Daejeon Okadaic Acid (okadaic Acid, Oa) Of Mouse Embryo Cells 3t3

Background and objective:In the oceans there are approximately 300 species of algae that can cause red tides, among which there are about 80 species can produce toxins. These toxins can poison and form a potential threat human through the fish, shellfish of marine life through enrichment transfer function. So governments attach great importance to the harm of red tide phycotoxins, and many countries have established red tides testing system.They made a series of research to the toxins toxicity mechanism and detection method of red tide phycotoxins which has made significant progress.However, so far, the studys of the impact of Prorocentrum lima Dorge to the ultrastructure, proliferation and apoptosis of mice cells are rare, and the studys mainly focus on freshwater algae, the remaining algae involving less. Therefore, it is necessary for us to have comprehensive research of the influence of harmful algal red tides to mamm alian cells, for providing accurate data on the detection of he toxicity of red tide phycotoxins and the prediction of red tide on cellular level.Methods:1. MTT assay on cell viability, by which the toxicity strength of OA was determined primarily.2. The cell count, detection of influence of different concentrations on OA cell count.3. Detection of cell apoptosis—Observation of cell morphological changes by Phase Contrast Microscope.—Observation of morphological changes of apoptosis by Acridine orange (AO) fluorescence staining combined with Laser Confocal Scanning Microscopy.—Detection of Reactive oxygen species (ROS), with fluorescence spectrophotome- ter detection intracellular ROS content.—Detection of the neutral red stain, observing cells to detect the intake of neutral red of cell apoptosis of the activity cell membranes changes with Inverted Microscopy.—Detection of Lactate dehydrogenase (LDH), detection intracellular LDH content change with lactate dehydrogenase kit combined with UV spectrophotometer.4. Detection of changes in cell cycle distribution—Flow cytometry was used to detect the changes about the cell cycle of 3T3 after treated with OA5. Detection of the activity of OA to caspase-3:analysis the activity change of caspase-3 enzyme intracellular with caspase-3 active immuosorbent. Results:1. The test results of MTT assay on cells livability—Influences of the extract of P. lima on viability of 3T3 cellWhen algae concentration in 0.2×103cells/ml, the extract of algae inhibit weakly to cellular. With algae concentration increases, the reduction of extract of algae on cell proliferation is gradually increasing, and positively correlated with time. OA can significantly inhibit 3T3 cell growth and presented dose dependent on 3T3 cell proliferation inhibiting effect obviously. Namely with the increase of the concentration of the drug, cell survival rate fall gradually.—OA can significantly inhibit 3T3 cell growth, and the proliferation inhibiting effect obviously presented dose dependent on 3T3 cell Namely with the increase of the concentration of the drug, cell survival rate fall gradually.2. The influence of OA on the internal and external form of 3T3 cell—Observing cells form changes with inverted differ microscopeResults show that:cells appear part and go round in 10 ng/mL, edge knits and cell membrane foaming phenomenon; Exposure in 50 ng/mL, most cells lose their spindle shape, the cells apart from each edge.We can see that a membrane-bound parcel apoptotic body forms. And exposure to 100 ng/mL, cells apoptotic body is visible and the emergence of different degrees of rupture.The results of condensed chromatin with Acridone acetamiprid orange (AO) dyeing combined with laser confocal microscopyThe result shows:after the OA dyeing, the cells dyeing enhancement, the fluorescence became more bright, the euchromatin became pyknotic form or round shape(early apoptosis); With the concentration increasing, cells and euchromatin is become pyknotic form or chromatin round shape, a typical cell apoptosis characteristics appears.3. The influence of OA on 3T3 cell membrane permeability—The neutral red dyeing experimentsThe result shows:OA act on the 3T3 cell 50ng/ml and 100ng/ml respectively processing 48 hours, compared with control group, the experimental group neutral red cell intake volume increased obviously, and accompanied by cells go round. This shows that with increasing concentration, membrane permeability increase.—Detection of membrane integrity with LDH kitThe result shows:OA act on the 3T3 cell 50ng/ml and 100ng/ml respectively processing 48 hours, compared with control group, in the medium of experimental group, LDH enzyme activity changes significantly (p<0.05, n=3). This suggests that high density dealing group of cells appeared necrosis phenomenon.4.The influence of OA on the apoptosis of 3T3 cell—Detection of intracellular ROS levelsThe result shows:after OA process 48 hours, the ROS in 3T3 cell erupt and gathered. Compared with control group, levels of ROS in 50ng/ml OA treatment groups from 4.408 elevated to 9.079, increased 2.05 times, and after 100ng/ml OA processing, ROS levels rise again to 22.6, increased 5.12 times as many.—Detection of Caspase-3 enzyme activityThe result shows:the Caspase-3 enzymes living immuosorbent detection analysis shows that, 50ng/ml of OA processing 3T3 cell for 48 hours, compared with control group, light absorption value in 405 nm 0.0425 rose to 0.133, active raised 2.9 times.—The result shows:OA can induce 3T3 cell apoptosis and blockade thecell cycle in G1 phase make cells can't enter S period.