Studies On The Apoptosis Mechanism Of Cattle Spleen Lymphocyte In Vitro Culture Induced By Fluorine

The excessive ingest of fluorine could cause the damage of organs and multifunction, and display systemic disease which caused by the change of skeleton. Among the damage of non-bone, the immunotoxicity of fluorine were gradually taken as important. The study took the cattle splenic lymphocyte cultured in vitro as a model and added different concentration of sodium fluoride (NaF) into the culture medium. The mechanisms on the fluorine-induced apoptosis of cattle splenic lymphocytes were revealed through detecting the activation of lymphocyte, apoptosis, antioxidative function, DNA damages, [Ca²⁺]i changes, activity of Caspase-3 and CaM and caspase-3 mRNA gene expression.The results showed as follows:1. the activity of cattle splenic lymphocyte and LDH in supernate were detected by the tapan blue method, MTT assay and dinitro-phenylhydrazine (DPNH) chromometry.The results revealed that IC50 of fluorine on cattle splenic lymphocytes cultured in vitro was 1134.321μmol/ L, fluorine could inhibit the activity of cattle splenic lymphocytes, and have toxic action on them.2. From the detection of the contents of nitric oxide (NO) and malondialdehyde (MDA), and the activity of nitric oxide synthase (NOS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), this study demonstrated that fluorine caused oxidative stress on cattle splenic lymphocytes, and exhibited its immunotoxicity on immunocytes.3. Cell growth condition and morphology changes were detected by AO/EB double fluorescence coloration and the inverted microscope. It revealed that cattle splenic lymphocytes apoptosis was induced by fluorine, and apoptosis ratio exhibited dosage effect.4. The effect of fluorine on DNA damage in cattle splenic lymphocytes were detected by the method of agarose gel electrophoresis and KCl-SDS precipitation. The results showed that fluorine could cause DNA damage of lymphocytes, and exhibited dosage effect.5. The change of△Ψm cattle splenic lymphocytes was detected by flow cytometry. It showed that fluorine induced apoptosis by decreasing△Ψmof cattle splenic lymphocytes.6. CaM mRNA expression and [Ca²⁺]i were separately detected by semiquantitative RT-PCR and fluorescence probe. The results proved that fluorine can reduce CaM mRNA expression, increase [Ca²⁺]i, interfere with the transport of [Ca²⁺]i and result in the unbalance of calcium homeostasis and then induce apoptosis.7. The expression level of Caspase-3 mRNA, and activity of Caspase-3 and Caspase-9 were detected by RT-PCR, and Caspase-3 and Caspase-9 colorimetric assay kit. It revealed that the increase of expression level of Caspase-3 mRNA and activity of Caspase-3 and Caspase-9 were the key factors to apoptosis induced by fluorine.The research elucidated the mechanism of fluorine on immunotoxicity at the molecular level from the aspects of cell morphous, DNA damage, apoptosis, enzymatic activity, and the expression of apoptosis genes. It provided the scientific theoretical foundation and experimental basis for exploring the mechanism of animal immunocell damage and immunotoxicity induced by fluorine.