Apoptosis Induction Of Carassius Auratus Lymphocytes By Bacterial Endotoxin In Vitro And A Preliminary Study On Its Mechanism

In the first part of our present study, the kidney lymphocytes from Carassius aruatus is choosed as the research material. These cells are induced by different LPS concentrations and under different time in vitro. The apoptotic effects of LPS are detected by various skills such as fluorescence staining, TUNEL reaction, DNA agarose gel electrophoresis, electron microscopy and fluorescence activated cell sorting (FACS). Our results demonstrated that fish lymphocytes would be apoptotic under different LPS concentrations(above 10μg/ml) and different time(above 6h) inducing, which is characterized by condensation and margination of chromatin, the formation of apoptotic bodies. The DNA-fragment by agarose gel electrophoresis showed the typical ladder pattern and DNA strand 3?-OH nick end was detected by TUNEL reaction. In addition, LPS induced lymphocyte apoptosis in time- and dose-dependent manner analysised by FACS with the PI staining.In part Ⅱ, with the evidence of lymphocytes apoptosis induced by LPS, we investigated whether mitochondrial apoptotic pathway regulated the lymphocyte apoptosis and what happened in apoptosis-related gene expression. The results showed that the intracellular ATP and mitochondria membrane potential (AT) decreased, but the O2" increased. So we deduced that mitochondrial apoptotic pathway determined the apoptosis of lymphocyte.In the further research, we proved lymphocyte mRNA expression level of apoptosis related gene induced by 100 U g/ml LPS at 6> 12> 24h were not in the same.Compared with that of the control, the results showed that the lymphocyte mRNA expression levels of TNFa were no changes and that of NOS were not remarkable different each induced group, but the Bel family gene bcl-2 was down-regulated and Bax is up-regulated increase consistently. The results of TNFa and NOS gene expression suggested that the mechanism of lymphocyte apoptosis induced by LPS is different from that of macrophage apoptosis.In all, the mechanism of lymphocyte apoptosis induced by LPS is regulated by mitochondrial apoptotic pathway and it is different from that of macrophage apoptosis.