氟损伤甲状腺机理研究

发布时间：2018-09-04 20:37

【摘要】： 氟是机体必需的微量元素,摄入适量能维持机体正常的生命活动,但它还是一种全身性毒物,其对机体内分泌腺的影响已成为近年来的研究热点。有实验表明氟能够引起甲状腺肿大,但是也有学者持反对意见。本实验分别复制SD大鼠氟中毒动物模型和氟化钠处理原代培养SD大鼠甲状腺细胞、大鼠甲状腺细胞系(fisher ratthyroid cell line,FRTL)。80只SD大鼠饮水中添加的氟化钠含量分别为0,50mg/L,100mg/L,200mg/L,原代培养的SD大鼠甲状腺细胞染氟化钠浓度分别为0,10μmol/L,100μmol/L,1000μmol/L,FRTL细胞染氟化钠浓度分别为0,1.25mg/L,2.5mg/L,5.0mg/L,10.0mg/L,20mg/L。运用电镜学、分子生物学、免疫学等技术研究氟对甲状腺激素代谢基因表达、功能蛋白表达和分布、细胞因子基因表达及分泌的影响,体外实验能解除下丘脑-腺垂体-甲状腺轴的影响,影响因素单一。本实验通过体内体外氟中毒实验阐述氟引起甲状腺肿大的分子机理,从而为揭示氟引起甲状腺损伤提供理论依据。实验成功的复制SD大鼠氟中毒动物模型和制备体外氟中毒甲状腺细胞样品,通过形态学观察及竞争放射性免疫法测定反映甲状腺细胞结构和分泌激素功能的一系列指标,结果发现氟引起甲状腺激素T_3、T_4合成和分泌障碍及直接影响整个甲状腺细胞的生长和发育过程。高氟(200mg/L)大鼠甲状腺血管增生和大面积增生结节,尤其氟对甲状腺细胞膜损伤严重;本实验通过甲状腺组织切片HE染色和扫描电镜等手段检测氟对甲状腺细胞的损伤,结果证明氟能够引起甲状腺肿大和一序列的损伤;运用RT-PCR、免疫细胞化学和免疫荧光组织化学等方法测定甲状腺相关功能酶类,以及部分相关蛋白和生长因子的表达与含量,结果发现甲状腺球蛋白(TG)、过氧化物酶(TPO)、钠碘转运体(NIS)、促甲状腺激素受体(TSHR)、血管内皮生长因子(VEGF)等与甲状腺功能密切相关的指标均表现异常;氟引起自由基代谢紊乱,致NO生成的功能酶以及相关生长因子(如iNOS和VEGF)表达受到影响发生显著变化,微血管增生,自由基和脂质过氧化物在甲状腺中蓄积,破坏了甲状腺滤泡上皮细胞结构和功能,干扰胶质的代谢,抑制甲状腺中碘的转运和有机化过程,干扰甲状腺过氧化物酶的活性,破坏甲状腺激素合成和分泌的各个环节,阻断了TSH对甲状腺细胞刺激作用,导致TSHR-cAMP无法发挥正常作用,甲状腺细胞功能下降。此外本实验研究FRTL细胞在外界氟的作用下NO分泌、细胞因子基因表达和分泌的影响。运用RT-PCR、ELISA检测氟刺激FRTL细胞72h后IL-6 mRNA、IL-8mRNA、IFN-γmRNA,TNF-a mRNA水平和细胞上清中VEGF分泌量;同时westernblot测定NF-κB p65蛋白水平,结果表明:氟刺激这些细胞因子的表达,一方面通过抑制甲状腺激素代谢基因表达而影响甲状腺细胞甲状腺激素的分泌;另一方面,氟刺激细胞因子和NF-κB p65表达导致甲状腺细胞自身抵抗氟能力下降,甲状腺细胞功能下降。氟化物影响甲状腺细胞的生长和发育,使细胞的形态发生改变,损伤程度与剂量有关,长期摄入过量氟可导致甲状腺发生结节性肿大;氟化物刺激iNOS、细胞因子和NF-κB p65异常的表达,这些因子通过抑制甲状腺激素代谢相关基因的表达进一步导致甲状腺细胞功能下降;氟化物造成甲状腺摄取和利用碘、甲状腺激素合成、贮存、分泌功能障碍,进而导致甲状腺结构和功能异常。
[Abstract]:Fluoride is an essential trace element in the body. It can maintain normal life activities of the body in moderate intake, but it is still a systemic poison. Its effect on the body's endocrine glands has become a research hotspot in recent years. The concentration of sodium fluoride in drinking water of 80 SD rats was 0,50 mg/L, 100 mg/L, 200 mg/L respectively. The concentration of sodium fluoride in primary cultured SD rat thyroid cells was 0,10, 100 um/L, 100 m ol/L, 100 m ol/L, respectively. The concentration of sodium fluoride in 0,1.25 mg/L,2.5 mg/L,5.0 mg/L,10.0 mg/L,20 mg/L of FRTL and FRTL cells was 0,1.25 mg/L,respectively. The molecular mechanism of goiter induced by fluoride was expounded by fluorosis experiment in vivo and in vitro, so as to provide theoretical basis for revealing the damage of thyroid caused by fluoride.
The animal model of SD rats with fluorosis was successfully reproduced and the thyroid cell samples were prepared in vitro. A series of indexes reflecting thyroid cell structure and hormone secretion function were determined by morphological observation and competitive radioimmunoassay. The results showed that fluoride caused disturbance of synthesis and secretion of thyroid hormone T_3 and T_4 and directly affected the whole body. The growth and development of thyroid cells.
Thyroid vascular hyperplasia and hyperplastic nodules of large area in rats with high fluoride (200mg/L), especially the damage of thyroid cell membrane caused by fluoride, were detected by HE staining and scanning electron microscopy. The results showed that fluoride could cause goiter and a series of damage. The expression and content of thyroid-related enzymes, some related proteins and growth factors were determined by cytochemistry and immunofluorescence histochemistry. The results showed that thyroglobulin (TG), peroxidase (TPO), sodium iodide transporter (NIS), thyroid stimulating hormone receptor (TSHR) and vascular endothelial growth factor (VEGF) were related to thyroid function. Fluoride causes the disorder of free radical metabolism, and the expression of NO-producing enzymes and related growth factors (such as iNOS and VEGF) is affected. Microvascular hyperplasia, free radicals and lipid peroxides accumulate in the thyroid gland, destroying the structure and function of thyroid follicular epithelial cells, interfering with glue. Metabolism of substance inhibits iodine transport and organization in thyroid, interferes with the activity of thyroid peroxidase, destroys the synthesis and secretion of thyroid hormones, and blocks the stimulation of TSH on thyroid cells, resulting in the failure of TSHR-cAMP to play a normal role and the decline of thyroid cell function.
In addition, the effects of fluoride on NO secretion, cytokine gene expression and secretion in FRTL cells were studied. The levels of IL-6 mRNA, IL-8 mRNA, IFN-gamma mRNA, TNF-a mRNA and the secretion of VEGF in the supernatant of FRTL cells 72 hours after fluoride stimulation were detected by RT-PCR and ELISA. On the one hand, stimulating the expression of these cytokines affects the secretion of thyroid hormones by inhibiting the expression of thyroid hormone metabolic genes; on the other hand, fluoride stimulates the expression of cytokines and NF-kappa B p65, which leads to the decrease of the ability of thyroid cells to resist fluoride and the function of thyroid cells.
Fluoride affects the growth and development of thyroid cells and changes the morphology of thyroid cells. The degree of damage is related to the dose. Long-term intake of excessive fluoride can lead to nodular enlargement of the thyroid. Fluoride stimulates the abnormal expression of iNOS, cytokines and NF-kappa B p65, which are progressing by inhibiting the expression of genes related to thyroid hormone metabolism. One step leads to a decline in thyroid cell function; fluoride causes thyroid uptake and utilization of iodine, thyroid hormone synthesis, storage, secretion dysfunction, and then lead to thyroid structure and function abnormalities.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

【相似文献】