烟雾提取物暴露对大鼠骨髓间充质干细胞凋亡的影响及其机制

目的:探讨烟雾提取物暴露对大鼠牙骨髓间充质干细胞(bone mesenchymal stem cells,BMMSC)凋亡的影响及其相关机制。方法:通过体外分离培养大鼠BMMSC并进行传代和鉴定,准备不同浓度的香烟烟雾提取物(cigarette smoke extract,CSE),分为0.5%低浓度CSE组、3.0%高浓度CSE组和空白对照组(无CSE组)。采用TUNE L染色法和流式细胞检测法检测不同组细胞的凋亡情况,检测氧化损伤指标苯二醛(MDA)及氧自由基(ROS)变化,并测定细胞线粒体膜电位及细胞内游离Ca2+浓度。结果:0.5%低浓度CSE组和3.0%高浓度CSE组大鼠BMMSC细胞凋亡情况较空白对照组明显增多,且3.0%高浓度CSE组细胞凋亡率也高于0.5%低浓度CSE组。此外,CSE组大鼠BMMSC中MDA含量和ROS水平较空白对照组显著升高,且两者水平在3.0%高浓度CSE组也都高于0.5%低浓度CSE组,组间差异有统计学意义(P<0.05)。0.5%低浓度和3.0%高浓度CSE组与空白对照组相比,大鼠BMMSC游离Ca2+浓度明显升高,差异有统计学意义(P<0.05);与空白对照组相比,0.5%低浓度CSE组和3.0%高浓度CSE组大鼠BMMSC内线粒体膜电位明显降低,差异有统计学意义(P<0.05)。结论:CSE能够促进大鼠BMMSC产生氧化自由基引起氧化损伤细胞膜和线粒体从而导致细胞凋亡。

Effects of smoke extract exposure on apoptosis of rat bone mesenchymal stem cells and its mechanism

Objective:To investigate the apoptosis and its related mechanisms of rat dental bone marrow mesenchymal stem cells(BMMSC)exposed to smoke extract.Methods:Different concentrations of cigarette smoke extract(CSE)were prepared by isolation and culture of BMMSC in vitro for passage and identification.The experiment was divided into three groups,namely 0.5%low concentration cigarette smoke extract(CSE)group,3.0% high concentration CSE group and blank control group,and no cigarette smoke extract group as the blank control group.TUNEL staining and flow cytometry were used to detect the apoptosis of different groups of cells,and then the changes of oxidative damage indicators of phthalaldehyde(MDA)and oxygen free radicals(ROS)were detected,and the mitochondrial membrane potential and intracellular freeness Ca2+concentration were measured.Results:The results of TUNEL and flow cytometry showed that the apoptosis of BMMSC cells in 0.5%low concentration CSE group and 3.0% high concentration CSE group was significantly increased compared with the blank control group,and the apoptosis rate of 3.0% high concentration CSE group was higher than 0.5% low concentration CSE group.In addition,MDA content and ROS levels in BMMSCs exposed to smoke extract were significantly higher than those in the blank control group,and 3.0%high concentration CSE group both levels were higher than 0.5% of low concentration CSE group and group differences were statistically significant(P<0.05).Compared with the blank control group,the concentration of free Ca2+in the BMMSCs of the 0.5% low concentration and 3.0% high concentration smoke extracts were significantly higher than that of the blank control group.There was significant statistical difference between the two groups(P<0.05).Compared with blank control group,the mitochondrial membrane potential of BMMSCs in 0.5%low concentration and 3.0% high concentration smoke extracts were significantly decreased,and there was a statistically significant difference(P<0.05).Conclusion:The smoke extract can promote the production of oxidative free radicals in rat BMMSCs,causing oxidative damage to cell membranes and mitochondria leading to apoptosis.