糖皮质激素致幼兔股骨头缺血坏死的作用机制研究

发布时间：2018-03-03 05:36

本文选题：股骨头缺血坏死　切入点：动物模型/幼兔　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的检测R-spondin1、DKK-1、β-catenin在幼兔激素性股骨头缺血坏死动物模型股骨头组织中的表达,比较不同剂量甲基泼尼松龙对人成骨细胞中R-spondin1的表达及人成骨细胞凋亡的影响,探讨糖皮质激素致幼兔股骨头缺血坏死的相关机制。方法1.幼兔股骨头坏死组(n=12):激素性股骨头缺血坏死新西兰幼兔;对照组(n=12):正常新西兰幼兔。应用Western Blot、RT-q PCR检测方法检测Rspondin1、DKK-1、β-catenin在各组股骨头中的表达情况,比较各相关蛋白及基因在各组中的表达差异,皮尔逊(Pearson)相关性检验分析R-spondin1与DKK-1、β-catenin的相关性。2.培养人成骨细胞并传代,激素干预组:分别加入(10-8mol/L、10-7mol/L、10-6mol/L)甲基泼尼松龙后,继续培养72h;空白组:不加甲基泼尼松龙,继续培养72h。Western Blot、RT-q PCR检测各组R-spondin1的表达情况,同期检测各组的细胞凋亡率。结果1.R-spondin1在幼兔股骨头坏死组与对照组中的蛋白表达量分别为0.179±0.028、0.471±0.104,差异具有统计学意义(t=9.38,P0.01);β-catenin在幼兔股骨头坏死组与对照组中的蛋白表达量分别为0.195±0.041、0.632±0.129,差异具有统计学意义(t=11.15,P0.01);Dkk1在幼兔股骨头坏死组与对照组中的蛋白表达量分别为0.713±0.161、0.181±0.029,差异具有统计学意义(t=-11.25,P0.01);在幼兔股骨头坏死组与对照组中,R-spondin1与β-catenin的蛋白表达呈正相关关系(r=0.89,P0.01),R-spondin1与Dkk1呈负相关关系(r=-0.945,P0.05)。2.R-spondin1在幼兔股骨头坏死组与对照组中的m RNA表达量分别为0.255±0.039、0.995±0.093,差异具有统计学意义(t=25.33,P0.01);β-catenin在幼兔股骨头坏死组与对照组中的m RNA表达量分别为0.216±0.036、1.055±0.083,差异具有统计学意义(t=32.49,P0.01);Dkk1在幼兔股骨头坏死组与对照组中的m RNA表达量分别为3.275±0.073、0.986±0.084,差异具有统计学意义(t=-10.32,P0.01);在幼兔股骨头坏死组与对照组中,R-spondin1与β-catenin的表达m RNA呈正相关关系(r=0.860,P0.01),R-spondin1与Dkk1呈负相关关系(r=-0.945,P0.01)。3.空白组与10-8mol/L、10-7mol/L、10-6mol/L三种不同剂量甲基泼尼松龙干预培养的细胞中R-spondin1蛋白表达量分别为0.622±0.105、0.441±0.125、0.194±0.038、0.112±0.019,R-spondin1蛋白表达量随甲基泼尼松龙剂量增大而降低,差异具有统计学意义(F=76.37,P0.01);R-spondin1m RNA表达量分别为0.999±0.168,0.897±0.131,0.624±0.101,0.323±0.033,R-spondin1m RNA表达量随甲基泼尼松龙剂量增大而降低,差异具有统计学意义(F=64.79,P0.01)。4.空白组与10-8mol/L组、10-7mol/L组、10-6mol/L组的细胞凋亡率(%)分别为7.41±1.11、13.14±1.92、25.01±5.21、31.99±9.48,细胞凋亡率随干预剂量增大而增大,差异具有统计学意义(F=40.80,P0.01)。结论1.糖皮质激素对人成骨细胞中R-spondin1的表达具有负调控作用,可能通过负调控R-spondin1表达促进人成骨细胞的凋亡。2.在糖皮质激素致幼兔股骨头缺血坏死模型中,R-spondin1与β-catenin表达正相关,与DKK-1表达负相关,R-spondin1可能负调控Wnt/β-catenin信号通路,促进成骨细胞的凋亡,导致幼兔激素性股骨头缺血坏死发生。
[Abstract]:Objective to detect the expression of R-spondin1, DKK-1, beta -catenin in rabbit steroid induced avascular necrosis of femoral head in animal model, comparison of different doses of methylprednisolone on the expression of human R-spondin1 and human bone cells, osteoblast apoptosis, and to explore the mechanism of glucocorticoid induced avascular necrosis of the femoral head in young rabbits methods 1. Rabbits Femoral Head Necrosis group (n=12): steroid induced femoral head necrosis rabbits; the control group (n=12): normal New Zealand rabbits. The application of Western Blot method for detection of RT-q PCR detection of Rspondin1, DKK-1, -catenin beta expression in each group in the femoral head, comparing the difference of the expression of related protein and the genes in each group, Pearson (Pearson) correlation analysis of R-spondin1 and DKK-1 in human osteoblasts and cultured.2. associated beta -catenin, hormone intervention group were added: (10-8mol/L, 10-7mol/L, 10 -6mol/L) methylprednisolone after cultured 72h; control group: without methylprednisolone, cultured for 72h.Western Blot expression was detected by R-spondin1 RT-q PCR, the apoptosis rate of each group. The results of earlier detection of 1.R-spondin1 in rabbit femoral head necrosis group and control group the protein expression were 0.179 + 0.028,0.471 + 0.104, the difference was statistically significant (t=9.38, P0.01); beta -catenin in rabbit femoral head necrosis group and control group the protein expression were 0.195 + 0.041,0.632 + 0.129, the difference was statistically significant (T, =11.15, P0.01); Dkk1 in rabbit femoral head necrosis group and control group the protein expression respectively. 0.713 + 0.161,0.181 + 0.029, the difference was statistically significant (t=-11.25, P0.01); in rabbit femoral head necrosis group and control group, R-spondin1 and beta -catenin protein expression was positively correlated (r=0.89, P0. 01), a negative correlation between R-spondin1 and Dkk1 (r=-0.945, P0.05).2.R-spondin1 in rabbit femoral head necrosis group and control group in M RNA expression were 0.255 + 0.039,0.995 + 0.093, the difference was statistically significant (t=25.33, P0.01); beta -catenin in rabbit femoral head necrosis group and control group. M RNA expression was 0.216 + 0.036,1.055 + 0.083, the difference was statistically significant (t=32.49, P0.01); Dkk1 in rabbit femoral head necrosis group and control group in M RNA expression were 3.275 + 0.073,0.986 + 0.084, the differences were statistically significant (t=-10.32, P0.01); in rabbit femoral head necrosis group and control group, the expression of M RNA was positively related to the relationship between R-spondin1 and beta -catenin (r=0.860, P0.01), a negative correlation between R-spondin1 and Dkk1 (r=-0.945, P0.01).3. control group and 10-8mol/L, 10-7mol/L, 10-6mol and /L in three different doses of methylprednisolone. The expression of R-spondin1 protein were 0.622 + 0.105,0.441 + 0.125,0.194 + 0.038,0.112 + 0.019 support cells, the expression of R-spondin1 protein with methyl prednisolone dose decreased, the difference was statistically significant (F=76.37, P0.01); R-spondin1m RNA expression were 0.999 + 0.168,0.897 + 0.131,0.624 + 0.101,0.323 + 0.033, R-spondin1m expression of RNA with methyl prednisolone dose decreased, the difference was statistically significant (F=64.79, P0.01).4. control group and 10-8mol/L group, 10-7mol/L group, 10-6mol/L group apoptosis rate (%) were 7.41 + 1.11,13.14 + 1.92,25.01 + 5.21,31.99 + 9.48, the apoptosis rate of intervention with the dose increased, the difference was statistically significant (F=40.80, P0.01 1.). Conclusion glucocorticoid negatively regulates human expression of R-spondin1 in osteoblasts, the expression of R-spondin1 may be through negative regulation To promote the apoptosis of.2. cells into bone in glucocorticoid induced avascular necrosis of femoral model young, R-spondin1 expression was positively correlated with beta -catenin, negatively correlated with the expression of DKK-1 and R-spondin1 may negatively regulate Wnt/ beta -catenin signaling pathway to promote cell apoptosis, leading to young rabbits with steroid induced avascular necrosis of femoral head.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R726.8

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