促肾上腺皮质激素对小鼠骨髓间充质干细胞成骨分化的影响  

Effect of adrenocorticotropic hormone on osteogenesis of mouse bone marrow mesenchymal stem cells

作　　者：尚国伟[1] 寇红伟[1] 姬彦辉 杨新林[2] 刘宏建[1] 崔全军[2] Shang Guowei;Kou Hongwei;Ji Yanhui;Yang Xinlin;Liu Hongjian;Cui Quanjun(Department of Orthopaedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450002,China;Orthopedic Laboratory of Medical College of Virginia University,Charlottesville 22903,USA)

机构地区：[1]郑州大学第一附属医院骨科,450052 [2]美国弗吉尼亚大学医院骨科实验室,夏洛特维尔,美国22903

出　　处：《中华实验外科杂志》2021年第5期868-870,共3页Chinese Journal of Experimental Surgery

摘　　要：目的观察促肾上腺皮质激素(ACTH)对小鼠骨髓间充质干细胞(D1细胞系)成骨分化的影响。方法将D1细胞分成6组,对照组、对照组+低浓度ACTH组、对照组+高浓度ACTH组、成骨组、成骨组+低浓度ACTH组、成骨组+高浓度ACTH组;各组孵育细胞6 d后通过茜素红染色法评价成骨分化;分别孵育细胞1、3、6 d后,采用聚合酶链反应(PCR)技术定量分析核心结合蛋白因子-2(Runx2)、碱性磷酸酶(ALP)、黑皮质素受体-3(MC3R)和血管内皮生长因子a(VEGFa)成骨相关基因的表达水平;孵育细胞3、6 d后,采取蛋白质印迹法(Western blot)技术分析成骨分化相关基因Runx2和骨涎蛋白(BSP)表达水平。两样本均数比较采用独立样本t检验,多样本均数比较采用单因素方差分析。结果RT-PCR检测结果发现,第3天成骨组+高浓度ACTH(1×10^(-8)mol/L)组黑色素3蛋白受体(MC3R)表达(1.33±0.01)低于成骨组(1.82±0.11),差异有统计学意义(t=5.039,P<0.05);第6天成骨组+高浓度ACTH(1×10^(-8)mol/L)组Runx2表达(0.72±0.01)低于成骨组(1.16±0.08),差异有统计学意义(t=5.542,P<0.05);成骨组+高浓度ACTH(1×10^(-8)mol/L)组血管内皮生长因子(VEGFa)表达(0.58±0.02)显著低于成骨组(2.23±0.06),差异有统计学意义(t=7.238,P<0.05);成骨组+高浓度ACTH(1×10^(-8)mol/L)组茜素红染色吸光度(A)值(0.270±0.012)显著低于成骨组(0.361±0.015),差异有统计学意义(t=4.435,P<0.05);在蛋白水平,Runx2、BSP基因蛋白表达都有下降的趋势;成骨组+低浓度ACTH(1×10-9 mol/L)组茜素红染色A值(0.363±0.013)同成骨组(0.360±0.016)比较差异无统计学意义(t=1.345,P>0.05),且对成骨基因和蛋白表达作用差异无统计学意义。结论高浓度ACTH(1×10^(-8)mol/L)对小鼠骨髓间充质干细胞成骨分化有抑制作用,而且在基因和蛋白水平对成骨特异性转录因子Runx2有明显的抑制作用。Objective To evaluate the influence of adrenocorticotropic hormone on osteogenic differentiation of a mouse bone marrow-derived multipotent cell line,D1.Methods D1 cells were divided into six groups:control group;control group treated with low concentration of adrenocorticotropic hormone(ACTH);control group treated with high concentration of ACTH;osteogenic medium group;osteogenic medium treated with low concentration of ACTH;osteogenic medium treated with high concentration of ACTH.Mineralization of mouse bone marrow-derived multipotent cell line(D1)cells were assessed by Alizarin Red staining at day 6;The gene expression of related transcription factor-2(Runx2),alkaline phosphatase(ALP),melanocortin receptor 3(MC3R)and vascular endothelial growth factor a(VEGFa)were detected by real-time quantitative polymerase chain reaction(Real-time PCR)when D1 cells were cultured at day 1,day 3 and day 6;Western blotting was used to detect the protein expression of Runx2 and BSP at day 3 and day 6.SPSS 17.0 software was used for all statistical analyses.All results are expressed as the mean±standard deviation.Student′s t-test and one-way analysis of variance were used for comparisons of means.Results At day 3,osteogenic medium treated with ACTH(1×10^(-8)mol/L)group(1.33±0.01)could suppress the gene expresssion of MC3R compared with osteogenic medium group(1.82±0.11)significantly(t=5.039,P<0.05);At day 6,compared with osteogenic medium group(1.16±0.08),the gene expression of Runx2(0.72±0.01)in osteogenic medium treated with ACTH(1×10^(-8)mol/L)group was reduced significantly(t=5.542,P<0.05),the gene expression of VEGFa(0.58±0.02)were downregulated by ACTH(1×10^(-8)mol/L)compared with osteogenic medium group(2.23±0.06)significantly(t=7.238,P<0.05);The mineralization of D1 cells in osteogenic medium treated with ACTH(1×10^(-8)mol/L)group(0.270±0.012)was decreased in comparison with osteogenic medium group(0.361±0.015)significantly(t=4.435,P<0.05);Western blotting analysis showed that the protein expression of Ru

关 键 词：骨髓间充质干细胞 成骨分化 促肾上腺皮质激素 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]