脂联素通过调节2型糖尿病大鼠骨微环境氧化应激水平对成骨细胞影响的实验研究

发布时间：2018-03-12 07:23

  本文选题：2型糖尿病　切入点：骨质疏松　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本实验旨在探讨2型糖尿病(DM)大鼠骨微环境氧化应激(OS)水平与成骨细胞(OB)分化调节因子Runx2的关系,同时观察脂联素(APN)干预是否可以通过改善骨微环境氧化应激影响成骨细胞分化,进一步探讨糖尿病性骨质疏松症(DOP)的发生机制及早期干预措施,为临床预防及治疗糖尿病性骨质疏松症提供新思路。方法:选择4周龄雄性SD大鼠随机分为正常对照组(n=18只)、糖尿病模型组(n=42只)。对照组予普通饲料喂养,模型组高胆固醇高糖喂养2个月,并一次性腹腔注射链脲佐菌素(STZ)30mg/kg建2型DM大鼠模型。剔除造模失败、死亡大鼠,建模成功的大鼠分为糖尿病组(DM组=18只)和脂联素干预组(APN组=18只),APN组予腹腔注射球形脂联素10μg/kg*d干预,分别于第4、8、12周末处死动物,迅速取出双侧股骨及胫骨,提取一侧股骨及胫骨骨髓液,经离心分别留取骨髓细胞及骨髓上清液,另一侧股骨经测定骨密度后脱钙包埋切片。ELISA、RT-PCR等方法检测Runx-2、BALP、AOPPs,骨组织免疫组化染色观察AGEs表达,HE染色观察骨质量(骨小梁结构、密度)及脂肪细胞量。结果:1.各组大鼠血糖水平比较随着喂养时间的延长,可见DM组大鼠血糖水平明显升高。APN干预后,血糖有所改善。2.三组大鼠骨髓上清液AOPP水平比较随着喂养时间延长,正常大鼠组骨髓上清液AOPP无明显变化,在DM组、APN干预组均升高(P0.05),但APN干预组升高幅度较DM组小。在4周时,DM组较正常对照组AOPP水平升高(P0.05),APN干预后较DM组无明显差异;8周及12周时DM组AOPP水平升高更显著(P0.05),APN干预后,8周时有轻微下降趋势,12周时较DM组下降且有统计学意义(P0.05)。3.三组大鼠骨组织免疫组化AGEs表达比较随着时间变化,正常大鼠组骨组织免疫组化表达AGEs无明显差异,在DM组、APN干预组表达均升高,(P0.05),但APN干预组升高幅度较DM组小。在4周时,DM组较正常对照组AGEs表达水平升高(P0.05),APN干预后较DM组降低(P0.05),8周及12周时DM组AGEs表达升高更显著(P0.05),APN干预后较DM组降低(P0.05),且12周较8周AGEs下降幅度显著增大。4.三组大鼠骨髓细胞Runx2 mRNA表达水平及骨髓上清液BALP水平比较随着饲养时间增加,正常对照组、DM组、APN干预组Runx2 mRNA、BALP水平均逐渐降低,且DM组下降幅度更显著(P0.05)。在4周时,两两组间比较均无明显统计学差异;8周时,DM组较正常对照组Runx2 mRNA、BALP水平开始降低,APN干预后较DM组,上述指标下降幅度减低,水平升高(P0.05);12周时,DM组较对照组明显降低,APN干预后较DM组上述指标升高(P0.05),且APN组与DM组间各指标的差异较8周时显著。5.三组大鼠股骨骨密度(BMD)比较4周与12周随时间变化,正常对照组、DM组、APN干预组三组BMD值降低;4周时三组两两比较BMD差异性较小无统计学意义,但是12周时,DM组较对照组BMD下降,APN干预后较DM组有所升高且有统计学意义(P0.05)。6.骨组织切片HE染色观察骨小梁结构、脂肪细胞含量比较成模后培养4周时,三组间骨小梁结构尚可,脂肪细胞量少;8周及12周时,三组骨小梁结构出现不同程度破坏,以DM组明显,骨小梁变细、变稀疏,部分骨小梁结构消失,髓腔扩大,出现较多脂肪细胞,APN干预后骨小梁结构有所恢复,脂肪细胞含量减少,且12周时上述变化更明显。结论:1.2型糖尿病大鼠骨髓微环境中氧化应激水平明显升高,可能对成骨分化的调节因子Runx2有直接抑制作用,可能引起成骨细胞分化减少,骨形成降低,结合AGEs水平偏高,骨胶原蛋白非酶糖化影响骨质及骨密度,均可能参与糖尿病性骨质疏松症的发生。2.脂联素可改善糖代谢,减少AGEs生成,并通过降低糖尿病大鼠骨微环境氧化应激水平,对骨骼可能起保护作用。具体机能尚需深入研究。
[Abstract]:Objective: To investigate the type 2 diabetes mellitus (DM) rat bone microenvironment of oxidative stress (OS) and the level of osteoblast (OB) differentiation regulator Runx2, adiponectin (APN) and observe whether intervention can improve the bone microenvironment effect of oxidative stress in osteoblast differentiation, further study of diabetic osteoporosis osteoporosis (DOP) in the pathogenesis and early intervention, to provide new ideas for clinical prevention and treatment of diabetic osteoporosis. Methods: 4 week old male SD rats were randomly divided into normal control group (n=18), diabetic model group (n=42). Control group was given normal diet, model group of high cholesterol and sugar for 2 months, and intraperitoneal injection of streptozotocin (STZ) 30mg/kg type 2 DM rat model. To eliminate the failure model, the death of rats of successful model rats were divided into diabetic group (DM group =18) and intervention group (APN group, adiponectin =18, APN) group were given intraperitoneal injection of globular adiponectin 10 g/kg*d were killed in the intervention, animal 4,8,12 weekend respectively, quickly remove the bilateral femur and tibia, femur and tibia bone marrow extraction side, bone marrow cells were collected by centrifugation and the supernatant of bone marrow, the other side of the femoral bone density after decalcified paraffin embedded.ELISA. RT-PCR method for detection of Runx-2, BALP, AOPPs, bone tissue immunohistochemical staining to observe the expression of AGEs, HE staining to observe the bone mass (trabecular structure, density) and fat cells. Results: the blood glucose level of 1. rats in each group were compared with the feeding time extension, the blood glucose level in DM group rats increased significantly.APN the prognosis, improve blood glucose level of AOPP bone marrow supernatant.2. three rats, compared with feeding time, normal rats bone marrow supernatant AOPP no significant changes in the DM group, APN intervention group were higher (P0.05), but the APN intervention group L 楂樺箙搴﹁緝DM缁勫皬.鍦，

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