發(fā)布時間：2018-03-23 17:24

  本文選題：D-半乳糖　切入點：晚期糖基化終末產(chǎn)物　出處：《重慶醫(yī)科大學學報》2017年12期

【摘要】：目的:建立糖基化終末產(chǎn)物(advanced glycation end products,AGEs)誘導小鼠皮膚成纖維細胞(NCTC clone 929,L929)衰老模型,并探討其相關生物學變化機制。方法:實驗選用L929細胞株為實驗對象,隨機分為5組:空白對照組(L929+完全培養(yǎng)基)、實驗組A(L929+0.05 g/L AGEs)、實驗組B(L929+0.1 g/L AGEs)、實驗組C(L929+0.2 g/L AGEs)、陽性對照組(L929+10 g/L D-半乳糖)。通過CCK-8檢測24、48、72 h各組細胞增殖情況;作用48 h后檢測β-半乳糖苷酶染色、細胞周期分析及衰老相關蛋白p16、CyclinD1表達水平驗證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)、過氧化氫酶(catalase,CAT)檢測細胞內(nèi)氧化應激水平。結果:CCK-8提示AGEs可抑制細胞增殖,刺激48 h細胞活力明顯下降。與空白對照組相比,AGEs呈濃度梯度性增高β-半乳糖苷酶活性(B:24±3.4,C:43±10.0,均P=0.000)和G1期阻滯率(A:69.90±2.13,B:74.74±1.49,C:79.29±3.38,均P=0.000),p16蛋白表達上調(diào)(B:0.53±0.20,C:0.63±0.11,P=0.001,P=0.000),CyclinD1蛋白表達下調(diào)(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);與陽性對照組相比,實驗組Aβ-半乳糖苷酶陽染細胞數(shù)和G1期細胞數(shù)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蛋白表達下調(diào)[(0.34±0.12)vs.(0.64±0.04),P=0.001],CyclinD1蛋白表達上調(diào)[(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];上述實驗結果提示實驗組B、C與陽性對照組比較差異沒有統(tǒng)計學意義(P0.05)。結論:0.1 g/L和0.2 g/L AGEs作用48 h均可建立L929體外衰老模型,這可能與細胞發(fā)生氧化應激及胞內(nèi)衰老相關蛋白p16、CyclinD1表達改變有關。
[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 鹵3.4% C: 43 鹵10.0) and G _ 1 phase arrest rate of A: 69.90 鹵2.13: B _ (74.74 鹵1.49C: 79.29 鹵3.38) increased significantly compared with the control group, and the expression of p16 protein increased significantly (P _ (0.53) 鹵0.20 C _ (0.63 鹵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) 鹵0.20 C _ (0.63 鹵0.20) P _ (0.001) P _ (0.000)) and the G _ 1 phase arrest rate (A: 69.90 鹵2.13) B _ (74.74) 鹵1.49 C _ (79.29 鹵3.38), respectively. B: B: 29.19 鹵6.67% C15.13 鹵5.21% P0. 000 A: 1.59 鹵0. 16 C: 1.20 鹵0. 20, increase in MDA and Ros levels. 1.19 鹵0. 08 B: 2 02 鹵0. 12 C: 2. 49 鹵0. 03, P0. 000: a: 1. 49 鹵0. 04 Bmember 1.81 鹵0. 34: C: 1.92 鹵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 鹵3.6)vs.(45.0 鹵8.2P0. 004 + P0. 004] p16 protein expression decreased [0. 34 鹵0.12)vs.(0.64 鹵0. 04 P0. 001] the expression of CyclinD1 protein increased [1. 32 鹵0.09)vs.(0.51 鹵0. 16 P0. 000] sod and CAT activity increased [39. 53 鹵4.24)vs.(27.09 鹵6. 25 P0. 0183.73 鹵0. 09P0. 000] and Ros decreased [1. 19 鹵0.08)vs.(2.39 鹵0. 07 + P0. 000 + 0.04)vs.(1.84 鹵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 鹵6. 25 鹵6. 25 鹵0. 093 鹵0. 093 鹵0. 090 鹵0. 093 鹵0. 090] and the level of reactive oxygen was decreased [1. 19 鹵0.08)vs.(2.39 鹵0. 07 鹵0. 070 鹵0. 020]. The results of the experiment indicated that the activity of CAT was increased in the experimental group [1. 32 鹵0.09)vs.(0.51 鹵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.
【作者單位】： 重慶醫(yī)科大學附屬第一醫(yī)院燒傷整形外科;重慶醫(yī)科大學附屬第一醫(yī)院神經(jīng)外科;
【基金】：重慶市衛(wèi)生局中醫(yī)藥科技資助項目(編號:ZY20132123)
【分類號】：R751

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