自噬在镉诱导大鼠成骨细胞凋亡中的作用
发布时间：2019-03-26 13:52
【摘要】：镉是广泛存在于环境中的重金属污染物,由于其累积毒性可对机体造成极大的损害。它可对机体的多个器官造成损害,其中肾脏和骨骼是镉主要的毒害器官。镉骨毒性相关的文献报道很多,但镉暴露对成骨细胞自噬和凋亡方面的报道较少。因此,本研究通过建立体外大鼠原代成骨细胞镉中毒模型,探讨了镉暴露对成骨细胞自噬和凋亡的影响以及自噬对凋亡的作用。以不同浓度的醋酸镉(0、1、2、5μmol/L)处理原代大鼠成骨细胞12h或2 μmol/L醋酸镉处理成骨细胞0、1、2、3、6、12 h,免疫印迹检测Bax、Bcl-2、Cleaved-PARP的蛋白表达,hoechst 33258染色观察细胞核的变化来研究镉能否诱导细胞凋亡；不同浓度的醋酸镉(0、1、2、5μmol/L)处理原代大鼠成骨细胞1h或2μmol/L 醋酸镉处理成骨细胞0、1、2、3、6、12 h,免疫印迹检测LC3-Ⅱ Beclin-1的表达量,MDC染色法、AO染色法、免疫荧光观察LC3的聚点现象来探究镉暴露细胞的自噬水平；镉、雷帕霉素(RAP)单独或共处理,氯喹(CQ)、镉单独或共处理及RNA干扰,免疫印迹测定LC3-Ⅱ的表达量、流式细胞术测定凋亡率来研究自噬对凋亡的作用。结果表明：①0、1、2、5 μmol/L醋酸镉处理大鼠成骨细胞12h或2 μmol/L醋酸镉处理细胞1、2、3、6、12 h,与对照组相比,染镉组Bax/Bcl-2比值显著或极显著升高(P0.05或P0.01),Cleaved-PARP蛋白表达量极显著上调(P0.01)；染镉能引起成骨细胞核形态的改变,出现核皱缩、染色质凝集等典型的凋亡特征,且随着染镉浓度的增强,发生核形态改变的细胞数增多。②与对照组相比,2μmol/L处理成骨细胞1h后,MDC荧光强度增强,出现明亮的点状结构；染镉组细胞出现明显的LC3聚点现象；0、1、2、5 μmol/L醋酸镉处理大鼠成骨细胞1h后,与对照组相比,1、2μmol/L染镉组AO染色红色荧光强度显著或极显著增强(P0.05或P0.01),而5 μmol/L染镉组红色荧光强度极显著下降(P0.01)；不同浓度的醋酸镉处理成骨细胞1h后,随着染镉浓度的增加,LC3-Ⅱ的表达量先增加、后降低,且与对照组相比,1、2 μmol/L染镉组LC3-Ⅱ表达量显著升高；Beclin-1蛋白表达的变化与LC3-Ⅱ基本一致。2 μmol/L醋酸镉处理成骨细胞不同时间后,随着染毒时间的延长,LC3-Ⅱ表达量先增加、后降低,且与对照组相比,染毒3h后(包括3h)LC3-Ⅱ表达量显著下调(p0.01)；Beclin-1蛋白表达的变化与LC3-Ⅱ相似。③与单独染镉组相比,RAP与镉联合处理组及CQ与镉联合处理组,LC3-Ⅱ蛋白表达量均显著升高；RAP与镉联合处理组细胞凋亡率极显著降低(p0.01),CQ与镉联合处理组细胞凋亡率极显著升高(p0.01)：Beclinl RNA干扰后,与单独染镉组相比,Beclin1 RNA干扰后染镉组的Beclin1、LC3-Ⅱ表达量均极显著降低(p0.01),细胞凋亡率也极显著升高(p0.01)。表明镉能诱导大鼠成骨细胞凋亡,引起细胞自噬水平的改变。镉能通过抑制自噬增加细胞凋亡,自噬对细胞具有保护作用。
[Abstract]:Cadmium is a heavy metal pollutant widely existing in the environment, which can cause great damage to the organism because of its cumulative toxicity. It can cause damage to multiple organs of the body, among which kidney and bone are the main toxic organs of cadmium. There are many reports about bone toxicity of cadmium, but there are few reports about autophagy and apoptosis of osteoblasts after cadmium exposure. In this study, the effects of cadmium exposure on autophagy and apoptosis of osteoblasts and the effect of autophagy on apoptosis were investigated by establishing the cadmium poisoning model of primary osteoblasts in vitro. Primary rat osteoblasts were treated with different concentrations of cadmium acetate (0, 1, 2, 5 渭 mol / L) for 12 hours or 2 渭 mol/L cadmium acetate for 0, 1, 2, 3, 6, 12 h. The protein expression of Bax,Bcl-2,Cleaved-PARP was detected by immunoblotting. Hoechst 33258 staining was used to observe the changes of nucleus in order to study whether cadmium could induce apoptosis. The primary rat osteoblasts were treated with different concentrations of cadmium acetate (0, 1, 2, 5 渭 mol / L) for 1 hour or 2 渭 mol / L cadmium acetate for 0, 1, 2, 3, 6, 12 h. The expression of LC3- 鈪，
本文编号：2447611
[Abstract]:Cadmium is a heavy metal pollutant widely existing in the environment, which can cause great damage to the organism because of its cumulative toxicity. It can cause damage to multiple organs of the body, among which kidney and bone are the main toxic organs of cadmium. There are many reports about bone toxicity of cadmium, but there are few reports about autophagy and apoptosis of osteoblasts after cadmium exposure. In this study, the effects of cadmium exposure on autophagy and apoptosis of osteoblasts and the effect of autophagy on apoptosis were investigated by establishing the cadmium poisoning model of primary osteoblasts in vitro. Primary rat osteoblasts were treated with different concentrations of cadmium acetate (0, 1, 2, 5 渭 mol / L) for 12 hours or 2 渭 mol/L cadmium acetate for 0, 1, 2, 3, 6, 12 h. The protein expression of Bax,Bcl-2,Cleaved-PARP was detected by immunoblotting. Hoechst 33258 staining was used to observe the changes of nucleus in order to study whether cadmium could induce apoptosis. The primary rat osteoblasts were treated with different concentrations of cadmium acetate (0, 1, 2, 5 渭 mol / L) for 1 hour or 2 渭 mol / L cadmium acetate for 0, 1, 2, 3, 6, 12 h. The expression of LC3- 鈪，
本文编号：2447611
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2447611.html
