摘要
目的:探讨体外尼古丁对大鼠髌腱肌腱干细胞(tendon-derived stem cells,TDSCs)的细胞活力、细胞早期凋亡以及肌腱相关基因表达的影响,揭示吸烟导致肌腱病变以及损伤肌腱延迟愈合的潜在细胞学发病机制。方法:无菌条件下取8周龄SD大鼠的髌腱,I型胶原酶消化分离获得单个有核细胞,以最适密度(50个/cm2)进行接种,细胞呈克隆样生长,获得原代TDSCs。基础培养细胞至第3代,将其分为两组,实验组用不同浓度的尼古丁培养基(10-7 M,10-6 M,10-5 M,10-4 M和10-2 M)干预,对照组继续采用基础培养基(0M)培养。处理6 h、24 h、48 h和72 h后分别应用四唑盐(MTT)比色法检测尼古丁对TDSCs体外存活的影响,72h后应用Annexin V-FITC/PI双染法检测尼古丁对TDSCs体外早期凋亡的影响,实时荧光定量PCR(Realtime PCR)检测肌腱相关基因Col1a1和Scx的表达。结果:与对照组相比,实验组不同浓度的尼古丁干预下,TDSCs的细胞活力呈下降趋势,呈时间依赖性和浓度依赖性,浓度高于10-4 M时作用显著,差异具有统计学意义(P<0.05)。流式细胞仪检测TDSCs早期凋亡(Annexin V+/PI-)的数量随着尼古丁浓度的增加而增加,差异具有统计学意义(P<0.05)。同时,Realtime PCR检测结果显示:与对照组相比,实验组中Col1a1和Scx的m RNA表达水平均降低,浓度高于10-5 M时作用显著,差异具有统计学意义(P<0.05)。结论:体外尼古丁可抑制大鼠髌腱TDSCs的细胞活力,导致TDSCs早期凋亡,同时抑制TDSCs中成肌腱分化转录、细胞外基质合成相关基因的表达。这可能是吸烟导致肌腱病变以及损伤肌腱延迟愈合的潜在细胞学发病机制。
Objective To explore the effects of nicotine on the cell viability, early cell apoptosis and expressions of tendon-related markers of patellar tendon-derived stem ceils (TDSCs)of rat. Methods The patellar tendons of 8-week old SD rat were collected, and their nucleated cells were isolated after type I collagenase digestion and vaccinated in culture dishes at optimal cell density (50 cells/cm2)for colonies,and the primary TDSCs were finally obtained and cultured to the third passage,and then they were divided into control and experimental groups. Different concentrations of nicotine ( 10^-7 M, 10^-6 M, 10^-5 M, 10^-4 M and 10^-2 M)were added to the basic culture medium of the experimental group. The cell viability was detected by MTY assay 6,24,48 and 72 hours after the experiment. Early cell apoptosis was determined with Annexin V-FITC/PI assay and expressions of tendon-related markers were detected by qRT-PCR after 72-hour culture. Results In comparison to the control group, cell viability of the experimental group decreased in a time- and dose- dependent manner,especially in 10^-2 M concentrations of nicotine. (P〈0.05). The percentage of early apoptotic TDSCs (Annexin V+/PI-)of the experimental group increased significantly in a dose-dependent manner (P〈 0.05). Expressions of Collal and Scx of the experimental group decreased,especially in 10^-5 M, 104 M and 10^-2 M nicotine concentrations (P〈0.05). Conclusion Nicotine could inhibit cell viability and expressions of tendon-related markers, and lead to early TDSCs apoptosis in vitro,which could be the potential pathogenesis of tendon injury and delayed tendon healing in smokers.
出处
《中国运动医学杂志》
CAS
北大核心
2015年第8期786-792,共7页
Chinese Journal of Sports Medicine
基金
国家自然科学基金青年基金项目(81201422)
江苏省自然科学基金青年基金项目(BK2012334)
东南大学基本科研业务费"创新基金"项目(3290002401)
国家大学生创新训练计划(1210286090)
中国博士后基金资助(2012M520983)
江苏省"六大人才高峰"资助项目(2013-WSW-054)
关键词
尼古丁
肌腱干细胞
肌腱病变
延迟愈合
成肌腱分化
nicotine, tendon-derived stem cells, tendon injury, delayed tendon healing, tenogenic differentiation