镉诱发肝细胞毒性及其胞内钙信号失调相互关系和硒的保护作用研究

发布时间：2018-06-28 06:11

本文选题：镉 + 肝细胞　；参考：《南京师范大学》2006年硕士论文

【摘要】：本文通过原代培养新生乳鼠肝细胞模型，运用细胞培养、荧光分光光度计、激光共聚焦显微镜和生化分析等生物学技术，系统研究了镉负荷诱发肝细胞损伤以及肝细胞存活性、胞内MDA含量、Ca~(2+)-Mg~(2+)-ATPase和Na~+-K~+-ATPase活性以及胞内Ca_i~(2+)含量的变化，细胞培养上清液中LDH及AST的活性、白蛋白及Ca~(2+)含量变化，较为深入地探讨了镉导致的肝细胞胞内Ca_i~(2+)稳态失衡的可能机制；同时，还观察了硒干预镉诱发鼠肝细胞存活及其MDA含量和培养液中LDH活性的变化以及硒对镉暴露肝细胞内Ca~(2+)稳态变化的影响。结果如下： 1 新生乳鼠肝细胞原代培养方法的建立无菌分离新生乳鼠肝脏组织，采用胶原蛋白酶消化法分离肝细胞并使用无血清培养液HepatoZYME-SFM对其进行体外培养，通过形态学及台盼蓝染色法鉴定，分离的肝细胞细胞完整，呈球形或椭球形，存活率在90％以上。说明该方法是比较理想的肝细胞培养法，为科学研究提供了细胞模型平台。 2 镉诱发肝细胞毒性及其胞内游离Ca~(2+)变化研究原代培养乳鼠肝细胞，分为三个镉处理组和正常对照组，分别加入5、10、25μM CdCl_2染毒以及等量D-hank's溶液。结果显示：实验后12h，CdCl_2导致肝细胞存活剂量依赖性下降；培养上清液LDH和AST的活力升高或显著升高，，并且白蛋白含量大大降低。在CdCl_2暴露12h后，细胞内MDA生成量增加处于较高水平，培养上清液Ca~(2+)含量显著下降。进一步观察显示，在CdCl_2暴露12h和24h后，肝细胞[Ca~(2+)]_i显著升高：胞内Ca~(2+)-Mg~(2+)-ATP酶及Na~+-K~+-ATP酶活力12h与对照组无明显差异，至24h下降或明显下降。提示：CdCl_2通过诱导肝细胞脂质过氧化和损伤，引发细胞存活下降和功能障碍而导致强烈毒性；镉暴露引发肝细胞[Ca~(2+)]_i异常增加可能是细胞损伤发展的重要机制，部分[Ca~(2+)]_i升高与胞外Ca~(2+)的进入有密切关系。 3 镉诱发肝细胞胞内游离Ca~(2+)堆积的机制探讨原代培养乳鼠肝细胞，用Fluo-3／AM对生长良好的原代培养肝细胞闭光孵育，然后清洗并重悬肝细胞于有钙HBSS或无钙HBSS中，同时设计加或不加2-APB抑制剂(100μM)的情况，用激光共聚焦显微镜观察CdCl_2(10μM)急性暴露引起的胞内钙离子荧光强度变化。结果显示：在仅有钙的HBSS中，CdCl_2诱导肝细胞[Ca~(2+)]_i明显上升，包括开始的缓慢上升期和随后的持续升高期；在有钙的HBSS+2-APB中，CdCl_2仅引起一个无开始上升期的缓缓升高；在无钙的
[Abstract]:In this paper, the hepatocyte injury induced by cadmium loading and the viability of hepatocytes were systematically studied by using biological techniques such as cell culture, fluorescence spectrophotometer, laser confocal microscopy and biochemical analysis. The changes of intracellular MDA content, the activities of Ca ~ (2) -mg ~ (2) -ATPase and Na ~ -K ~ (-ATPase), the contents of Ca ~ (2), LDH and AST in supernatant of cell culture, and the changes of albumin and Ca ~ (2) contents were observed. The possible mechanism of cai ~ (2) homeostasis in hepatocytes induced by cadmium was discussed. The effects of selenium on cadmium induced hepatocyte survival and the changes of MDA content and LDH activity in culture medium were also observed. The effects of selenium on the changes of Ca ~ (2) homeostasis in hepatocytes exposed to cadmium were also observed. The results are as follows: 1 Establishment of primary culture method of neonatal rat hepatocytes Aseptic isolation of liver tissue from newborn rat Hepatocytes were isolated by collagenase digestion and cultured in vitro with serum-free medium HepatoZYME-SFM. The isolated hepatocytes were identified by morphology and trypan blue staining. The isolated hepatocytes were spherical or ellipsoid, and the survival rate was over 90%. It shows that this method is an ideal method for hepatocyte culture. 2 cadmium induced hepatocytotoxicity and its intracellular free Ca ~ (2) changes. Primary culture of neonatal rat hepatocytes, The rats were divided into three cadmium treatment groups and normal control group, which were treated with 5 ~ 10 ~ 10 渭 m CD _ Cl _ 2 and D-hankos solution respectively. The results showed that CDCl2 induced a dose-dependent decrease in the survival of hepatocytes, the activities of LDH and AST in the supernatant increased or increased significantly, and the albumin content decreased significantly. After exposure to CDCl2 for 12 h, the content of MDA increased at a high level, and the content of Ca2 in culture supernatant decreased significantly. Further observation showed that after CDCl2 exposure for 12 h and 24 h, [Ca2] tii in hepatocytes increased significantly: there was no significant difference in intracellular Ca ~ (2) -Mg- (2) -ATPase activity and Na ~ -K ~ -ATPase activity at 24 h after exposure to CDCl2, but decreased or decreased significantly at 24 h. The results suggest that the increase of [Ca ~ (2)] I in hepatocytes induced by cadmium exposure may be an important mechanism for the development of hepatocyte injury, which is caused by inducing lipid peroxidation and injury of hepatocytes, resulting in the decrease of cell survival and dysfunction of hepatocyte function, and the abnormal increase of [Ca ~ (2)] I in hepatocytes induced by cadmium exposure. The increase of partial [Ca ~ (2)] I is closely related to the entry of extracellular Ca ~ (2). 3 the mechanism of cadmium induced intracellular free Ca ~ (2) accumulation in hepatocytes Primary culture of neonatal rat hepatocytes, The cultured primary cultured hepatocytes were incubated with Fluo-3 / AM in closed light, then washed and suspended in the presence or absence of calcium HBSS, and the condition of adding or not adding 2-APB inhibitor (100 渭 M) was designed. The changes of intracellular calcium fluorescence intensity induced by CDCl2 (10 渭 M) acute exposure were observed by laser confocal microscopy. The results showed that CDCL _ 2 induced a marked increase of [Ca ~ (2)] _ I in the calcium-only HBSS, including the initial slow ascending period and the subsequent continuous rising period; in the calcium HBSS _ 2-APB, CDCL _ 2 only caused a slow rise in the non-initial ascendant phase; in the calcium free HBSS _ 2-APB, CDCL _ 2 induced a slow rise; in the calcium free HBSS _ 2-APB, CDCL _ 2 induced a slow rise in the liver cell [Ca ~ (2)].
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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