脂联素对2型糖尿病大鼠骨髓微环境氧化应激水平及骨吸收影响的实验研究

发布时间：2018-03-10 03:16

  本文选题：2型糖尿病　切入点：氧化应激　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:通过探讨脂联素对2型糖尿病(T2DM)大鼠骨髓中抗酒石酸酸性磷酸酶(TRAP)、骨保护素(OPG)与NF-κB受体活化配体(RANKL)表达水平的影响,结合脂联素对骨髓微环境中糖基化终末产物(AGEs)及晚期氧化蛋白产物(AOPP)的影响,探讨脂联素是否可以通过调节氧化应激水平对糖尿病骨代谢起一定的作用。方法:60只清洁级6周龄雄性SD大鼠随机分为正常对照组(N组,n=18)及2型糖尿病模型组42只。正常对照组予普通饲料喂养,2型糖尿病模型组先以高脂高糖饲料喂养8周,随后按体重给予以一次性腹腔注射浓度为1%的链脲佐菌素(STZ)30mg/kg,以建立2型糖尿病大鼠模型。于建模成功后,选取造模成功的SD大鼠36只,随机分为糖尿病组(DM组,n=18)及糖尿病脂联素干预组(APN,n=18),APN组予皮下注射脂联素10μg/kg·d干预,普通组及糖尿病组大鼠给予等量生理盐水。分别于干预后第4、8、12周末随机选取6只符合标准的大鼠,麻醉后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,另一侧股骨测定骨密度后脱钙包埋切片。ELISA、RT-PCR等方法检测TRAP、RANKL、OPG、AOPP、AGEs,骨组织HE染色观察骨小梁密度。结果:1.随着饲养时间的延长,可见糖尿病组大鼠血糖水平明显升高,而脂联素干预组大鼠血糖水平较糖尿病组大鼠低(P0.05)。2.随饲养时间延长,正常对照组大鼠骨组织免疫组化表达AGEs水平无明显变化,DM组及APN组大鼠骨组织AGEs逐渐升高;三组间比较表明,4周时,DM组大鼠较N组大鼠AGEs水平明显升高(P0.05),脂联素干预后,APN组大鼠较DM组明显减低,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周两组间AGEs下降幅度显著增大。3.随着饲养时间延长,N组大鼠骨髓上清液中AOPP无明显变化,TRAP水平逐渐升高,DM组及APN组大鼠AOPP及TRAP水平均逐渐升高;4周时,三组大鼠TRAP表达水平无明显差异,DM组较N组大鼠AOPP水平明显升高(P0.05),APN组较DM组无明显差异。8周时,与N组比较,DM组T2DM大鼠AOPP及TRAP水平明显升高(P0.05),APN组大鼠骨髓上清液AOPP及TRAP水平较DM组明显降低(P0.05),12周时,两组间AOPP及TRAP下降幅度增大(P0.05)。4.随着喂养时间延长,各组大鼠骨髓细胞中OPG mRNA表达水平呈逐渐降低趋势,RANKL mRNA表达水平呈逐渐增高趋势。4周时,三组大鼠OPG mRNA、RANKL mRNA表达水平无明显差异,8周时,与N组比较,DM组大鼠骨髓细胞OPG mRNA表达水平显著降低,RANKL mRNA表达水平显著升高(P0.05),脂联素干预后,APN组大鼠骨髓细胞OPG mRNA表达水平较DM组明显升高,RANKL mRNA表达水平较DM组显著降低,12周时,两组间比较变化幅度更加明显。OPG/RANKL值与OPG mRNA表达水平变化趋势一致(P0.001)。5.随着时间延长,各组大鼠股骨颈BMD指标均逐渐减低,以DM组明显;4周时,三组大鼠BMD无明显差异,骨小梁结构尚无明显变化,12周时,DM组较N组BMD水平明显减低,骨小梁破坏严重,APN组较DM组BMD水平明显升高,骨小梁结构有所改善(P0.001)。结论:1.2型糖尿病大鼠糖代谢紊乱致骨髓微环境中糖基化终末产物生成增多,氧化应激程度加重,可能使2型糖尿病大鼠破骨细胞信号转导通路中OPG/RANKL比值降低,结合TRAP显著升高,表明破骨细胞分化生成增多,可促进骨吸收过程,破坏骨小梁,影响骨质量及骨密度,加速骨丢失。2.外源性脂联素可能通过减少AGEs的生成,使糖尿病大鼠骨髓微环境中氧化应激水平降低,升高OPG表达,抑制RANKL表达,降低2型糖尿病大鼠骨髓中破骨细胞分化,从而对骨代谢起正向的保护作用。
[Abstract]:Objective: To investigate adiponectin in type 2 diabetes mellitus (T2DM) rat tartrate resistant acid phosphatase in bone marrow (TRAP), osteoprotegerin (OPG) and NF- kappa B ligand receptor activation (RANKL) on the expression of adiponectin on bone marrow microenvironment, combined with advanced glycation end products (AGEs) and advanced oxidation protein products (AOPP) effect, investigate whether adiponectin can be regulated by oxidative stress plays a role in diabetic bone metabolism. Methods: 60 male 6 week old male SD rats were randomly divided into normal control group (group N, n=18) and type 2 diabetes model group with 42 rats in normal control group. Given common forage, type 2 diabetic model group with high-fat and high sugar diet for 8 weeks, then according to the weight given by intraperitoneal injection of 1% of the concentration of streptozotocin (30mg/kg, STZ) in type 2 diabetic rat model was established. After the success of Yu Jianmo, select the successful model of SD 36 rats were randomly divided into diabetic group (DM group, n=18) and diabetic group (APN, n=18, adiponectin), APN group received subcutaneous injection of adiponectin 10 g/kg - D group, normal group and diabetic group rats were given saline respectively. In the intervention after 4,8,12 weeks 6 rabbits were randomly selected for standard the rat, anesthesia was quickly removed after 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, RANKL, OPG methods were used to detect TRAP, AOPP, AGEs, observe the trabecular bone density bone tissue HE staining. Results: 1. with the feeding time, blood glucose levels seen in diabetic group rats increased significantly, while the blood glucose level of adiponectin rats compared with diabetic group rats (P0.05.2.) with low feeding time, the normal control group rats from bone tissue 鐤�缁勫寲琛ㄨ揪AGEs姘村钩鏃犳槑鏄惧彉鍖，

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