激素性股骨头坏死和酒精性股骨头坏死的病理学差异及其分子机制研究

Pathological differences between steroid-induced and alcohol-induced osteonecrosis of the femoral head and their molecular mechanisms

目的:探讨激素性股骨头坏死(osteonecrosis of the femoral head,ONFH)和酒精性ONFH的病理学差异及其分子机制。方法:纳入接受人工全髋关节置换术治疗的ONFH患者的股骨头60个,根据患者发生ONFH的病因,将股骨头分为激素性ONFH组和酒精性ONFH组。将股骨头做冠状面逐层切片后,观察2组股骨头的分区情况及病理特点;于股骨头正中切片上分别取坏死区、硬化区、正常区的骨组织,处理后采用扫描电镜观察2组股骨头不同区域骨组织的结构特点;分离股骨头坏死区、硬化区、正常区的骨组织,采用蛋白印迹法检测2组股骨头各区骨组织中血小板-内皮细胞粘附分子-1(platelet endothelial cell adhesion molecule-1,PECAM-1)、内皮粘蛋白(endomucin,EMCN)、血小板衍生生长因子(platelet derived growth factor,PDGF)-BB、表皮生长因子样结构域8(epidermal growth factor-like domain 8,EGFL8)及骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)的表达量。结果:股骨头切片显示,激素性ONFH组股骨头坏死区与硬化区交界处有新鲜肉芽组织,酒精性ONFH组股骨头坏死区与硬化区交界处无新鲜肉芽组织,有纤维组织填充。扫描电镜图像显示,激素性ONFH组股骨头的坏死区骨细胞胞体呈现粗糙、溶解样,周围有树突包裹,有骨小管分布;硬化区骨细胞胞体较正常骨细胞略小,树突缩短、数量减少,有骨小管分布;正常区有丰富的骨小管分布。酒精性ONFH组股骨头的坏死区骨细胞萎缩,甚至消失,未见树突与骨小管分布;硬化区骨细胞萎缩、体积较小,树突稀疏,骨小管小、数量少;正常区骨小管较小、数量较少。激素性ONFH组股骨头坏死区、硬化区、正常区骨组织中PECAM-1、EMCN、EGFL8,坏死区、硬化区骨组织中PDGF-BB,硬化区骨组织中BMP-2的蛋白表达量均高于酒精性ONFH组(PECAM-1:1.970±0.053,0.493±0.076,t=0.453,P=0.000;0.797±0.047,0.467±0.025,t=1.910,P=0.000;2.207±0.045,1.443±0.118,t=4.993,P=0.000;EMCN:1.277±0.038,1.077±0.025,t=1.087,P=0.002;2.220±0.106,0.270±0.036,t=4.655,P=0.000;2.400±0.046,0.473±0.126,t=2.494,P=0.000;EGFL8:4.443±0.313,1.043±0.091,t=4.106,P=0.000;1.833±0.096,0.090±0.030,t=2.969,P=0.000;0.553±0.110,0.130±0.010,t=8.892,P=0.021;PDGF-BB:1.413±0.040,0.223±0.006,t=4.654,P=0.000;0.980±0.036,0.193±0.015,t=2.579,P=0.000;BMP-2:0.417±0.023,0.270±0.036,t=0.810,P=0.004),坏死区骨组织中BMP-2的蛋白表达量低于酒精性ONFH组(0.343±0.070,1.160±0.056,t=0.116,P=0.000);激素性ONFH组股骨头正常区骨组织中PDGF-BB、BMP-2的蛋白表达量与酒精性ONFH组比较,组间差异均无统计学意义(0.250±0.056,0.287±0.031,t=1.062,P=0.374;0.263±0.025,0.320±0.036,t=0.604,P=0.089)。结论:与激素性ONFH相比,酒精性ONFH的股骨头坏死区与硬化区交界处无新鲜肉芽组织、有纤维组织填充,坏死区、硬化区的骨细胞明显萎缩,各区骨小管更小、数量更少,这种病理学差异与股骨头不同区域骨组织中血管生成蛋白和骨代谢相关蛋白的表达有关。

Objective:To explore the pathological differences between steroid-induced osteonecrosis of the femoral head(ONFH)and alcohol-induced ONFH,and to explore their molecular mechanisms.Methods:Sixty femoral heads from ONFH patients who underwent artificial total hip arthroplasty were enrolled in the study and were divided into steroid-induced ONFH group and alcohol-induced ONFH group according to the etiology of ONFH.The femoral heads were sectioned in coronal plane layer by layer,and their subregion and pathological characteristics were observed.The bone tissues from the necrotic zone,sclerotic zone and normal zone of the femoral head were harvested from the median section of the femoral head,and their structural characteristic were observed in the 2 groups using a scanning electron microscope after processing.The bone tissues from the necrotic zone,sclerotic zone and normal zone were isolated,and the protein expression levels of platelet endothelial cell adhesion molecule-1(PECAM-1),endomucin(EMCN),platelet-derived growth factor(PDGF)-BB,epider-mal growth factor-like domain 8(EGFL8)and bone morphogenetic protein-2(BMP-2)in the bone tissues of each zone were detected using Western blot.Results:The femoral head sections showed that there were fresh granulation tissues at the junction of the necrotic zone and sclerotic zone in the steroid-induced ONFH group,while no fresh granulation tissues were observed,but fibrous tissues were found in alco-hol-induced ONFH group.The scanning electron microscopy images showed that,in steroid-induced ONFH group,the osteocytes in the nec-rotic zone displayed as rough,dissolved cell bodies surrounded by dendrites,with distributed bone canaliculi.The osteocytes in the sclerotic zone displayed as slightly smaller cell bodies compared with that of normal osteocytes,with shorter and fewer dendrites and distributed bone canaliculi.The abundant bone canaliculi were found in the normal zone.While in alcohol-induced ONFH group,the osteocytes in the necrotic zone were atrophied or even disappeared,and no dendrites or bone canaliculi were observed.The osteocytes in the sclerotic zone were al-so atrophied,manifesting as a smaller volume with sparse dendrites and small and few bone canaliculi,meanwhile,smaller and fewer bone canaliculi were found in the normal zone.The PECAM-1,EMCN and EGFL8 in the bone tissues of the necroticzon zone,sclerotic zone and normal zone,and PDGF-BB in the bone tissues of the necrotic zone and sclerotic zone,and BMP-2 in the bone tissues of the sclerotic zone were all highly expressed in the steroid-induced ONFH group compared to alcohol-induced ONFH group(PECAM-1:1.970±0.053 vs 0.493±0.076,t=0.453,P=0.000;0.797±0.047 vs 0.467±0.025,t=1.910,P=0.000;2.207±0.045 vs 1.443±0.118,t=4.993,P=0.000;EMCN:1.277±0.038 vs 1.077±0.025,t=1.087,P=0.002;2.220±0.106 vs 0.270±0.036,t=4.655,P=0.000;2.400±0.046 vs 0.473±0.126,t=2.494,P=0.000;EGFL8:4.443±0.313 vs 1.043±0.091,t=4.106,P=0.000;1.833±0.096 vs 0.090±0.030,t=2.969,P=0.000;0.553±0.110 vs 0.130±0.010,t=8.892,P=0.021;PDGF-BB:1.413±0.040 vs 0.223±0.006,t=4.654,P=0.000;0.980±0.036 vs 0.193±0.015,t=2.579,P=0.000;BMP-2:0.417±0.023 vs 0.270±0.036,t=0.810,P=0.004),while the BMP-2 in the bone tissues of the necrotic zone was lowly expressed in the steroid-induced ONFH group compared to the alcohol-induced ONFH group(0.343±0.070 vs 1.160±0.056,t=0.116,P=0.000).There was no statistical difference in the protein expression levels of PDGF-BB and BMP-2 in the bone tissues of normal zone between steroid-induced ONFH group and alcohol-induced ONFH group(0.250±0.056 vs 0.287±0.031,t=1.062,P=0.374;0.263±0.025 vs 0.320±0.036,t=0.604,P=0.089).Conclusion:Compared to steroid-induced ONFH patients,the fibrous tissues can be observed,but the fresh granulation tissues can not be observed at the junction of the necrotic zone and sclerotic zone of the femoral head in alcohol-induced ONFH patients,moreover,the osteocytes in the necrotic zone and sclerotic zone are significantly atrophied and the bone canaliculi in each zone are smaller and fewer.These pathological differences are related to the expression of vascular growth proteins and bone metabolism-related proteins in the bone tissues of different zones of the femoral head.