The Researches On Endoplasmic Stress Related Apoptosis Of Cadmium On Chicken Hepatoytes

Cadmium is a kind of environmental chemical pollutant, it is accumulation in animal and plant through water, air and soil which was polluted by cadmium. And liver was the initial part of cadmium accumulation and toxicity produce in the bady. Cadmium had a toxicity effect of liver in animals, which could induce endoplasmic reticulum stress in cell. Research of endoplasmic reticulum stress was a hot topic in foreign country, but research about hepatocytes injuries of cadmium was rare, especially, research on endoplasmic reticulum stress related apoptosis in poultry hepatocytes induced by cadmium has not been studied.In this study, Coding sequence of GRP78gene was cloned by the methods of molecular biology. GRP78was expressed in prokaryotic system by genetic engineering technology. The polyclonal antibody was prepared using expressed fusion protein. Chicken hepatocytes was taken as a model, cultivated in the Williams’E for24h with the final concentration of CdCl2,which was0,2.5,5,10,20μmol/L respectively. To discuss the endoplasmic reticulum stress related apoptosis mechanism of cadmium on chicken hepatocytes, we detected the cell viability, the changes of apoptotic morphology, DNA fragmentation, calcium homeostasis, expression of GRP78mRNA and protein and the expression level of GRP94, CaM, Bak, Bcl-2, CaM mRNA. The results showed as follows:1Fusion proteins of GRP78were expressed in prokaryotic system. The polyclonal antibody was prepared by using expressed fusion proteins.2The result of IC50of cadmium on chicken hepatocytes for24h by the MTT assay was15.59±0.89μmol/L, It revealed that cadmium had a toxic effect to chicken hepatocytes.3Different growth period chicken hepatocytes were detected by inverted microscope. AO/EB double fluorescence coloration and DNA Ladder indicated that chicken hepatocytes apoptosis was induced by CdCl2accompanied with dose-effect relation.4Cadmium increased calcium ion concentration within chicken hepatocytes [Ca2+]i by Fura-3/AM fluorescent probe and decreased the expression of CaM mRNA. This indicated that disequilibrium of calcium homeostasis played a role of cadmium induced endoplasmic reticulum stress related apoptosis on chicken hepatocytes.5The expression of GRP78protein in cytolymph was detected by western blot, expression quantity alteration of GRP78and GRP94mRNA were examined by real-time fluorescence quantitative PCR. The expression of GRP78and GRP94was increased with dose-effect relation. It indicated that GRP78and GRP94could protect cell from endoplasmic reticulum stress related apoptosis.6The expression of Bak, Bcl-2, Caspase-3mRNA were examined by real-time fluorescence quantitative PCR. The expression of Bak mRNA was decreased while the expression of Bcl-2, Caspase-3mRNA was increased. It indicated that CdCl2could induce he apoptotic of the chicken hepatocytes.Our study discussed the mechanism of cadmium-induced endoplasmic reticulum stress related apoptosis of chicken hepatocytes on the aspect of cell morphous, apoptosis, calcium homeostasis, endoplasmic reticulum stress, calcium homeostasis and gene of apoptosis etc.