糖基化终末产物对小鼠皮肤成纤维细胞衰老的影响

发布时间：2018-03-23 17:24

本文选题：D-半乳糖　切入点：晚期糖基化终末产物　出处：《重庆医科大学学报》2017年12期

【摘要】：目的:建立糖基化终末产物(advanced glycation end products,AGEs)诱导小鼠皮肤成纤维细胞(NCTC clone 929,L929)衰老模型,并探讨其相关生物学变化机制。方法:实验选用L929细胞株为实验对象,随机分为5组:空白对照组(L929+完全培养基)、实验组A(L929+0.05 g/L AGEs)、实验组B(L929+0.1 g/L AGEs)、实验组C(L929+0.2 g/L AGEs)、阳性对照组(L929+10 g/L D-半乳糖)。通过CCK-8检测24、48、72 h各组细胞增殖情况;作用48 h后检测β-半乳糖苷酶染色、细胞周期分析及衰老相关蛋白p16、CyclinD1表达水平验证L929衰老模型成立;活性氧和超氧化物歧化酶(superoxide dismutate,SOD)、丙二醛(malondialdehyde,MDA)、过氧化氢酶(catalase,CAT)检测细胞内氧化应激水平。结果:CCK-8提示AGEs可抑制细胞增殖,刺激48 h细胞活力明显下降。与空白对照组相比,AGEs呈浓度梯度性增高β-半乳糖苷酶活性(B:24±3.4,C:43±10.0,均P=0.000)和G1期阻滞率(A:69.90±2.13,B:74.74±1.49,C:79.29±3.38,均P=0.000),p16蛋白表达上调(B:0.53±0.20,C:0.63±0.11,P=0.001,P=0.000),CyclinD1蛋白表达下调(B:1.07±0.14,C:0.63±0.11,P=0.01,P=0.000),SOD及CAT活性降低(B:29.19±6.67,C:15.13±5.21,P=0.001,P=0.000;A:3.73±0.42,B:1.59±0.16,C:1.20±0.20,均P=0.000),MDA和活性氧水平增高(A:1.19±0.08,B:2.02±0.12,C:2.49±0.03,均P=0.000;A:1.49±0.04,B:1.81±0.34,C:1.92±0.04,P=0.006,P=0.000,P=0.000);与阳性对照组相比,实验组Aβ-半乳糖苷酶阳染细胞数和G1期细胞数降低[(12.0±3.6)vs.(45.0±8.2),P=0.004;(69.90±2.13)vs.(76.59±0.61),P=0.001],p16蛋白表达下调[(0.34±0.12)vs.(0.64±0.04),P=0.001],CyclinD1蛋白表达上调[(1.32±0.09)vs.(0.51±0.16),P=0.000],SOD及CAT活性增高[(39.53±4.24)vs.(27.09±6.25),P=0.018;(3.73±0.42)vs.(1.64±0.09),P=0.000],MDA和活性氧水平降低[(1.19±0.08)vs.(2.39±0.07),P=0.000;(1.47±0.04)vs.(1.84±0.09),P=0.020];上述实验结果提示实验组B、C与阳性对照组比较差异没有统计学意义(P0.05)。结论:0.1 g/L和0.2 g/L AGEs作用48 h均可建立L929体外衰老模型,这可能与细胞发生氧化应激及胞内衰老相关蛋白p16、CyclinD1表达改变有关。
[Abstract]:Objective: to establish advanced glycation end products (ages) -induced senescence model of mouse skin fibroblasts (NCTC clone 929 and L929), and to explore the mechanism of its biological changes. Methods: L929 cell line was selected as experimental object. Five groups were randomly divided into five groups: blank control group (L929), A(L929 0.05g / L ages, B(L929 0.1g / L ages, C(L929 0.2g / L ages, positive control group (L92910 g / L Dgalactose). Cell proliferation was detected by CCK-8. 48 h after treatment, 尾 -galactosidase staining, cell cycle analysis and expression of senescence-associated protein p16cyclin D1 were detected to verify the establishment of L929 aging model. Reactive oxygen species (Ros) and superoxide dismutate (SOD), malondialdehyde (malondialdehyde), catalase (catalase) were used to detect intracellular oxidative stress. The activity of 尾 -galactosidase B: 24 卤3.4% C: 43 卤10.0) and G _ 1 phase arrest rate of A: 69.90 卤2.13: B _ (74.74 卤1.49C: 79.29 卤3.38) increased significantly compared with the control group, and the expression of p16 protein increased significantly (P _ (0.53) 卤0.20 C _ (0.63 卤0.11) P _ (0.001) P _ (0.000) D _ (1)) and the activity of CAT decreased as compared with that of the control group (P _ (0.53) 卤0.20 C _ (0.63 卤0.20) P _ (0.001) P _ (0.000)) and the G _ 1 phase arrest rate (A: 69.90 卤2.13) B _ (74.74) 卤1.49 C _ (79.29 卤3.38), respectively. B: B: 29.19 卤6.67% C15.13 卤5.21% P0. 000 A: 1.59 卤0. 16 C: 1.20 卤0. 20, increase in MDA and Ros levels. 1.19 卤0. 08 B: 2 02 卤0. 12 C: 2. 49 卤0. 03, P0. 000: a: 1. 49 卤0. 04 Bmember 1.81 卤0. 34: C: 1.92 卤0. 006P0. 000 compared with the positive control group. In the experimental group, the number of A 尾 -galactosidase positive staining cells and G1 phase cells decreased [12.0 卤3.6)vs.(45.0 卤8.2P0. 004 + P0. 004] p16 protein expression decreased [0. 34 卤0.12)vs.(0.64 卤0. 04 P0. 001] the expression of CyclinD1 protein increased [1. 32 卤0.09)vs.(0.51 卤0. 16 P0. 000] sod and CAT activity increased [39. 53 卤4.24)vs.(27.09 卤6. 25 P0. 0183.73 卤0. 09P0. 000] and Ros decreased [1. 19 卤0.08)vs.(2.39 卤0. 07 + P0. 000 + 0.04)vs.(1.84 卤0. 020]; the above results indicated that the activity of CAT in the experimental group was significantly higher than that in the control group [39. 53 卤6. 25 卤6. 25 卤0. 093 卤0. 093 卤0. 090 卤0. 093 卤0. 090] and the level of reactive oxygen was decreased [1. 19 卤0.08)vs.(2.39 卤0. 07 卤0. 070 卤0. 020]. The results of the experiment indicated that the activity of CAT was increased in the experimental group [1. 32 卤0.09)vs.(0.51 卤0. 16] sod and CAT activity. Conclusion 0. 1 g / L and 0. 2 g / L AGEs could induce the aging model of L929 in vitro after 48 h treatment with 0. 1 g / L and 0. 2 g / L AGEs. This may be related to the changes of cell oxidative stress and the expression of p16 cyclin D1.
【作者单位】：重庆医科大学附属第一医院烧伤整形外科;重庆医科大学附属第一医院神经外科;
【基金】：重庆市卫生局中医药科技资助项目(编号:ZY20132123)
【分类号】：R751

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