planted and attached to the plates. Most of the cells exhibited fibroblast-like spindle shape. Compared with that of empty control, proliferation of cells treated with dexamethasone became slow. Under the inverted phase-contrast microscope, cytoplasmic lipid droplets, i.e. triglyceride-containing vesicles, were observed in the cells treated with dexamethasone. The cells became larger and transferred to round or polygonal shape. (2) Inhibition of the proliferation of MSCs by Dexamethasone: compared with that of control group,the OD value of group treated with 10-8 mol/L Dex, groups treated with 10-7 mol/L Dex, groups treated with 10-6 mol/L Dex group was respectively 0.289±0.023、0.221±0.015,0.154±0.013, compared with the control group(0.352±0.012),the difference was significant (P<0.05). (3) Dexamethasone can directly stimulate the differentiation of MSCs into adipocytes: the positive rates of adipocytes in four groups (empty control, group treated with 10-8 mol/L Dex, groups treated with 10-7 mol/L Dex, groups treated with 10-6 mol/L Dex) was respectively 1.8±0.7,46.2±3.2,77.1±3.4,83.6±2.8(%). Compared with that of control group,the difference between them was significant (P<0.05).These results suggested that dexamethasone could not only inhibit the proliferation of bone marrow mesenchymal stem cells directly, but also induce the differentiation of MSCs into adipocytes, which was similar to the pathologic changes of steroid-induced osteonecrosis, and helpful for clarifying the pathobiological mechanism of osteonecrosis induced by steroid.To sum up, changes of these indexes in steroid-induced avascular necrosis of femoral head animal model and bone marrow mesenchymal stem cells were observed from the whole level of animals and cellular level of in vitro in this study. They were both independent and interrelated with each other by the glucocorticoid. We observed that blood perfusion decreased gradually during the progress of osteonecrosis with dynamic contrast-anhanced MRI. The expression of VEGF and BMP-2 protein in bone tissue is inhibited by glucocorticoid during the steroid-induced necrosis of the femoral head,then angiogenesis and new bone tissue are restrained and so the degree of ischemia of the local environment in bone tissue become serious,which explains the decrease in femoral blood perfusion. Otherwise,it was observed that dexamethasone can directly induce bone marrow mesenchymal stem cells differentiation into a large number of adipocytes by the ways of cell and morphology. This might lead to an increase in marrow fat volume of the femoral head following administration of high dose of steroids; eventually an increase of intraosseous pressure occured, with a decrease of vascular perfusion. Without sufficient repair of the necrotic bone the final result is avascular necrosis of the femoral head.更多还原