摘要
目的:糖尿病患者体内晚期糖基化终末产物(advanced glycation end products,AGEs)的过量生成会影响成骨细胞的正常功能,而这一过程可能与成骨细胞的自噬有关。本实验研究AGEs对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化过程中自噬能力的影响。方法:将大鼠BMSCs体外分离与培养,并用不同质量浓度(0、50、100、200及400 mg/L)的AGEs处理不同时间(3、6、12、24、48及72 h),检测各组细胞增殖活性,采用透射电镜观察各组细胞内自噬体形成,real-time PCR检测各组细胞Beclin1和LC3的m RNA表达水平,蛋白质印迹法检测各组细胞Beclin1和LC3的蛋白质表达水平。随后加入自噬促进剂或抑制剂,观察成骨相关基因的表达。结果:与0 mg/L组比较,50和100 mg/L AGEs组BMSCs增殖活性均升高(均P<0.01),自噬体形成的数量增多,自噬相关基因LC3、Beclin1的mRNA和蛋白质表达均增加(均P<0.01)。200和400 mg/L AGEs组BMSCs增殖活性降低,自噬体形成数量减少,自噬相关基因LC3、Beclin1的mRNA和蛋白质表达减少。100 mg/L AGEs组碱性磷酸酶染色深于0 mg/LAGEs组,成骨相关基因的mRNA表达增加(P<0.01),加入自噬抑制剂后结果得到逆转;200 mg/L AGEs组碱性磷酸酶染色浅于0 mg/L AGEs组,成骨相关基因的mRNA表达减少(P<0.01),加入自噬促进剂后结果得到逆转。结论:低浓度的AGEs通过促进自噬增强BMSCs的增殖活性,促进其成骨分化;高浓度的AGEs通过抑制自噬降低BMSCs的增殖活性,抑制其成骨分化。不同浓度的AGEs,对BMSCs成骨分化过程中自噬能力的影响不同。
Objective:Excessive production of AGEs in diabetic patients will affect the normal function of osteoblasts,and this process may be related to autophagy of osteoblasts.This study aims to explore the effect of advanced glycation end products(AGEs)on autophagic activity during osteogenic differentiation in rat bone marrow mesenchymal stem cells(BMSCs).Methods:BMSCs were isolated and cultured in vitro,treated with different concentrations(0,50,100,200,and 400 mg/L)of AGEs for different time(3,6,12,24,48,and 72 h).The proliferation activity was detected by CCK-8 method.The mRNA and protein expression levels of Beclin1 and LC3 in cells were detected by real-time PCR and Western blotting,respectively.The autophagic vacuoles were observed under the transmission electron microscope.The cells were treated with autophagy promoter rapamycin or autophagy inhibitor 3MA.After 7 days of osteogenic induction,we performed alkaline phosphatase(ALP)staining and real-time PCR to detect the mRNA expression levels of osteogenesisrelated genes.Results:In the low-concentration groups,the proliferation activity in BMSCs was increased(P<0.01),the mRNA and protein expressions of autophagy-related genes LC3 and Beclin1 were increased(both P<0.01).The number of autophagosome also was increased.In the high-concentration groups,the results were just the opposite.In the lowconcentration groups,the ALP staining was deeper than that of the 0 mg/L AGEs group,and the mRNA expressions of the osteogenic related genes were increased(P<0.01).But the results were reversed in the presence of autophagy inhibitor 3MA.In the highconcentration groups,the ALP staining was lighter than that of the 0 mg/L AGEs group,and the mRNA expressions of the osteogenic related genes were decreased(P<0.01).After the addition of the autophagy promoter rapamycin,the results were reversed.Conclusion:Low concentration of AGEs can enhance the proliferative activity of BMSCs and promote osteogenic differentiation by accelerating autophagy.High concentration of AGEs can suppress t
作者
罗丹
胡赟
唐宇
丁晓倩
李彩玉
郑雷蕾
LUO Dan;HU Yun;TANG Yu;DING Xiaoqian;LI Caiyu;ZHENG Leilei(Department of Orthodontics,Stomatological Hospital of Chongqing Medical University,Chongqing 401145;Chongqing Key Laboratory of Oral Diseases and Biomedicine Science,Chongqing 401145;Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education,Chongqing 401145;Department of Pediatric Stomatology,Stomatological Hospital of Chongqing Medical University,Chongqing 401145,China)
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2021年第4期361-367,共7页
Journal of Central South University ：Medical Science
基金
国家自然科学基金(81470772)
重庆市科卫联合医学科研重点项目(2018ZDXM020)
重庆市自然科学基金(cstc2016jcyjA0238)。
