【摘要】： 氟是機(jī)體必需的微量元素,攝入適量能維持機(jī)體正常的生命活動(dòng),但它還是一種全身性毒物,其對(duì)機(jī)體內(nèi)分泌腺的影響已成為近年來(lái)的研究熱點(diǎn)。有實(shí)驗(yàn)表明氟能夠引起甲狀腺腫大,但是也有學(xué)者持反對(duì)意見(jiàn)。本實(shí)驗(yàn)分別復(fù)制SD大鼠氟中毒動(dòng)物模型和氟化鈉處理原代培養(yǎng)SD大鼠甲狀腺細(xì)胞、大鼠甲狀腺細(xì)胞系(fisher ratthyroid cell line,FRTL)。80只SD大鼠飲水中添加的氟化鈉含量分別為0,50mg/L,100mg/L,200mg/L,原代培養(yǎng)的SD大鼠甲狀腺細(xì)胞染氟化鈉濃度分別為0,10μmol/L,100μmol/L,1000μmol/L,FRTL細(xì)胞染氟化鈉濃度分別為0,1.25mg/L,2.5mg/L,5.0mg/L,10.0mg/L,20mg/L。運(yùn)用電鏡學(xué)、分子生物學(xué)、免疫學(xué)等技術(shù)研究氟對(duì)甲狀腺激素代謝基因表達(dá)、功能蛋白表達(dá)和分布、細(xì)胞因子基因表達(dá)及分泌的影響,體外實(shí)驗(yàn)?zāi)芙獬虑鹉X-腺垂體-甲狀腺軸的影響,影響因素單一。本實(shí)驗(yàn)通過(guò)體內(nèi)體外氟中毒實(shí)驗(yàn)闡述氟引起甲狀腺腫大的分子機(jī)理,從而為揭示氟引起甲狀腺損傷提供理論依據(jù)。 實(shí)驗(yàn)成功的復(fù)制SD大鼠氟中毒動(dòng)物模型和制備體外氟中毒甲狀腺細(xì)胞樣品,通過(guò)形態(tài)學(xué)觀察及競(jìng)爭(zhēng)放射性免疫法測(cè)定反映甲狀腺細(xì)胞結(jié)構(gòu)和分泌激素功能的一系列指標(biāo),結(jié)果發(fā)現(xiàn)氟引起甲狀腺激素T_3、T_4合成和分泌障礙及直接影響整個(gè)甲狀腺細(xì)胞的生長(zhǎng)和發(fā)育過(guò)程。 高氟(200mg/L)大鼠甲狀腺血管增生和大面積增生結(jié)節(jié),尤其氟對(duì)甲狀腺細(xì)胞膜損傷嚴(yán)重;本實(shí)驗(yàn)通過(guò)甲狀腺組織切片HE染色和掃描電鏡等手段檢測(cè)氟對(duì)甲狀腺細(xì)胞的損傷,結(jié)果證明氟能夠引起甲狀腺腫大和一序列的損傷;運(yùn)用RT-PCR、免疫細(xì)胞化學(xué)和免疫熒光組織化學(xué)等方法測(cè)定甲狀腺相關(guān)功能酶類,以及部分相關(guān)蛋白和生長(zhǎng)因子的表達(dá)與含量,結(jié)果發(fā)現(xiàn)甲狀腺球蛋白(TG)、過(guò)氧化物酶(TPO)、鈉碘轉(zhuǎn)運(yùn)體(NIS)、促甲狀腺激素受體(TSHR)、血管內(nèi)皮生長(zhǎng)因子(VEGF)等與甲狀腺功能密切相關(guān)的指標(biāo)均表現(xiàn)異常;氟引起自由基代謝紊亂,致NO生成的功能酶以及相關(guān)生長(zhǎng)因子(如iNOS和VEGF)表達(dá)受到影響發(fā)生顯著變化,微血管增生,自由基和脂質(zhì)過(guò)氧化物在甲狀腺中蓄積,破壞了甲狀腺濾泡上皮細(xì)胞結(jié)構(gòu)和功能,干擾膠質(zhì)的代謝,抑制甲狀腺中碘的轉(zhuǎn)運(yùn)和有機(jī)化過(guò)程,干擾甲狀腺過(guò)氧化物酶的活性,破壞甲狀腺激素合成和分泌的各個(gè)環(huán)節(jié),阻斷了TSH對(duì)甲狀腺細(xì)胞刺激作用,導(dǎo)致TSHR-cAMP無(wú)法發(fā)揮正常作用,甲狀腺細(xì)胞功能下降。 此外本實(shí)驗(yàn)研究FRTL細(xì)胞在外界氟的作用下NO分泌、細(xì)胞因子基因表達(dá)和分泌的影響。運(yùn)用RT-PCR、ELISA檢測(cè)氟刺激FRTL細(xì)胞72h后IL-6 mRNA、IL-8mRNA、IFN-γmRNA,TNF-a mRNA水平和細(xì)胞上清中VEGF分泌量;同時(shí)westernblot測(cè)定NF-κB p65蛋白水平,結(jié)果表明:氟刺激這些細(xì)胞因子的表達(dá),一方面通過(guò)抑制甲狀腺激素代謝基因表達(dá)而影響甲狀腺細(xì)胞甲狀腺激素的分泌;另一方面,氟刺激細(xì)胞因子和NF-κB p65表達(dá)導(dǎo)致甲狀腺細(xì)胞自身抵抗氟能力下降,甲狀腺細(xì)胞功能下降。 氟化物影響甲狀腺細(xì)胞的生長(zhǎng)和發(fā)育,使細(xì)胞的形態(tài)發(fā)生改變,損傷程度與劑量有關(guān),長(zhǎng)期攝入過(guò)量氟可導(dǎo)致甲狀腺發(fā)生結(jié)節(jié)性腫大;氟化物刺激iNOS、細(xì)胞因子和NF-κB p65異常的表達(dá),這些因子通過(guò)抑制甲狀腺激素代謝相關(guān)基因的表達(dá)進(jìn)一步導(dǎo)致甲狀腺細(xì)胞功能下降;氟化物造成甲狀腺攝取和利用碘、甲狀腺激素合成、貯存、分泌功能障礙,進(jìn)而導(dǎo)致甲狀腺結(jié)構(gòu)和功能異常。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

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