氟对大鼠肝脏功能和形态的影响及与氧化应激关系的实验研究

【作者】 郭晓英；

【导师】 孙贵范；

【作者基本信息】 中国医科大学 ， 劳动卫生与环境卫生学， 2003， 博士

【摘要】 氟及其化合物在自然界中分布极为广泛，且广泛地应用于化学、医学、农业等领域中。氟是机体生命活动所必需的微量元素之一，具有多方面的生理作用。但如果机体长期摄入过量的氟，则可导致氟在体内蓄积，引起氟中毒，即以氟斑牙和氟骨症为主要特征的慢性全身性疾病。地方性氟中毒是严重危害我国人民身体健康的地方病，在我国分布面积广，受威胁人口多。其作用机制至今尚不十分清楚。目前，全国地氟病病情仍然比较严重，防治任务繁重，因此深入研究氟对机体的毒性作用是摆在我们面前的一项迫切任务。 肝脏是机体新陈代谢和解毒功能得以完成的重要器官，过量的氟进入体内必然对肝脏产生毒性作用。人群流行病学调查和动物实验均表明氟中毒可引起肝脏组织结构和功能异常。本研究以自由基理论为基础，从体内实验和体外实验两个方面探讨氧化应激在氟化物所致的肝损伤中的可能作用。 目的 1．探讨氧化应激在亚慢性氟中毒大鼠肝脏损伤中的作用。分析氟中毒大鼠肝功能生化指标、过氧化指标及肝结构损害指标的改变及其相关关系，并说明氧化损伤在氟性肝损伤中的可能作用。 2．以原代培养的大鼠肝细胞为研究对象，用各种浓度的氟化钠(NaF)处理肝细胞，从形态学、酶学和生物化学角度观察氟化钠对肝细胞的毒性作用及其可能机制。 方法 1．体内实验 将Wistar大鼠随机分为4组，用含氟化钠(NaF)0mg／L、50mg／L、100mg／L、150mg／L的蒸馏水喂饲大鼠3个月后，分别检测血氟、尿氟含量，肝脏中脂质过氧化产物丙二醛(MDA)的含量及抗氧化酶超氧化物歧化酶(SOD)、谷胧甘肤过氧化物酶(GSH一入)活性和抗氧化物质谷胧甘肤(GsH)含量的变化，同时检测血清中谷丙转氨酶(SGPT)、谷草转氨酶(SGoT)的活性，并观察肝脏形态学及超微结构的改变。 2.体外实验 以原代培养的大鼠肝细胞为研究对象，用各种浓度的氟化钠(NaF)处理肝细胞24小时，MTr比色法测定氟化钠对肝细胞的细胞毒性作用。在倒置显微镜下直接观察细胞的形态学改变并在电镜下观察细胞超微结构的变化。收集细胞培养上清液，测定ALT和AST活性和MDA含量。用上述指标来评价氟化钠对肝细胞的毒性作用及其可能机制。结果 (一)体内实验 1.三个月后，实验组大鼠尿氟、血氟含量显著高于对照组(p<0.01)，实验组所有大鼠均有不同程度的氟斑牙形成，表明已成功地复制出了亚慢性氟中毒大鼠模型。 2.实验组大鼠SGPT、SGOT活性随饮水氟含量增加逐渐升高，具有剂量一效应关系(r二0.569，p二0.001;r二0.624，p<0.01)，15om扩L染氟组SG钾活性显著高于对照组(p<0.01)，loom岁L组、150m扩L染氟组SGOT活性显著高于对照组(p<0.01)。 3.实验组大鼠肝脏MDA含量随染氟剂量增加而逐渐升高，150m岁L组大鼠肝脏MDA含量显著高于对照组(p<0.01)，MDA与染氟剂量、血氟之间存在显著的正相关关系(r二0.567，p<0.01;r二0.559，p<0·01)。SOD、GsH一入活性随染毒剂量增加逐渐降低，与血氟之间存在显著的负相关关系(r二一0.563，p<0·01;r=一0.419，p<0.05)。10om扩L、15om酬L组大鼠肝脏SOD活性显著低于正常对照组(p<0.05);实验组大鼠肝脏GSH含量显著低于正常对照组，GSH与染氟剂量、血氟之间存在显著的负相关关系(r二一0.848，v<0.01;r==一0.799，p<0·01)。 4.MDA与SGPT二者之间存在显著的正相关关系(r二0.460，p=0 .007)。 5.光镜下对照组大鼠肝小叶结构完整，肝细胞与肝血窦沿中央静脉呈放射状排列，细胞形态规则。实验组大鼠肝细胞索排列紊乱，肝小叶中央静脉和肝窦扩张、充血，有假小叶形成，小叶周边性脂肪变性，肝内可见散在的点状坏死灶，肝汇管区静脉淤血，间质结缔组织增生。透射电镜检查可见对照组大鼠肝细胞界限清晰，细胞核圆且居中，核仁明显。细胞质内可见丰富的粗面内质网、线粒体和糖原颗粒，少见脂滴。肝细胞界面处可见毛细胆管，毛细胆管腔内有微绒毛。染氟组大鼠肝细胞细胞界限不清，细胞内染色质浓缩、边集，呈半月形聚集于核膜下;线粒体可见明显肿胀，蜡断裂或消失;内质网扩张;可见较多的白色脂滴;毛细胆管扩张，腔面微绒毛减少。 (二)体外实验 1.采用半原位酶分离方法获得的大鼠新鲜游离肝细胞呈圆形，透亮，有立体感。培养Zh后开始贴壁，24h后肝细胞由圆形转变为多角形，胞体变平变薄，体积明显增大，细胞间出现岛状连接，许多肝细胞呈双核。透射电镜显示细胞胞浆内存在多种丰富的细胞器，其中线粒体丰富，内质网、核蛋白体发达。滑面内质网附近常见糖原颗粒。细胞核圆形，位于细胞中央，有1一2个核仁。肝细胞膜上有大量微绒毛，细胞间有特征性的紧密连接。以上结果证明所分离的细胞为大鼠肝实质细胞。 2.采用MTT比色试验评价氟化钠对原代培养大鼠肝细胞存活率的影响。在1.5一5~oFL浓度范围内，氟化钠染毒24h后可使原代培养大鼠肝细胞的细胞存活率显著下降，并呈剂量一效应关系。Ic50为3 .58~FL。 3.倒置显微镜下观察发现未经NaF处理的原?更多还原

【Abstract】 The distribution of fluorine and fluoride in nature is very wide and they are used widely in chemistry, medicine, agriculture and et al. Fluorine is one of the necessary trace elements for the body and it has many physiological effects. But if it is ingested excessively over a prolonged period, it can accumulate in the body and adversely influence many tissues and organs characterized by a vast array of symptoms and pathological changes in addition to the well-known effects on skeleton and teeth. Endemic fluorosis is a serious disease that threatened the health of people in our country. It distributes widely and there are a lot of people threatened by it. But the mechanism is still unclear. The situation of fluorosis is very serious in China and further study on its toxicity is a pressing task.Liver is an important metabolism and detoxication organ for the body. Excessive fluoride ingestion inevitably induces toxicity to the liver. Epidemiological and animal studies indicate that fluorosis induced the histology and function of liver abnormal. This study combines the studies done before and the theory on free radical to explore the role of oxidative stress in the hepatotoxicity induced by fluoride.Objectives1. To study the role of oxidative stress in the hepatotoxicity induced by sub-chronic fluorosis. Analyses the changes of the indices of the liver function, lipid peroxidation, the structure damages and the correlation among them.2. Primary cultured rat hepatocytes were cultured with various concentrations of sodium fluoride for 24 hours to study the toxicity and possible mechanism induced by fluoride from morphological, enzymological and biochemical changes.Methods1. Animal experimentsWistar rats were randomly divided into four groups. The rats were given various concentration of sodium fluoride in drinking water for three months. The content of fluoride in urine and serum were assayed. The contents of malondial-dehyde and GSH and the activities of superoxide (SOD) , GSH-Px, serum glu-tamate pyruvate transaminase (SGPT) and serum glutamate oxalate transaminase (SGOT) were also assayed. Meanwhile observed the morphological and ultra-structure changes.2. Cell cultureThe primary cultured rat hepatocytes were cultured with various concentration of sodium fluoride for 24 hours to observe its effects on the cell viability (examined by MTT assay) , morphological changes, activity of AST, ALT and contents of MDA in the medium. Using these indices to explore the toxicity of sodium fluoride and its possible mechanism.ResultsAnimal experiments1. After three months with the various indicated concentrations of fluoride in their drinking water, the rats had fluoride levels in their urine and serum that were significantly higher than those of the control group and dental fluorosis were observed in the rats of the experimental groups. All manifestations indicated that fluorosis rats model were produced successfully.2. The activities of SGPT and SGOT were elevated in dose-dependent manner following NaF treatment. The activity of SGPT in 150 mg/L group was significantly higher than the control group ( p <0. 01). The activities of SGOT in 100 mg/L and 150 mg/L group were significantly higher than the control group3. The content of MDA in 150 mg/L group increased significantly compared with the control group. There was a positive correlation between MDA and the fluoride content in serum (r =0.559 ,p <0.01) . The activities of SOD and GSH-Px decreased with the increased concentration of fluoride. The activities of SOD in 100 mg/L and 150 mg/L decreased significantly compared with the control group (p<0.05) . There was a negative correlation between SOD, GSH-Px and the fluoride content in serum ( r = - 0.563, p<0.01; r =- 0.419, p< 0.05). The contents of GSH in the experimental groups were significantly decreased compared with the control group. There was a negative correlation between GSH and dose and blood fluoride ( r = -0.848, p<0.01; r = - 0.799, p<0.01).4.更多还原