糖皮质激素对骨微血管内皮细胞和成骨细胞的影响

Effect of glucocorticoid on bone microvascular endothelial cells and osteoblasts

目的观察和比较不同浓度糖皮质激素(GC)对骨微血管内皮细胞(BMECs)和成骨细胞(OBs)活性和凋亡的影响。方法从Sprague-Dawley大鼠股骨中分离和培养BMECs和OBs,通过血管性血友病因子(vWF)免疫荧光染色鉴定BMECs,碱性磷酸酶(ALP)染色和茜素红S染色鉴定OBs。分别用0.0、0.1、0.2、0.3和0.4 mg/ml的GC处理细胞,细胞计数试剂盒(CCK-8)法检测BMECs和OBs的活性,膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙锭(PI)染色和原位缺口末端标记法(TUNEL)/4’,6-二脒基-2-苯基吲哚(DAPI)染色检测BMECs和OBs的凋亡,SPSS 19.0进行数据分析。结果随着GC浓度逐渐升高,BMECs和OBs的活性呈剂量依赖性下降,当GC=0.2 mg/ml时,BMECs活性为(74.511±3.109)%,OBs活性为(92.750±6.344)%,OBs活性高于BMECs(t=5.106,P<0.01)。0.2 mg/ml GC处理的BMECs凋亡率为(17.766±1.393)%,与0 mg/ml组比较差异有统计学意义(t=17.150,P<0.01),而0.2 mg/ml GC处理的OBs凋亡率为(4.932±0.616)%,与0 mg/ml组比较差异无统计学意义(t=1.324,P>0.05)。当GC浓度=0.2 mg/ml时,BMECs的TUNEL阳性细胞比率为(18.765±1.393)%,OBs的TUNEL阳性细胞比率为(5.295±1.371)%,BMECs的TUNEL阳性细胞数高于OBs(t=13.784,P<0.01)。结论与OBs比较,GC可更明显地降低BMECs的活性和增加其凋亡。

Objective To evaluate and compare the effects of different concentrations of glucocorti-coids(GC)on the activity and apoptosis of bone microvascular endothelial cells(BMECs)and osteoblasts(OBs).Methods BMECs and OBs were isolated and cultured from the femus of Sprague-Dawley rats.The BMECs were identified by von willebrand factor(vWF)immunofluorescence staining,and the OBs were identified by alkaline phosphatase(ALP)staining and alizarin red S staining.Cells were treated with 0,0.1,0.2,0.3 and 0.4 mg/ml GC,respectively.Cell counting kit-8(CCK-8)assay was used to detect the activity of BMECs and OBs,and Annexin V-fluoresceine isothiocyanate(FITC)/propidium iodide(PI)staining and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)/4',6-diamidino-2-phenylindole(DAPI)staining were used to detect the apoptosis of BMECs and OBs.The SPSS 19.O was used for data analysis.Results With the increase of CC concentration,the activity of BMECs and OBs decreased in a dose-dependent manner.When GC=O.2 mg/ml,the activity of BMECs and OBs was(74.511±3.109)%and(92.750±6.344)%respectively.The activity of 0Bs was higher than that of BMECs(t=5.106,P<0.01).The apoptosis rate of BMECs treated with 0.2 mg/ml GC was(17.766±1.393)%,and the difference was statistically significant compared with 0 mg/ml GC group(t=17.150,P<0.01).However,the apoptosis rate of OBs treated with 0.2 mg/ml GC was(4.932±0.616)%,and there was no significant difference compared with 0 mg/ml GC group(t=1.324,P>0.05).When the concentration of GC was 0.2 mg/ml,the ratio of TUNEL-positive cells in BMECs and OBs was(18.765±1.393)%and(5.295±1.371)%,respectively.The number of TUNEL positive cells in BMECs was grea-ter than that in OBs(t=13.784,P<0.01).Conclusion Compared with OBs,GC can significantly reduce the activity and increase the apoptosis of BMECs.