Fluroide Induce Apoptosis In Primacy Cultures Of Hepatocytes From Rat And The Expression Of Bax

Improved two -step collagenase perfusion method was employed to isolate rat hepatocytcs in this study ,and the primary culture hepatocytes were used as a model to investigate the role of sodium fluoride in cytotoxicity and induction of apoptosis with varying concentrations from 0 to 400mg/L for different periods(12, 24h) .The results were showed as follows:1. Hepatocytes of rat were isolate efficiently by employed improved two-step collagenase perfusion,the number of cell over 2×107 per rat and the viability of hepatocytes up to 90%;2. Antiproliferative effect of NaF in hepatocytes of rat were measured by the MTT assay and the concentration requied for 50% inhibition of growth (IC50) for 12h of treatment is 153.85mg/L, IC50 of 24h is 114.43mg/ L.3. Agarose gels electrophorese analysis of DNA extracted from NaF-treated hepatocytes of rat revealed that fragmentation of nuclear DNA was induced and increased in a dose and time-dependent manner,but the control group DNA ladder cann't be observed. The results ensure that NaF induced apoptosis in hepatocytes of rat in low concentration.4. Western blot analysis showed that the hepatocytes of rat exposure to 25-400mg/L of NaF increased the expression of bax gene in a dose and time-dependent.Conclusion: The cytotoxicity of NaF can be obsevered even at lowconcentration in hepatocytcs of rat,and the value of IC50 incubated by NaF for 12h is!53.85mg/L, IC50 of 24h is 114.43 mg/L. DNA ladder was observed by agarose gels electrophorese analysis and it showed that the toxicity of NaF is induce apoptosis at low concentration (25mg/L) . The result will offer some data to consummate the standard dosage of NaF added in feed and foodstuff. Western blot analysis revealed that the expression of bax increased when treated with NaF.lt suggest that NaF induced apoptosis through the up-regulation of Bax.This study will provide some experimental data to uncover the molecular toxicologymechanism of fluoride , put forward a new way to prevention and cure the fluorosis.