硫酸铟细胞毒性机制及茶多酚拮抗作用的研究

发布时间：2018-05-29 14:04

本文选题：硫酸铟 + 凋亡　；参考：《苏州大学》2012年硕士论文

【摘要】：目的：铟(indium,In)是一种稀有金属元素,属于第ⅢA族。其低熔点、高沸点、传导性好,具有广泛的使用空间。它的主要用途是：生产铟锡氧化物(ITO)靶材(占整个铟用量的60%～70%)。经各种途径吸收入血的铟可与血浆蛋白(转铁蛋白、α。球蛋白和白蛋白)结合,迅速转运到软组织和骨骼。胶状体的铟则不与血浆蛋白结合,但可被白细胞吞噬后送到肝和脾的网状内皮系统。进入体内的铟主要蓄积在骨骼；皮下注射后,大部分的铟蓄积在皮肤和肌肉内；腹腔注射时,铟多蓄积在肠系膜和肝脏,然后转移到脾、肾和骨骼等。金属铟基本无毒或低毒,而其化合物对人体具有毒性作用。其中,又以可溶性钢盐毒性最大。本实验所用硫酸铟(indium sulfate)为具有代表性的铟可溶性化合物。本研究旨在探讨体外实验条件下硫酸铟的毒性,从氧化应激(oxidative stress)的角度研究硫酸铟引起的L929细胞损伤及毒性机制,并进一步探讨茶多酚对该损伤的拮抗作用,以了解还原性物质是否具有硫酸钢毒性拮抗作用,为今后铟环境暴露的预防提供相关资料。方法：本研究分为三个部分。第一部分：以L929细胞作为实验系统,首先采用四甲基偶氮唑盐法(MTT比色法)探讨不同浓度的硫酸铟对L929细胞存活率的影响,了解浓度。时间-毒性效应之间的关系,并为后期深入研究筛选硫酸铟染毒剂量；继而采用碘化丙啶(PI)染色,观察细胞周期分布状况,了解硫酸铟对L929细胞生长状态的影响,进一步确定硫酸铟对L929细胞的细胞毒性。第二部分：在第一部分确定的硫酸铟染毒浓度剂量范围内,用Annexin V-FITC/PI凋亡试剂盒,检测硫酸铟对细胞凋亡的影响,探讨硫酸铟导致细胞损伤的可能机制。观察硫酸铟是否导致细胞内总蛋白含量、超氧化物歧化酶(T-SOD)活力、丙二醛(MDA)含量变化。第三部分：针对硫酸铟导致细胞损伤的可能机制,探讨茶多酚的拮抗作用。探讨茶多酚存在条件下,硫酸铟对L929细胞存活率、细胞凋亡率、细胞内T-SOD活力和细胞内MDA含量水平的相应变化。结果：硫酸铟从3.0mmol/L(作用24h)、6.0mmol/L(作用2h)开始即对L929细胞生长产生明显抑制作用,细胞存活率呈浓度-时间依赖性降低；在硫酸铟作用下,细胞周期分布变化明显,G1期细胞减少,G2/M期和S期细胞明显增多。在硫酸铟的作用下,细胞凋亡结果显示硫酸钢对L929细胞具有促进凋亡的作用；细胞内T-SOD活力减弱,MDA含量增多,差异具有统计学意义(P0.05)。在茶多酚存在的条件下,0.2mg/ml的茶多酚在一定程度上对硫酸铟引起的L929细胞损伤具有一定的保护作用；1.0mg/ml的茶多酚能够明显的降低硫酸铟导致的细胞凋亡率升高的作用；1.0mg/ml的茶多酚对硫酸铟所致L929细胞内T-SOD活力减弱,0.2mg/ml的茶多酚对细胞内MDA含量增加均具有一定的拮抗作用,差异具有统计学意义(P0.05)。结论：本研究结果显示：硫酸铟低浓度、短时间即可明显抑制L929细胞增值；并且可以导致细胞周期分布的改变。硫酸铟可引起细胞凋亡率的升高、T-SOD活力减弱及MDA含量增加,表明硫酸铟对L929细胞产生氧化损伤作用。茶多酚可以在一定程度上拮抗该损伤,适宜剂量可以起到较好的拮抗作用。综合结果,本文认为硫酸铟引起细胞脂质过氧化,继而引发细胞周期的变化和细胞凋亡率的升高。
[Abstract]:Objective: indium (In) is a rare metal element, belonging to the third A group. Its low melting point, high boiling point, good conductivity, and wide use space. Its main use is to produce indium tin oxide (ITO) target (60% to 70% of the total indium). Indium can be absorbed into blood by various routes and plasma protein (transferrin, alpha. Globulin) The indium can be transported to the soft tissue and bone quickly, and the indium of the colloidal body is not combined with the plasma protein, but can be swallowed to the reticuloendothelial system of the liver and spleen after being phagocytic by white cells. The indium entering the body is mainly accumulated in the bone; after subcutaneous injection, most indium is accumulated in skin and muscles; indium accumulates more in the intestines when intraperitoneal injection. The mesangial and liver, then transferred to the spleen, kidney and bone, etc., metal indium is essentially nontoxic or low toxic, and its compounds are toxic to the human body. Among them, the toxicity of soluble steel salt is the most. Indium sulphate (indium sulfate) is a representative indium soluble compound used in this experiment.
The purpose of this study is to investigate the toxicity of indium sulphate in vitro, and to study the mechanism of L929 cell damage and toxicity induced by indium sulfate from the angle of oxidative stress (oxidative stress), and to further explore the antagonism of tea polyphenols to the damage to understand whether the reductive substances have the toxic antagonism of sulphuric acid steel and for the future indium environment storm. The prevention of exposure provides relevant information.
Methods: This study was divided into three parts. Part 1: using L929 cells as the experimental system, the effect of indium sulphate on the survival rate of L929 cells with different concentrations of indium sulfate with different concentrations of AZO (MTT colorimetric method) was first investigated, and the relationship between the concentration and the time toxicity effect was investigated, and the dosage of indium sulfate was screened in the later period. Then the cell cycle distribution was observed with propidium iodide (PI), and the effect of indium sulphate on the growth state of L929 cells was investigated, and the cytotoxicity of indium sulphate to L929 cells was further determined. The second part: in the first part of the dose range of indium sulfate dye concentration, Annexin V-FITC/PI apoptosis kit was used to detect indium sulphate. The effect of cell apoptosis and the possible mechanism of indium sulphate causing cell damage. Observe whether indium sulphate leads to the total protein content in cells, the activity of superoxide dismutase (T-SOD) and the content of malondialdehyde (MDA). The third part: the antagonistic effect of indium sulfate on cell damage and the antagonism of tea polyphenols. Under the conditions, indium sulphate can change the survival rate, apoptosis rate, intracellular T-SOD activity and intracellular MDA level of L929 cells.
Results: indium sulfate could inhibit the growth of L929 cells from 3.0mmol/L (action 24h) and 6.0mmol/L (action 2H). The cell survival rate was decreased in concentration time dependence; in the action of indium sulfate, the cell cycle distribution changed obviously, the G1 phase cells decreased, the G2/ M phase and the S phase cells increased obviously. Under the action of indium sulphate, cells The apoptosis results showed that sulphuric acid steel could promote apoptosis of L929 cells, the intracellular T-SOD activity was weakened, and the content of MDA increased, the difference was statistically significant (P0.05). In the presence of tea polyphenols, 0.2mg/ml tea polyphenols had certain protective effects on L929 fine cell damage caused by indium sulphate; 1.0mg/ml tea Polyphenols can obviously reduce the effect of indium sulphate on the increase of apoptosis rate. The activity of T-SOD in L929 cells induced by indium sulfate in 1.0mg/ml is weakened, and the tea polyphenols of 0.2mg/ml have a certain antagonistic effect on the increase of MDA content in the cells, and the difference is statistically significant (P0.05).
Conclusion: the results of this study show that the low concentration of indium sulfate can inhibit the proliferation of L929 cells in a short time, and can lead to the change of cell cycle distribution. Indium sulfate can cause the increase of cell apoptosis rate, decrease the activity of T-SOD and increase the content of MDA, which indicates that indium sulfate can produce oxidative damage to L929 cells. In a certain degree, the appropriate dose can be antagonistic to the damage. In this paper, indium sulfate is considered to cause lipid peroxidation, which leads to the change of cell cycle and the increase of cell apoptosis rate.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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