【作者】 王艳凤；

【导师】 林洪金；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 氟过量摄入可引起机体多器官、多功能的损害,表现为以骨骼改变为主的全身性疾病。在非骨相损害中,氟的免疫毒性日渐受到重视。本课题以体外培养牛脾脏淋巴细胞为研究对象,在培养液中加入不同浓度氟化钠（NaF）,通过对牛脾脏淋巴细胞活性、细胞凋亡、抗氧化功能、DNA损伤、细胞内游离Ca²⁺浓度、Caspase-3与Caspase-9活性及CaM、Caspase-3 mRNA表达水平的检测,旨在探讨氟致牛脾脏淋巴细胞凋亡的机制。研究结果表明:1.应用台盼蓝拒染试验、四唑盐比色法（MTT）、二硝基苯肼比色法检测了牛脾脏淋巴细胞的活性和培养上清液中乳酸脱氢酶（LDH）活性。结果表明,氟对牛脾脏淋巴细胞的半数抑制浓度（IC50）为1134.32μmol/L,氟能够抑制体外培养牛脾脏淋巴细胞的活性,并对牛脾脏淋巴细胞具有毒性作用。2.对染氟牛脾脏淋巴细胞内一氧化氮（NO）、丙二醛（MDA）含量及一氧化氮合酶（NOS）、超氧化物歧化酶（SOD）与谷胱甘肽过氧化物酶（GSH-Px）活性的检测。结果表明,氟能够引发牛脾脏淋巴细胞内氧化胁迫,对免疫细胞具有毒性作用。3.用吖啶橙/溴化乙锭（AO/EB）双荧光染色荧光显微镜下、倒置显微镜下观察不同浓度NaF诱导牛脾脏淋巴细胞凋亡的形态学变化及细胞生长情况。结果表明,氟能够诱导牛脾脏淋巴细胞凋亡,且凋亡率具有剂量依赖性。4.应用琼脂糖凝胶电泳、KCl-SDS沉淀法检测氟对牛脾脏淋巴细胞DNA的损伤作用。结果表明,氟能够引发淋巴细胞DNA损伤,并呈现剂量效应。5.应用流式细胞术检测牛脾脏淋巴细胞Δψm变化。结果表明,氟可通过引起牛脾脏淋巴细胞Δψm降低而诱导细胞凋亡。6.应用半定量RT-PCR法和荧光探针法检测细胞内CaM mRNA表达水平和游离Ca²⁺浓度。结果表明,氟能够引起牛脾脏淋巴细胞内CaM mRNA表达水平下降,游离Ca²⁺浓度升高,干扰细胞Ca²⁺的转运,导致细胞钙稳态失衡而诱导细胞凋亡。7.应用半定量RT-PCR方法检测细胞Caspase-3 mRNA表达水平,比色检测Caspase-3、Caspase-9活性。结果表明,牛脾脏淋巴细胞内Caspase-3 mRNA表达及Caspase-3、Caspase-9活性增加是氟引起牛脾脏淋巴细胞凋亡的关键因素。本研究以体外培养染氟牛脾脏淋巴细胞为研究对象,从细胞的形态、DNA损伤、酶活性及凋亡基因表达水平等角度入手,从分子水平上揭示了氟免疫毒性机制。该研究结果可为揭示氟致动物免疫细胞损伤和免疫毒性机制提供科学的理论基础和试验依据。更多还原

【Abstract】 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.更多还原