Studies On Lead、cadmium-induced Siberian Tiger Fibroblast Cells Apoptosis And Its Apoptotic Mechanisms

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.