缝隙连接在镉致BRL 3A细胞凋亡中的作用

发布时间：2018-11-03 14:19

【摘要】：缝隙连接(OJ)介导相邻细胞间的物质交换和信息交流,在调控细胞生长、增殖、转化和凋亡的稳态平衡中起着重要作用。镉是一种重要的工业和环境污染物,可诱导多种细胞凋亡。为探讨镉对大鼠肝细胞(BRL 3A)缝隙连接通讯功能的影响及缝隙连接在镉致BRL 3A细胞凋亡中的作用,本实验以不同浓度的醋酸镉(0、2.5、10 μmol/L)作用BRL 3A细胞12 h或10 μmol/L醋酸镉、5 μmol/L 18β-甘草次酸(18 P-glycyrrhizic acid, GA)单独或联合作用细胞9 h,采用实时分析技术(RTCA)测定细胞指数(CI),显微镜观察细胞形态,划痕染料标记示踪法(SL/DT)检测细胞缝隙连接通讯(GJIC),激光共聚焦观察缝隙连接蛋白Cx43分布,hoechst 33258染核,流式细胞术测定胞内Ca2+浓度([Ca2+],)和细胞凋亡率,免疫印迹测定Cx32、Cx43、p-Cx43、Bax、Bcl-2、caspase-3和MAPK成员ERK、JNK和p38的活化。结果表明：①0、2.5、10 μpmol/L镉暴露12 h后,CI值显著或极显著降低(P0.05或P0.01),细胞间荧光黄(LY)两侧扩散距离极显著减少(P0.01),Cx43在细胞膜上分布减少、胞内增多,Cx32、Cx43和p-Cx43表达显著或极显著降低(P0.05或P0.01)。②10 μmol/L镉暴露9 h后,LY两侧扩散距离明显减少,Cx32表达无显著变化(P0.05),Cx43表达极显著降低(P0.01),p-Cx43表达极显著升高(P0.01),胞内Ca2+浓度([Ca2+].)极显著升高(P0.01)。缝隙连接阻滞剂(GA)和镉联合作用对镉引起的Cx32、Cx43和p-Cx43表达变化无明显影响(P0.05),但能增强镉引起的[Ca2+]。的升高(P0.01)。③10μmol/L镉暴露9 h后,可引起BRL 3A细胞核形态的变化,出现核皱缩、核碎裂等典型的凋亡特征,并增加肝细胞的凋亡率。GA和镉联合作用可增强镉诱导的胞核形态的改变、细胞凋亡率升高和Bax/Bcl-2比值和cleaved caspase-3/procaspase-3比值升高(P0.05或P0.01)。镉可诱导ERK、JNK和p38的磷酸化水平极显著上升(P0.01)。GA和镉联合作用能极显著增加镉引起的ERK和p38磷酸化水平的升高(P0.01),但对JNK的磷酸化水平无显著影响(P0.05)。结论,镉对BRL 3A细胞有明显的细胞毒性,可抑制GJIC,其机制可能是镉诱导BRL3A细胞Cx43分布异常,Cx32和Cx43表达及Cx43磷酸化水平的改变,以及[Ca2+],升高。缝隙连接阻滞后可增强镉对肝细胞的毒性损伤,促进镉诱导的细胞凋亡,这可能与凋亡相关蛋白的调控,以及ERK和p38信号途径有关,故缝隙连接在镉致肝细胞凋亡中起着重要作用
[Abstract]:Gap junction (OJ) mediates material exchange and information exchange between adjacent cells, which plays an important role in regulating the homeostasis of cell growth, proliferation, transformation and apoptosis. Cadmium is an important industrial and environmental pollutant, which can induce apoptosis of many kinds of cells. To investigate the effect of cadmium on the function of gap junction communication in rat hepatocytes (BRL 3A) and the role of gap junction in the apoptosis of BRL 3A cells induced by cadmium. In this study, cadmium acetate (10 渭 mol/L) at different concentrations of cadmium acetate was used to treat BRL 3A cells for 12 h or 10 渭 mol/L, and 5 渭 mol/L 18 尾 -glycyrrhetinic acid (18 P-glycyrrhizic acid, GA) alone or in combination for 9 h). The cell morphology was observed by (CI), microscope with real time analysis technique (RTCA), and the distribution of gap junction protein Cx43 was observed by scratch dye tracer (SL/DT) and (GJIC), laser confocal analysis. Hoechst 33258 was stained with nucleus, intracellular Ca2 concentration ([Ca2],) and apoptosis rate were measured by flow cytometry. The activation of ERK,JNK and p38 in Cx32,Cx43,p-Cx43,Bax,Bcl-2,caspase-3 and MAPK members were detected by Western blot. The results showed that after exposure to 10 渭 pmol/L cadmium for 12 h, the CI value decreased significantly (P0.05 or P0.01), the diffusion distance between the two sides of the fluorescent yellow (LY) decreased significantly (P0.01), and the distribution of Cx43 on the cell membrane decreased. The expression of Cx32,Cx43 and p-Cx43 decreased significantly (P0.05 or P0.01). After 9 h of cadmium exposure at 210 渭 mol/L, the diffusion distance on both sides of LY decreased significantly, but the expression of Cx32 did not change significantly (P0.05). The expression of Cx43 was significantly decreased (P0.01), the expression of p-Cx43 was significantly increased (P0.01), and the intracellular Ca2 concentration ([Ca2]) was significantly increased (P0.01). Very significantly increased (P0.01). The combined action of gap junction blocker (GA) and cadmium had no significant effect on the changes of Cx32,Cx43 and p-Cx43 expression induced by cadmium (P0.05), but could enhance [Ca2] induced by cadmium. After exposure to cadmium at 310 渭 mol/L for 9 h, the nuclear morphology of BRL 3A cells was changed, and typical apoptotic features such as nuclear shrinkage and nuclear fragmentation were observed. Combined effects of GA and cadmium could enhance the changes of nuclear morphology induced by cadmium, increase the rate of apoptosis and increase the ratio of Bax/Bcl-2 and cleaved caspase-3/procaspase-3 (P0.05 or P0.01). Cadmium could significantly increase the phosphorylation level of ERK,JNK and p38 (P0.01). GA combined with cadmium could increase the phosphorylation level of ERK and p38 induced by CD (P0.01). But there was no significant effect on the phosphorylation level of JNK (P0.05). Conclusion: cadmium has obvious cytotoxicity to BRL 3A cells, and the mechanism of inhibiting GJIC, may be the abnormal distribution of Cx43, the changes of Cx32 and Cx43 expression and Cx43 phosphorylation, and the increase of [Ca2] in BRL3A cells induced by cadmium. The blocking of gap junction can enhance the toxicity of cadmium to hepatocytes and promote the apoptosis induced by cadmium, which may be related to the regulation of apoptosis-related proteins and the signaling pathway of ERK and p38. So gap junction plays an important role in cadmium induced hepatocyte apoptosis.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.87

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