自噬在鎘誘導(dǎo)大鼠成骨細胞凋亡中的作用
發(fā)布時間：2019-03-26 13:52
【摘要】：鎘是廣泛存在于環(huán)境中的重金屬污染物,由于其累積毒性可對機體造成極大的損害。它可對機體的多個器官造成損害,其中腎臟和骨骼是鎘主要的毒害器官。鎘骨毒性相關(guān)的文獻報道很多,但鎘暴露對成骨細胞自噬和凋亡方面的報道較少。因此,本研究通過建立體外大鼠原代成骨細胞鎘中毒模型,探討了鎘暴露對成骨細胞自噬和凋亡的影響以及自噬對凋亡的作用。以不同濃度的醋酸鎘(0、1、2、5μmol/L)處理原代大鼠成骨細胞12h或2 μmol/L醋酸鎘處理成骨細胞0、1、2、3、6、12 h,免疫印跡檢測Bax、Bcl-2、Cleaved-PARP的蛋白表達,hoechst 33258染色觀察細胞核的變化來研究鎘能否誘導(dǎo)細胞凋亡；不同濃度的醋酸鎘(0、1、2、5μmol/L)處理原代大鼠成骨細胞1h或2μmol/L 醋酸鎘處理成骨細胞0、1、2、3、6、12 h,免疫印跡檢測LC3-Ⅱ Beclin-1的表達量,MDC染色法、AO染色法、免疫熒光觀察LC3的聚點現(xiàn)象來探究鎘暴露細胞的自噬水平；鎘、雷帕霉素(RAP)單獨或共處理,氯喹(CQ)、鎘單獨或共處理及RNA干擾,免疫印跡測定LC3-Ⅱ的表達量、流式細胞術(shù)測定凋亡率來研究自噬對凋亡的作用。結(jié)果表明：①0、1、2、5 μmol/L醋酸鎘處理大鼠成骨細胞12h或2 μmol/L醋酸鎘處理細胞1、2、3、6、12 h,與對照組相比,染鎘組Bax/Bcl-2比值顯著或極顯著升高(P0.05或P0.01),Cleaved-PARP蛋白表達量極顯著上調(diào)(P0.01)；染鎘能引起成骨細胞核形態(tài)的改變,出現(xiàn)核皺縮、染色質(zhì)凝集等典型的凋亡特征,且隨著染鎘濃度的增強,發(fā)生核形態(tài)改變的細胞數(shù)增多。②與對照組相比,2μmol/L處理成骨細胞1h后,MDC熒光強度增強,出現(xiàn)明亮的點狀結(jié)構(gòu)；染鎘組細胞出現(xiàn)明顯的LC3聚點現(xiàn)象；0、1、2、5 μmol/L醋酸鎘處理大鼠成骨細胞1h后,與對照組相比,1、2μmol/L染鎘組AO染色紅色熒光強度顯著或極顯著增強(P0.05或P0.01),而5 μmol/L染鎘組紅色熒光強度極顯著下降(P0.01)；不同濃度的醋酸鎘處理成骨細胞1h后,隨著染鎘濃度的增加,LC3-Ⅱ的表達量先增加、后降低,且與對照組相比,1、2 μmol/L染鎘組LC3-Ⅱ表達量顯著升高；Beclin-1蛋白表達的變化與LC3-Ⅱ基本一致。2 μmol/L醋酸鎘處理成骨細胞不同時間后,隨著染毒時間的延長,LC3-Ⅱ表達量先增加、后降低,且與對照組相比,染毒3h后(包括3h)LC3-Ⅱ表達量顯著下調(diào)(p0.01)；Beclin-1蛋白表達的變化與LC3-Ⅱ相似。③與單獨染鎘組相比,RAP與鎘聯(lián)合處理組及CQ與鎘聯(lián)合處理組,LC3-Ⅱ蛋白表達量均顯著升高；RAP與鎘聯(lián)合處理組細胞凋亡率極顯著降低(p0.01),CQ與鎘聯(lián)合處理組細胞凋亡率極顯著升高(p0.01)：Beclinl RNA干擾后,與單獨染鎘組相比,Beclin1 RNA干擾后染鎘組的Beclin1、LC3-Ⅱ表達量均極顯著降低(p0.01),細胞凋亡率也極顯著升高(p0.01)。表明鎘能誘導(dǎo)大鼠成骨細胞凋亡,引起細胞自噬水平的改變。鎘能通過抑制自噬增加細胞凋亡,自噬對細胞具有保護作用。
[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，
本文編號：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，
本文編號：2447611
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