糖基化终末产物对小鼠皮肤成纤维细胞衰老的影响

刘露; 龙媛媛; 郭宗雨; 唐维; 果磊

doi:10.13406/j.cnki.cyxb.001409

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糖基化终末产物对小鼠皮肤成纤维细胞衰老的影响

皮肤病学 | 更新时间：2024-01-17

- 糖基化终末产物对小鼠皮肤成纤维细胞衰老的影响
- Effects of advanced glycation end products on aging of L929
- 重庆医科大学学报 2017年42卷第12期页码：1540-1546
- 作者机构：
  
  1. 重庆医科大学附属第一医院烧伤整形外科
  2. 重庆医科大学附属第一医院神经外科
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.001409
  中图分类号： R751
- 纸质出版日期：2017，
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刘露, 龙媛媛, 郭宗雨, 等. 糖基化终末产物对小鼠皮肤成纤维细胞衰老的影响[J]. 重庆医科大学学报, 2017,42(12):1540-1546.

Liu Lu, Long Yuanyuan, Guo Zongyu, et al. Effects of advanced glycation end products on aging of L929[J]. Journal of Chongqing Medical University, 2017,42(12):1540-1546.
刘露, 龙媛媛, 郭宗雨, 等. 糖基化终末产物对小鼠皮肤成纤维细胞衰老的影响[J]. 重庆医科大学学报, 2017,42(12):1540-1546. DOI： 10.13406/j.cnki.cyxb.001409.

Liu Lu, Long Yuanyuan, Guo Zongyu, et al. Effects of advanced glycation end products on aging of L929[J]. Journal of Chongqing Medical University, 2017,42(12):1540-1546. DOI： 10.13406/j.cnki.cyxb.001409.

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(P>0.05)。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 the aging model of NCTC clone 929（L929）induced by advanced glycation end products（AGEs）and to explore its biological mechanism.Methods:In this study

L929 was randomly divided into five groups :blank control group（cultivated L929 with common incubation media）

experimental group A（L929+AGEs

0.05 g/L）

experimental group B（L929+AGEs

0.1 g/L）

experimental group C（L929+AGEs

0.2 g/L）

positive control group（L929+D-galactose

10 g/L）.Cell proliferation was detected by CCK-8 at 24

72 hours.The establishment of aging model in L929 was evaluated by senescence aging β-galactosidase（SA-β-gal）staining and cell cycle assay

and the expressions of p16 and CyclinD1 were detected by Western blot.The levels of reactive oxygen species associated with oxidative stress in the cells

and aging indicators such as superoxide dismutase（SOD）

malondialdehyde（MDA）and catalase（CAT）were examined.Results:CCK8 showed that AGEs could inhibit L929 proliferation and cell viability was decreased significantly after being cultivated for 48 hours.Compared with blank control group

AGEs increased the activity of β-galactosidase（B:24.0±3.4

C:43.0±10.0

P =0.000）and the blocking rate of phase G1（A:69.90±2.13

B:74.74±1.49

C:79.29±3.38

P=0.000）and protein expression of p16（B:0.53±0.20

C:0.63±0.11

P=0.001

P=0.000） in a dose-dependent manner

while AGEs decreased CyclinD1 expression[（1.32±0.09） vs.（0.51±0.16）

P=0.000].AGEs decreased the activity of SOD and CAT（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

P=0.000）and increased the level of MDA and reactive oxygen species [（1.19±0.08）vs.（2.39±0.07）

P=0.000;（1.47±0.04）vs.（1.84±0.09）

P=0.020].The number of cells in phase G1 and β-galactosidase positive staining cells was significantly lower in experimental group A than in positive control group [（12.0±3.6）vs.（45.0±8.2）

P=0.004;（69.90±2.13）vs.（76.59±0.61）

P=0.001].The expression of p16 protein was significantly decreased in experimental group A than in positive control group[（0.34±0.12）vs.（0.64±0.04）

P=0.001]

while the expression of CyclinD1 protein was higher than that in positive control group[（1.32±0.09）vs.（0.51±0.16）

P=0.000].The activities of SOD and CAT were significantly higher than those of positive control group[（39.53±4.24）vs.（27.09±6.25）

P=0.018;（3.73±0.42）vs.（1.64±0.09）

P=0.000]

while the levels of MDA and reactive oxygen species were significantly lower than those in positive control group[（1.19±0.08）vs.（2.39±0.07）

P=0.000;（1.47±0.04）vs.（1.84±0.09）

P=0.02].There was no significant difference between experimental B and C and positive control group in these respects（P>0.05）.Conclusion:0.1 g/L and 0.2 g/L AGEs can establish aging L929 in vitro model

which may be related to the changes of oxidative stress and senescence-associated protein expression of p16 and CyclinD1.

关键词

D-半乳糖晚期糖基化终末产物衰老氧化应激

Keywords

D-galactoseadvanced glycation end productsagingoxidative stress

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