Autophagy And Related Proteins To Cadmium Induced Liver Were Studied

Some study results have indicated that cadmium (>20μM) can induce apoptosis in HEK293and WRL68cells, but it is still unclear if cadmium could induce autophagy in the cells. Therefore, we have studied Cd-induced autophagy and investigated the relationship between autophagy and apoptosis in the cells. There were three parts in our research:1. We constructed eukaryotic expression plasmid expressing GFP-LC3B gene, that could provide a basis for establishment of method detecting autophagy;2. We established Cd-induced autophagy model in HEK293and WRL68cells;3. We explored Cd-induced autophagy and apoptosis in HEK293cells.In present study, by means of cell culture, PCR technology, calcium phosphate transfection, fluorescence microscopy, morphological observation, transmission electron microscopy, flow cytometry, and Western blot analysis, etc., we constructed pcDNA3.1-GFP-LC3B eukaryotic expression plasmid, and detected the expression of LC3B-II, ERK1/2and AKT induced by cadmium (0～10μM) in HEK293cells. Apoptosis of cell induced by cadmium was evaluated by flow cytometry. The effects of3-MA on Cd-induced autophagy and apoptosis were primarily discussed. The HEK293cells exposed to cadmium (0～2.0μM) for40days were used to detect the relationship between autophagy and transformation. The results were summarized as follows:1. The LC3B gene was amplified by PCR, and the recombinant expression plasmid pcDNA3.1-GFP-LC3B was successfully constructed and expressed in HEK293and WRL68cells.2. Autophagy was induced in HEK293and WRL68cells which were incubated with different doses of cadmium (0～10μM), because that the expression of LC3B-II protein was increased and the cytoplasmic GFP-LC3B punctuates were observed under the fluorescence microscope, and autophagic vacuoles were observed with electron microscope.3. The ERK1/2and AKT were activated in HEK293cells. The addition of3-MA enhanced the expression of cleaved Caspase-3protein. Then apoptosis was induced in the concentrations of Cd-induced autophagy with flow cytometry.Our experiment showed that cadmium (0～10μM) could induce autophagy in HEK293and WRL68cells, and it was accompanied with apoptosis. Autophagy may inhibit apoptosis in certain concentrations of cadmium. The ERK1/2and AKT were associated with the activation of autophagy in HEK293cells.