【作者】 王会；

【导师】 刘娣；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2013， 博士

【摘要】 东北虎,是世界上虎中体型最大的亚种,野生种群现主要分布于中国东北、俄罗斯远东和朝鲜北部。在我国,东北虎曾广泛分布于东北各林区,20世纪初受到人类的直接猎杀和生存环境的恶化,野生种群数量锐减,分布区逐渐缩小,现已成为世界上最为濒危的大型猫科动物之一。随着现代工农业和交通的快速发展,东北虎的生境受到严重的威胁,尤其是环境中的重金属铅、镉的污染,对东北虎的生长、发育和疾病的发生都会产生重要的影响。铅、镉可诱导各类细胞发生凋亡并对多个系统造成损伤。首先围绕东北虎成纤维细胞展开生物学特性研究。细胞复苏后进行了形态学、细胞活率、细胞生长动力学、同工酶酶谱分析以及荧光蛋白基因表达等细胞质量检测与生物学特性研究。结果显示,东北虎成纤维细胞具有典型的成纤维细胞形态特征,细胞冻存前和复苏后的活率分别为96.34%±2.24%,93.72%±2.77%,差异不显著(P>0.05)。细胞生长曲线呈“S”型曲线,同工酶酶谱表明该细胞和实验室其它物种细胞不存在交叉污染。细胞具有接受外源基因表达的能力。前期保存的东北虎成纤维细胞系为高质量的、符合标准的、典型的成纤维细胞,以此作为实验材料研究铅、镉对东北虎成纤维细胞的凋亡作用及其机制。通过细胞毒性分析,激光共聚焦显微镜和透射电了显微镜观察凋亡细胞显微及亚显微结构变化,彗星实验分析DNA损伤情况,流式细胞术检测细胞亚二倍体峰和凋亡率一系列实验,结果显示,35μM,64μM,125μM,250μM浓度的Pb(Ac)2和1.2μM,2.4μM,4.8μM,9.6μM浓度的CdCl2在12-48h时间内对东北虎成纤维细胞具有一定的毒性；东北虎成纤维细胞在铅、镉作用下发生特征性的凋亡形态学改变,如胞质固缩,染色质浓缩,核膜边缘化,线粒体肿胀,崩解等现象,但铅并未对细胞骨架微丝的分布、形态结构造成明显的损坏,而镉对东北虎成纤维细胞骨架微丝的分布、形态结构造成明显的损坏,微丝分布松散,顶端缩短、缺失：彗星实验表明铅、镉可使东北虎成纤维细胞DNA损伤,呈现出明显的拖尾现象；亚二倍体峰和细胞凋亡率检测结果表明,铅、镉可诱导东北虎成纤维细胞出现明显的二倍体峰,且表现出浓度依赖性,凋亡率也呈现出浓度依赖性。通过细胞周期的测定,线粒体膜电位的检测,胞内钙离子稳态分析,胞内活性氧的检测,胞外钙离子流速监测,胞外钾离子流速监测,Caspase-3、Caspase-8及Caspase-9酶活性的检测,RT-PCR检测等一系列实验,结果显示,铅、镉均可使细胞增殖阻滞在G0/G1期,干扰了细胞增殖活动从而诱导其凋亡；铅、镉可使东北虎成纤维细胞线粒体跨膜电位下降,结合电子显微镜观察结果可知线粒体肿胀,崩解,铅、镉破坏线粒体的功能,最终导致凋亡发生；铅、镉可打破胞内钙稳态,激活钙的凋亡信号转导作用,胞内钙离子浓度的的增加是由于内源性的钙库释放所引起的,而非吸收胞外的钙离子而升高的；铅、镉可破坏细胞氧化-抗氧化平衡,导致细胞氧化损伤；铅、镉处理东北虎成纤维细胞凋亡早期促使了钾离子外流,破坏胞质渗透压,这一研究结果为认识铅、镉诱导细胞凋亡的内在机制提供了新的证据。铅、镉诱导细胞凋亡过程中Caspase-3、Caspase-8及Caspase-9酶活性呈上升的趋势,呈浓度依赖性；基因Bax与Bcl-2表达量之比值随铅、镉浓度增加而上升,Caspase-3,Caspase-8, Fas, P53表达量也随铅、镉浓度增大呈上升趋势。铅、镉通过阻滞DNA合成,破坏线粒体的功能,干扰钙稳态,氧化损伤,破坏胞质渗透压以及基因调控,介导Caspase来诱导东北虎成纤维细胞凋亡的。一定浓度的铅、镉在一定的作用时间内均对东北虎成纤维细胞具有一定的细胞毒性,并可造成凋亡作用。铅、镉对东北虎成纤维细胞的凋亡作用机制也具有相似性。更多还原

【Abstract】 The Siberian tiger was a subspecies of the largest size tiger in the world. Wild populations are mainly distributed in Northeast China, the northern part of the Russian Far East and North Korea. In China, the Siberian tiger has been widely distributed in the Northeast forest areas. The early20th century, because the human hunting and the deterioration of the living environment, the sharp drop in wild populations, distribution narrowing, it has become one of the world’s most endangered felid. With the rapid development of modern industry, agriculture and transport, Siberian tiger has been suffered serious threat. Especially in the environment of the heavy metals lead, cadmium pollution, have an important impact on the growth, development of the Siberian tiger and the occurrence of the disease. Lead, cadmium can induce various types of cell apoptosis and cause damage of multiple systems.First expand the biological characteristics of the Siberian tiger fibroblasts. Cell quality detection and biological characteristics of study were completed after cell recovery, such as, cell morphology, cell survival rate, cell growth kinetics, isoenzyme fluorescent protein gene expression. The results show that the Siberian tiger fibroblast cells has typical fibroblast morphology characteristical, living cell rate before freezing and recovery were96.34%±2.24%,93.72%±2.77%, the difference was not significant (P>0.05). Cell growth curve was "S" shaped curve. Isoenzyme results show that the cells and other species cells there is no cross-contamination. Cell has the ability to accept the expression of foreign genes.After assurance pre-save the Siberian tiger fibroblast cell line was high-quality, standards-compliant, typical fibroblast, as experimental materials study apoptosis effects and mechanism for lead and cadmium on Siberian tiger fibroblast.Via ytotoxicity assay, confocal microscopy and transmission electron microscopy observation of apoptotic cells microscopic and submicroscopic structural changes, comet assay analysis of DNA damage, flow cytometry detect cell hypodiploid peaks and apoptosis rate. The results show that32μuM,64μM,125μM,250μM Pb(Ac)2and1.2μM,2.4μM,4.8μM,9.6μM CdCl2have a certain toxicity on Siberian tiger fibroblasts at12-48h.Occurrence of characteristic morphological changes of apoptosis, for example, cytoplasm pyknosis, chromatin condensation, nuclear membrane marginalized, mitochondrial swelling and disintegration. But the lead did not cause significant damage to cytoskeletal microfilaments distribution, morphology. While cadmium into the distribution of the fiber cell cytoskeletal filaments Siberian tiger, the morphology caused significant damage, microfilament distribution loose, top shortened and missing. The comet assay showed that lead and cadmium can be damage Siberian tiger fibroblast DNA, showing a significant tailing phenomenon. The detection results of hypodiploid peak and apoptosis rate show that lead and cadmium can induce the Siberian tiger fibroblast appear obvious diploid peak, and showed a concentration-dependent manner. The apoptosis rate also showed a concentration-dependent manner.Via determination of cell cycle, detection of mitochondrial membrane potential, steady-state analysis of intracellular calcium, detection of intracellular reactive oxygen species, monitor of extracellular calcium flow, monitor of extracellular potassium ions flow, detection of Caspase-3、Caspase-8and Caspase-9activity, RT-PCR analysis. The results show that lead and cadmium can arrest cell proliferation in G0/G1phase, interfere with cell proliferation thereby induced apoptosis. Mitochondrial membrane potential decline, combined electron microscopy results of mitochondrial swelling, disintegration, lead and cadmium destroy the mitochondrial function, ultimately lead to apoptosis. Lead and cadmium can break of intracellular calcium homeostasis, activated calcium signal transduction. The increase of intracellular calcium concentration was caused endogenous calcium store release, rather than absorption of the extracellular calcium ions elevated. Lead and cadmium can damage the cells oxidation-antioxidant balance, lead to cellular oxidative damage. The early apoptosis potassium ion efflux, destruction of cytoplasmic osmolality, the results provided new evidence for understanding of lead and cadmium induced apoptosis intrinsic mechanisms. Caspase-3, Caspase-8and Caspase-9activity showed an upward trend in a concentration-dependent manner. Gene Bax and Bcl-2expression amount ratio with lead and cadmium concentration increased rise. Caspase-3, Caspase-8, Fas, P53expression levels also increased with the concentrations of lead and cadmium upward trend. In short, lead and cadmium-induced apoptosis by block DNA synthesis, destruction of mitochondrial function, interfere with calcium homeostasis, oxidative damage, destruction of cytoplasmic osmolality and gene regulation, caspase-mediated induce Siberian tiger fibroblast apoptosis.Considering, a certain concentration of lead and cadmium has certain cytotoxic on Siberian tiger fibroblasts in the role of time, and can cause apoptosis. Lead and cadmium also has similarities apoptosis mechanism on Siberian tiger fibroblast cells.更多还原