巨噬细胞移动抑制因子对激素性股骨头缺血性坏死血管修复影响的体外实验研究

发布时间：2018-01-27 08:52

本文关键词： 激素性股骨头缺血性坏死巨噬细胞移动抑制因子血管修复体外实验　出处：《中国修复重建外科杂志》2016年08期 　论文类型：期刊论文

【摘要】：目的通过检测体外缺氧/激素作用下内皮细胞(endothelial cells,ECs)增殖、迁徙以及巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)和VEGF的表达变化,探讨激素性股骨头缺血性坏死(steroid-induced avascular necrosis of femoral head,SANFH)血管修复障碍发生机制。方法取第3代人脐静脉ECs(human umbilical vein ECs,HUVECs)进行分组干预。正常对照组(A组)细胞不作任何干预;地塞米松组(B组):用含1.0×10~(-6) mol/L地塞米松的完全培养液干预;缺氧培养组(C组):细胞在低氧细胞培养罐内培养;地塞米松+缺氧培养组(D组):细胞在低氧细胞培养罐内培养,同时用含1.0×10~(-6) mol/L地塞米松的完全培养液干预。干预培养后24 h,行Alamar Blue细胞活性检测及活/死细胞染色观测细胞增殖情况,细胞骨架染色观察细胞骨架形态;采用划痕实验比较各组细胞迁徙能力,ELISA法检测各组细胞MIF及VEGF表达水平。结果干预培养后24 h,Alamar Blue细胞活性检测及活/死细胞染色示C组细胞活性最好且活细胞数多,D组细胞活性最差、活细胞数最少,组间比较差异均有统计学意义(P0.05)。细胞骨架染色见,A、B组细胞形态正常;C组细胞大量增殖,细胞内见大量分泌颗粒;D组细胞骨架形态异常。划痕实验示C组细胞迁移能力最强,D组最弱。各组MIF和VEGF累积浓度随时间延长均显著增高。各时间点,C组MIF累积浓度显著高于其他各组(P0.05)。干预培养24 h内,各组组内1~8 h时MIF阶段浓度显著低于0~1 h及8~24 h时(P0.05);且C组0~1 h和8~24 h时MIF阶段浓度显著高于其他组(P0.05)。干预培养2 h内,各组组内0.5~1 h MIF阶段浓度显著高于其他各时间段(P0.05)。8、24 h时C组VEGF累积浓度显著高于其他各组(P0.05)。干预24 h内,C、D组8~24 h时VEGF阶段浓度显著高于其他各时间段(P0.05);干预培养2 h内,各组组内各时间段及组间VEGF阶段浓度比较,差异均无统计学意义(P0.05)。结论缺氧条件下ECs增殖及迁徙能力增强,MIF及VEGF分泌增加;而高浓度地塞米松会抑制上述过程,引发血管修复障碍,导致SANFH发生、发展。
[Abstract]:Objective to detect the proliferation of endothelial cells (ECs) induced by hypoxia / hormone in vitro. The expression of macrophage migration inhibitory factor MIF and VEGF in migration and macrophage migration suppressor. To investigate steroid-induced avascular necrosis of femoral head. Methods the third generation of human umbilical vein ECs(human umbilical vein ECs was used. HUVECs were divided into groups. The normal control group (group A) was not treated with any intervention. Dexamethasone group (group B) was treated with mol/L dexamethasone (1.0 脳 10 ~ (-1)) dexamethasone. Anoxic culture group (group C): cells were cultured in hypoxic cell culture tank; Dexamethasone anoxic culture group (group D): cells were cultured in hypoxic cell culture tank. At the same time, mol/L dexamethasone containing 1.0 脳 10 ~ (-1) -6) was used for 24 h after intervention. The activity of Alamar Blue cells was detected and the cell proliferation was observed by living / dead cell staining. The cytoskeleton morphology was observed by cytoskeleton staining. The expression of MIF and VEGF in the cells of each group were detected by scratch test and Elisa. Results 24 hours after intervention, the expression of MIF and VEGF were detected. Alamar Blue cell activity test and live / dead cell staining showed that C group had the best cell activity, and group D had the worst cell activity and the number of living cells was the least. The difference between the two groups was statistically significant (P 0.05). The cytoskeleton staining showed that the morphology of the cells was normal in group B. In group C, a large number of cells proliferated and a large number of secretory granules were found in the cells. The morphology of cytoskeleton in group D was abnormal. Scratch test showed that the ability of cell migration in group C was the strongest and the accumulation of MIF and VEGF in group D was the weakest. The cumulative concentrations of MIF and VEGF increased significantly with time. The cumulative concentration of MIF in group C was significantly higher than that in other groups (P 0.05). The concentration of MIF at 1h and 8h in each group was significantly lower than that at 0 h and 824 h, respectively. The concentration of MIF in group C was significantly higher than that in other groups at 0 h and 824 h. The concentration of 0. 5 h MIF in each group was significantly higher than that in other time periods (P 0. 05. 8). The cumulative concentration of VEGF in group C was significantly higher than that in other groups at 24 h. The concentration of VEGF phase in group D was significantly higher than that in other time periods at 824 h. Within 2 hours of intervention, there was no significant difference in the concentration of VEGF in each time period and between groups. Conclusion the proliferation and migration ability of ECs was enhanced under hypoxia condition. [WT5 "HZ] [WT5" HZ] [WT5 "BZ] [WT5" BZ]. The secretion of MIF and VEGF increased. High concentration of dexamethasone can inhibit these processes, causing vascular repair disorders, leading to the occurrence and development of SANFH.
【作者单位】：西安交通大学第二附属医院骨一科;
【基金】：国家自然科学基金资助项目(81301562) 西安交通大学第二附属医院人才培养专项科研基金资助项目[RC(XM)201501]~~
【分类号】：R681.8
【正文快照】： 激素性股骨头缺血性坏死(steroid-induced avas-cular necrosis of femoral head,SANFH)是骨科常见病、多发病[1]。近年研究发现,SANFH临床标本中可观察到严重微血管损伤,骨细胞缺血性坏死,新生血管减少,血管修复障碍等病理现象[2-4]。糖皮质激素(glucocorticoids,Glc)诱发的

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