硫辛酸对氢醌所致V79细胞线粒体膜电位损伤的保护性实验研究

发布时间：2018-05-18 09:55

本文选题：氢醌 + 硫辛酸　；参考：《中国医科大学》2008年硕士论文

【摘要】： 前言近年来,随着人们生活水平的提高,室内装修与装饰已很普遍,使室内污染成为人们关注的问题。儿童少年正处于快速生长发育期,与成人比较,室内污染对儿童少年健康的危害更加严重。儿童患呼吸系统疾病尤其是哮喘的发病率逐年升高,究其原因可能与环境污染程度加重,室内外空气中含有的粉尘,二氧化碳,甲醛,苯,氨以及香烟的烟雾等有关。然而,这些污染物造成的肺损伤机制尚不十分清楚。本研究以广泛存在于油漆及装饰材料中苯的主要代谢产物氢醌(Hydroquinone,HQ)为毒物,研究其对肺组织的毒性机制,并探讨有效的预防和干预方法。 HQ是苯在体内的重要代谢产物,在自然界中广泛存在,同时也是香烟烟雾和焦油中的主要成分之一。HQ在体内代谢过程中可产生大量自由基导致支气管和肺的损伤,但目前确切机制尚不十分清楚。已知线粒体在外来化学物所致的细胞损伤,尤其是氧化损伤中为主要的靶细胞器,线粒体损伤是导致多种疾病发生的主要病理机制之一。硫辛酸(R-α-Lipoic Acid,LA)是目前备受关注的抗氧化剂,已经广泛用于糖尿病并发症的预防和治疗中。本文采用噻唑蓝(MTT)和线粒体特异性探针(5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide,JC-1)的方法,检测氢醌对体外培养的中国仓鼠肺细胞(V79)的毒性作用,并重点研究HQ对线粒体膜电位(Mitochondrial membrane potential,△Ψm)的影响,同时选择硫辛酸(R-α-Lipoic Acid,LA)作为抗氧化剂,进行干预性研究,为进一步研究HQ的病理发生机制,采取有效的预防与治疗措施提供理论依据。研究目的 1、探讨不同剂量HQ对V79细胞的毒性作用。 2、探讨能引起V79细胞毒性作用的HQ剂量对V79细胞线粒体膜电位的影响。 3、采用LA进行干预性研究,确定LA的适宜干预剂量及可能保护机制。实验材料和方法一、实验材料中国仓鼠肺细胞(V79),HQ,LA,DMEM高糖培养液,胎牛血清,胰蛋白酶-EDTA,JC-1,MTT。二、实验方法 1、细胞的培养与分组 V79细胞用细胞培养液(DMEM培养液含10%胎牛血清,100U/mL青霉素和100μg/ml链霉素)置37℃,5%CO_2培养箱中,每2天换液1次。将处于对数生长期的V79细胞按10~5个/ml的细胞密度接种到96孔板、6孔板和培养瓶中,培养24h后换成含1%胎牛血清的培养液,并分组施加以下处理因素:①对照组:单纯培养液培养。②HQ组:HQ终浓度为10,20,30,40,50,100,150μmol/L。③LA组:LA终浓度为50,100μmol/L。④LA+HQ组1:LA终浓度为50,100μmol/L;HQ终浓度为50μmol/L。⑤LA+HQ组2:LA浓度同④组,HQ终浓度为100μmol/L。继续培养24h。 2、细胞存活率的测定 MTT法 3、线粒体膜电位的测定流式细胞术和倒置荧光显微镜 4、统计分析采用SPSS11.5统计软件中单因素方差分析处理,数据用(?)±s表示,检验标注α=0.05。实验结果一、不同浓度HQ对V79细胞存活率的影响与对照组比较,当HQ为40μmol/L时,V79细胞存活率明显下降,差异有统计学意义(P＜0.05);并且HQ在40～150μmol/L范围内,随着HQ浓度增高,V79细胞存活率下降,呈剂量-效应关系。二、LA对HQ所致V79细胞存活率下降的干预作用与对照组比较,LA为50和100μmol/L时,对细胞存活率没有影响,但此浓度下的LA可以明显拮抗50和100μmol/L HQ所引起的V79细胞存活率的下降,差异有统计学意义(P＜0.05)。三、LA对HQ所致V79细胞线粒体膜电位影响的干预作用对照组V79细胞线粒体膜电位为1.12,当50、100μmol/L的HQ作用于V79细胞时线粒体膜电位明显降低,分别为0.22和0.03;50μmol/L的LA单独作用于V79细胞的线粒体膜电位为0.95;与单纯HQ组比较,50μmol/L的LA能明显拮抗50、100μmol/L的HQ对V79细胞线粒体膜电位造成的损伤作用,使其线粒体膜电位分别上升至0.79和0.63。结论 1、对线粒体的损伤可能是HQ引起V79细胞毒性的重要途径之一。 2、一定浓度的LA对HQ引起的V79细胞毒性和线粒体损伤有保护作用。
[Abstract]:Preface
In recent years, with the improvement of people's living standards, indoor decoration and decoration have become very common, making indoor pollution a problem of concern. Children and adolescents are in a rapid growth period. Compared with adults, indoor pollution has more serious harm to children and adolescents. The incidence of respiratory diseases, especially asthma, is rising year by year. High levels of pollution may be associated with environmental pollution, including dust, carbon dioxide, formaldehyde, benzene, ammonia, and cigarette smoke. However, the mechanism of lung injury caused by these pollutants is not very clear. This study is widely found in the main metabolite of benzene in paint and decoration materials (Hydroquino Ne (HQ) was used as a toxicant to study its toxic mechanism on lung tissue and to explore effective prevention and intervention methods.
HQ is an important metabolite of benzene in the body. It exists widely in nature, and it is also one of the main components of cigarette smoke and tar..HQ can produce a large number of free radicals in the metabolic process of the body, causing damage to the bronchus and lungs. But the exact mechanism is not yet very clear. Injury, especially in oxidative damage, is one of the main target organelles, and mitochondrial damage is one of the main pathological mechanisms that lead to a variety of diseases. R- alpha -Lipoic Acid (LA) is a popular antioxidant and has been widely used in the prevention and treatment of diabetic complications. This article uses thiazolium (MTT) and mitochondrial specific exploration. The toxic effects of hydroquinone on Chinese hamster lung cells (V79) in vitro were detected by 5,5', 6,6'-Tetrachloro-1,1', 3,3'-tetraethyl-imidacarbocyanine iodide, JC-1, and the effect of HQ on the mitochondrial membrane potential (Mitochondrial membrane potential, delta m) was selected as the anti - lipoic acid. An intervention study was conducted to provide a theoretical basis for further study of the pathogenesis of HQ and effective prevention and treatment measures.
research objective
1, the toxic effects of different doses of HQ on V79 cells were investigated.
2, to explore the effect of HQ dose on mitochondrial membrane potential of V79 cells induced by V79 cytotoxicity.
3, use LA to conduct an intervention study to determine the appropriate dose and possible protective mechanism of LA.
Experimental materials and methods
First, experimental materials
Chinese hamster lung cells (V79), HQ, LA, DMEM high glucose medium, fetal bovine serum, trypsin -EDTA, JC-1, MTT.
Two, experimental method
1, cell culture and grouping
V79 cells were cultured in cell culture medium (DMEM culture medium containing 10% fetal bovine serum, 100U/mL penicillin and 100 g/ml streptomycin) at 37 degrees, 5%CO_2 incubator, and 1 times every 2 days.
The V79 cells at the logarithmic growth period were inoculated into 96 orifice plates, 6 orifice plates and culture bottles according to the cell density of 10~5 /ml. After culture 24h, the culture medium containing 1% fetal bovine serum was replaced by the following treatment factors: (1) the control group: pure culture medium. Group HQ: HQ terminal concentration was 10,20,30,40,50100150 u mol/L. (LA group): LA final concentration was 5. 0100, mol/L. 4. The final concentration of 1:LA in group LA+HQ was 50100 mol/L; the final concentration of HQ was 50 mol/L. mol/L.; 2:LA concentration in LA+HQ group was same as that in group 4, and HQ at 100 mol/L. mol/L. continued to cultivate 24h..
2, determination of cell survival rate
MTT method
3, determination of mitochondrial membrane potential
Flow cytometry and inverted fluorescence microscopy
4, statistical analysis
The data were analyzed by one-way ANOVA in SPSS11.5 statistical software. The data were expressed by (+) s, and the test was labeled with alpha =0.05..
experimental result
The effect of different concentrations of HQ on the survival rate of V79 cells
Compared with the control group, the survival rate of V79 cells decreased significantly when HQ was 40 mu mol/L, and the difference was statistically significant (P < 0.05), and the survival rate of V79 cells decreased with the increase of HQ concentration in the range of 40~150 micron mol/L, which showed a dose effect relationship.
Two, the intervention effect of LA on the decrease of V79 cell survival rate induced by HQ.
Compared with the control group, when LA was 50 and 100 mu mol/L, there was no effect on the cell survival rate, but the LA under this concentration could obviously antagonize the decrease of the survival rate of V79 cells caused by 50 and 100 mu mol/L HQ, and the difference was statistically significant (P < 0.05).
Three, the effect of LA on the mitochondrial membrane potential of V79 cells induced by HQ.
The mitochondrial membrane potential of the V79 cells in the control group was 1.12, and the mitochondrial membrane potential decreased significantly when the 50100 mol/L HQ acted on the V79 cells, respectively, 0.22 and 0.03, and the mitochondrial membrane potential of 50 mu LA alone on V79 cells was 0.95. Compared with the simple HQ group, 50 micron LA could clearly antagonize the mitochondrial membrane of the 50100 mu mol/L HQ cells. The potential damage caused by electric potential increased the mitochondrial membrane potential to 0.79 and 0.63. respectively.
conclusion
1, mitochondrial damage may be one of the important ways for HQ to cause V79 cytotoxicity.
2, a certain concentration of LA has protective effects on HQ induced V79 cytotoxicity and mitochondrial damage.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

【参考文献】