【作者】 陈晶；

【导师】 钟才高；

【作者基本信息】 中南大学 ， 卫生毒理学， 2010， 硕士

【摘要】 目的：在体外试验系统,初步探讨Cr(Ⅵ)诱导肝细胞凋亡与线粒体能量代谢障碍的关系,为进一步阐明Cr(Ⅵ)诱导肝细胞凋亡的机制提供线索。方法：以L-02肝细胞为受试细胞,通过MTT试验检测不同浓度单独Cr(Ⅵ)及单独ATP染毒对L-02肝细胞存活率的影响,筛选确定低、中、高Cr(Ⅵ)作用浓度及ATP有效干预浓度即Cr(Ⅵ)处理浓度为2.5μmol/L,10μmol/L, 40μmol/L及ATP浓度为1μmol/L,染毒时间为24h。通过流式细胞仪检测细胞凋亡率及DNA-Ladder检测细胞凋亡,反相高效液相色谱检测细胞内ATP、ADP、AMP含量变化,氧电极检测细胞线粒体呼吸功能(ST3、ST4、RCR、P/O),荧光分光光度法检测线粒体通透性转运孔(PTP)及跨膜电位(△ψm)、细胞内活性氧(ROS),酶标仪检测细胞内caspase-3的活性。结果：1.Cr(Ⅵ)引起L-02肝细胞存活率降低：Cr(Ⅵ)在2.5-100pmol/L处理浓度范围内,能明显引起L-02肝细胞存活率的降低(P<0.05),处理浓度和细胞存活率之间存在负相关(r=-0.909,P=0.00)选取2.5μmol/L、10μmol/L和40μmol/LCr(VI)浓度和细胞生存率最高的ATP浓度1μmol/L。2.Cr(Ⅵ)导致肝细胞凋亡：与对照组相比,Cr(Ⅵ)处理组细胞凋亡率均明显上升,加入ATP可明显减轻中剂量组细胞凋亡率,而在高剂量组引起细胞凋亡率上升,坏死率下降(P<0.05)。3.Cr(Ⅵ)引起肝细胞DNA损伤：中、高剂量Cr(Ⅵ)处理组诱导细胞DNA发生断裂,加入ATP联合处理组,细胞DNA断裂均明显减轻。4.Cr(Ⅵ)对肝细胞caspase-3表达的影响：与对照组相比,其他各组caspase-3活性均明显升高(P<0.05)。ATP加入高剂量Cr(Ⅵ)处理组caspase-3活性明显降低(P<0.05)。5.Cr(Ⅵ)所致肝细胞氧化应激：与对照组相比,除了ATP处理组外,其他各组ROS水平均有所升高(P<0.05)。6.Cr(Ⅵ)所致细胞线粒体损伤：与对照组相比,各处理组线粒体PTP孔开放度和线粒体膜电位(△ψm)下降均有所增加(P<0.05)。加入ATP联合处理,中剂量、高剂量组与相同浓度的Cr(Ⅵ)单独处理组相比,PTP孔开放度降低,膜电位下降率降低(P<0.05)。7.Cr(Ⅵ)对细胞ATP、ADP、AMP的影响：与对照组相比,低剂量Cr(Ⅵ)组中ATP、ADP、TAN略有上升,中、高剂量Cr(Ⅵ)处理组则明显降低(P<0.05),加入ATP的各处理组,以上各指标均与相应浓度的Cr(Ⅵ)单独处理组有差别(P<0.05)。与对照组相比,细胞能荷(Ec)在中剂量Cr(Ⅵ)单独处理组上升,高剂量Cr(Ⅵ)单独处理组下降(P<0.05)。8.Cr(Ⅵ)和ATP对肝细胞细胞呼吸功能影响：与对照组相比,各Cr(Ⅵ)处理组RCR明显降低,40μmol/LCr(VI)+ATP与相同浓度的Cr(Ⅵ)处理组相比明显升高(P<0.05)。与对照组相比,Cr(Ⅵ)处理组P/O均明显降低(P<0.05)。结论：体外试验系统中,Cr(Ⅵ)可诱导肝细胞发生凋亡和线粒体能量代谢障碍,二者呈明显的相关性。ATP能减轻Cr(Ⅵ)所致的线粒体损伤,使线粒体呼吸功能恢复,降低caspase3的活性,减轻DNA损伤。在Cr(Ⅵ)为2.5μmol/L-10μmol/L浓度范围,ATP能明显减轻Cr(Ⅵ)诱导的细胞凋亡,当Cr(Ⅵ)浓度为40μmol/L时,ATP能使Cr(Ⅵ)诱导的细胞坏死率下降,细胞凋亡率增加。更多还原

【Abstract】 Objective:To explore the relationship between the energy metabolism dysfunction and t apoptosis induced by Cr(Ⅵ) he L-02 hepatocytes and provide clues for the further research in the mechanism of Cr (Ⅵ)-induced hepatocyte apoptosis.Methods:The effects of Cr(Ⅵ) single treated or ATP single treated on cell survival rate were assessed by the reductions of tetrazolium dye (MTT) in cultured L-02 hepatocytes. L-02 hepatocytes in all tests were respectively incubated with 2.5μmol/L, 10μmol/L,40μmol/L concentrations of Cr(Ⅵ) or/and 1μmol/L ATP for 24h according to cell survival rate.The cellular apoptosis and necrosis ratios.AnnexinV-FITC/ PI double stained analysed by FCM; DNA damage was observed by using the DNA-Ladder. The mitochondrial permeability transition pore (PTP) and the mitochondrial transmembrane potential (△ψm) were determined by the fluorospectrophotometric method ;the activity of caspase-3 was tested by the Microplate Reader. The levels of cellular ATP, ADP, AMP content as well as the changes of ATP/ADP ratio and the energy charge (Ec) were detected by high performance liquid chromatography (HPLC), The respiratory function of mitochondria was detected by oxygen electrode.Results:1. The concentration-dependent decrease in cell survival rate of Cr(Ⅵ)-treated L-02 cells was observed. The relationship between concentration and survival rate was significantly negative correlation (r=-0.909, P＜0.05). The 2.5μmol/L,10μmol/L,40μmol/LCr(Ⅵ) concentration and 1μmol/L ATP were chosen.2. Compared to the control group, the apoptosis ratio of all the Cr(Ⅵ)-treated groups increased markedly (P<0.05). Compared to the same concentration of Cr(Ⅵ), the apoptosis ratio in the 10μmol/L Cr(Ⅵ)+ATP group decreased, while the apoptosis ratio in the 4μmol/L Cr(Ⅵ)+ATP group increased but the necrosis ratio decreased significantly (P＜0.05).3. DNA damage was observed in the intermediate dose group[10μmol/L Cr(Ⅵ)] and maximum group[40μmol/L Cr(VI)], but in the intermediate combined group[10μmol/L Cr(Ⅵ)+ATP] and the maximum combined group[40μmol/L Cr(Ⅵ)+ATP] the DNA breakage reduced compared to the same concentration of Cr(Ⅵ) single group (P＜0.05).4. Compared to the control group, the caspase-3 activity increased remarkably in other groups (P＜0.05), and after treated with ATP together, the caspase-3 activity of the maximum group decreased significantly (P＜0.05).5. The ROS in all groups except ATP treated group rose significantly compared to the control group (P＜0.05).6. The mitochondrial openness of permeability transition pore (PTP) and the rate of mitochondrial membrane potential (△ψm) declining significantly increased in all groups compared to the control group(P＜0.05). ATP markedly improved them in the intermediate combined group and maximum combined group compared to the same concentration of Cr(VI) groups(P＜0.05).7. The contents of ATP,ADP and total adenine nucleotide (TAN) significantly increased in the minimum group but declined in the intermediate dose group and the maximum group compared to the control group(P＜0.05).While the energy charge (Ec) significantly increased in the intermediate dose group but decreased in the maximum compared with the control group (p＜0.05).8. In all the Cr(VI) single treated groups, RCR had significantly declined compared to the control group(P＜0.05), ATP apparently improved the RCR in the maximum group compared to the same concerntration of the Cr(VI) group, and the P/O declined in all the Cr(VI)-treated groups compared to the control group(P＜0.05),.Conclusions:Cr(VI) can induce L-02 hepatocyte apoptosis and mitochondrial energy metabolism dysfunction. Energy metabolism dysfunction has a close relationship with the Cr(Ⅵ)-induced hepatocyte apoptosis process. ATP can repair the damage of mitochondrial respiration function, depress the caspase-3 activity, and alleviate the mitochondria and DNA damage in Cr(Ⅵ)-treated hepatocyte. ATP can protect hepatocytes from Cr(Ⅵ)-induced apoptosis at the lower dose(2.5μmol/L-10μmol/L),but increase the apoptosis and decrease the necrosis in the 40μmol/L group.更多还原