人股骨头不同区域骨微血管内皮细胞11β-羟基类固醇脱氢酶表达的差异

Genetic expression differences of 11 beta-hydroxysteroid dehydrogenase in the bone microvascular endothelial cells derived from different regions of the human femoral head

目的:探讨人股骨头内不同区域骨微血管内皮细胞(BMECs)11β-羟基类固醇脱氢酶(11β-HSD)基因表达水平及活化差异。方法:选用于2017年1月至2018年6月在中日友好医院接受髋关节置换术切除的人新鲜股骨头组织标本,分别对人股骨头松质骨区与软骨下骨区的BMECs进行分离、纯化、鉴定和培养,用一系列低浓度梯度氢化可的松(0、0.03、0.06、0.10 mg/ml)分别对两个区域BMECs进行干预,观察股骨头不同区域的BMECs的细胞表型与功能状态,划痕实验检测细胞迁移能力,观察血管生成能力。应用实时荧光定量聚合酶链反应(RT-PCR)法检测比较不同部位BMECs内11β-HSD1、11β-HSD2 mRNA的表达,采用Western蛋白印迹法检测11β-HSD1、11β-HSD2蛋白表达情况。结果:随着氢化可的松浓度升高,股骨头松质骨区和软骨下骨区BMECs 11β-HSD1 mRNA及蛋白相对表达量均明显增加,软骨下骨区BMECs 11β-HSD1 mRNA及蛋白相对表达量明显低于松质骨区(均P<0.05)。11β-HSD2 mRNA及蛋白相对表达量在股骨头松质骨区BMECs表现为先缓慢减少,然后在0.10 mg/ml时略微增加,而在软骨下骨区表现与之相反,软骨下骨区11β-HSD2 mRNA及蛋白相对表达量比值略高于松质骨区(均P<0.05),但在0.10 mg/ml时两区域差异无统计学意义(0.123±0.018比0.126±0.021、0.577±0.231比0.609±0.174,t=1.380、0.409,均P>0.05)。0.06 mg/ml氢化可的松处理后不同时间,股骨头不同区域BMECs划痕闭合率、管腔数、出芽数和小管分支长度差异均无统计学意义(均P>0.05)。结论:人股骨头不同区域BMECs 11β-HSD表达存在明显差异,松质骨区BMECs 11β-HSD1高度表达而11β-HSD2却低表达,软骨下骨区则表现相反,这有助于解释激素性骨坏死的病理特征及发病机制。

Objective To investigate the expression levels and activation differences of 11beta-hydroxysteroid dehydrogenase(11beta-HSD)gene in bone microvascular endothelial cells(BMECs)in different regions of human femoral head.Methods Tissue specimens of femoral heads were obtained from hip arthroplasty carried out in China-Japan Friendship Hospital from January 2017 to June 2018.And the BMECs we isolated,purified,identified and cultured from different regions of the human femoral head:in the subchondral and cancellous bone regions.The BMECs from the two regions were intervened by hydrocortisone with a series of low concentration gradients(0,0.03,0.06,0.10 mg/ml)respectively.The cell phenotype and functional status of BMECs and cell migration were detected by scratch experiments,and the angiogenesis in different regions of the femoral head was observed.The mRNA and protein expression of 11beta-HSD1,11beta-HSD2 in BMECs were detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and Western-blot method,respectively.Results With the increase of the concentration of hydrocortisone,the 11beta-HSD1 mRNA and protein expression of BMECs in the subchondral and cancellous bone regions of the femoral head increased significantly,and the 11beta-HSD1 mRNA and protein expression of BMECs in the subchondral bone region was significantly lower than those in cancellous bone region(all P<0.05).The 11beta-HSD2 mRNA and protein expression of BMECs in the cancellous bone region showed a slow decrease first and then increased slightly at 0.10 mg/ml,while the expression in the subchondral bone region was the opposite.The 11beta-HSD2 mRNA and protein expression of BMECs in subchondral bone region was slightly higher than those in cancellous bone region(all P<0.05),but there was no significant statistical difference between the two regions at 0.10 mg/ml(0.123±0.018 vs 0.126±0.021,0.577±0.231 vs 0.609±0.174,t=1.380,0.409,both P>0.05).At different times of the 0.06 mg/ml hydrocortisone intervention,there was no significant differences in scratch closure rate,the number of BMECs lumen,the number of buds and the length of tubule branches in different regions of the femoral head(all P>0.05).Conclusion The 11beta-HSD expression of BMECs in different regions of human femoral head is significantly different.The 11beta-HSD1 is high-expressed,but 11beta-HSD2 is low-expressed in BMECs of the cancellous bone region,and those are opposite in the subchondral bone region,which helps to explain the pathological characteristics and pathogenesis of hormonal osteonecrosis.