本文選題：氫醌 + 硫辛酸��； 參考：《中國(guó)醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 前言 近年來(lái),隨著人們生活水平的提高,室內(nèi)裝修與裝飾已很普遍,使室內(nèi)污染成為人們關(guān)注的問(wèn)題。兒童少年正處于快速生長(zhǎng)發(fā)育期,與成人比較,室內(nèi)污染對(duì)兒童少年健康的危害更加嚴(yán)重。兒童患呼吸系統(tǒng)疾病尤其是哮喘的發(fā)病率逐年升高,究其原因可能與環(huán)境污染程度加重,室內(nèi)外空氣中含有的粉塵,二氧化碳,甲醛,苯,氨以及香煙的煙霧等有關(guān)。然而,這些污染物造成的肺損傷機(jī)制尚不十分清楚。本研究以廣泛存在于油漆及裝飾材料中苯的主要代謝產(chǎn)物氫醌(Hydroquinone,HQ)為毒物,研究其對(duì)肺組織的毒性機(jī)制,并探討有效的預(yù)防和干預(yù)方法。 HQ是苯在體內(nèi)的重要代謝產(chǎn)物,在自然界中廣泛存在,同時(shí)也是香煙煙霧和焦油中的主要成分之一。HQ在體內(nèi)代謝過(guò)程中可產(chǎn)生大量自由基導(dǎo)致支氣管和肺的損傷,但目前確切機(jī)制尚不十分清楚。已知線粒體在外來(lái)化學(xué)物所致的細(xì)胞損傷,尤其是氧化損傷中為主要的靶細(xì)胞器,線粒體損傷是導(dǎo)致多種疾病發(fā)生的主要病理機(jī)制之一。硫辛酸(R-α-Lipoic Acid,LA)是目前備受關(guān)注的抗氧化劑,已經(jīng)廣泛用于糖尿病并發(fā)癥的預(yù)防和治療中。本文采用噻唑藍(lán)(MTT)和線粒體特異性探針(5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide,JC-1)的方法,檢測(cè)氫醌對(duì)體外培養(yǎng)的中國(guó)倉(cāng)鼠肺細(xì)胞(V79)的毒性作用,并重點(diǎn)研究HQ對(duì)線粒體膜電位(Mitochondrial membrane potential,△Ψm)的影響,同時(shí)選擇硫辛酸(R-α-Lipoic Acid,LA)作為抗氧化劑,進(jìn)行干預(yù)性研究,為進(jìn)一步研究HQ的病理發(fā)生機(jī)制,采取有效的預(yù)防與治療措施提供理論依據(jù)。 研究目的 1、探討不同劑量HQ對(duì)V79細(xì)胞的毒性作用。 2、探討能引起V79細(xì)胞毒性作用的HQ劑量對(duì)V79細(xì)胞線粒體膜電位的影響。 3、采用LA進(jìn)行干預(yù)性研究,確定LA的適宜干預(yù)劑量及可能保護(hù)機(jī)制。 實(shí)驗(yàn)材料和方法 一、實(shí)驗(yàn)材料 中國(guó)倉(cāng)鼠肺細(xì)胞(V79),HQ,LA,DMEM高糖培養(yǎng)液,胎牛血清,胰蛋白酶-EDTA,JC-1,MTT。 二、實(shí)驗(yàn)方法 1、細(xì)胞的培養(yǎng)與分組 V79細(xì)胞用細(xì)胞培養(yǎng)液(DMEM培養(yǎng)液含10%胎牛血清,100U/mL青霉素和100μg/ml鏈霉素)置37℃,5%CO_2培養(yǎng)箱中,每2天換液1次。 將處于對(duì)數(shù)生長(zhǎng)期的V79細(xì)胞按10~5個(gè)/ml的細(xì)胞密度接種到96孔板、6孔板和培養(yǎng)瓶中,培養(yǎng)24h后換成含1%胎牛血清的培養(yǎng)液,并分組施加以下處理因素:①對(duì)照組:單純培養(yǎng)液培養(yǎng)。②HQ組:HQ終濃度為10,20,30,40,50,100,150μmol/L。③LA組:LA終濃度為50,100μmol/L。④LA+HQ組1:LA終濃度為50,100μmol/L;HQ終濃度為50μmol/L。⑤LA+HQ組2:LA濃度同④組,HQ終濃度為100μmol/L。繼續(xù)培養(yǎng)24h。 2、細(xì)胞存活率的測(cè)定 MTT法 3、線粒體膜電位的測(cè)定 流式細(xì)胞術(shù)和倒置熒光顯微鏡 4、統(tǒng)計(jì)分析 采用SPSS11.5統(tǒng)計(jì)軟件中單因素方差分析處理,數(shù)據(jù)用(?)±s表示,檢驗(yàn)標(biāo)注α=0.05。 實(shí)驗(yàn)結(jié)果 一、不同濃度HQ對(duì)V79細(xì)胞存活率的影響 與對(duì)照組比較,當(dāng)HQ為40μmol/L時(shí),V79細(xì)胞存活率明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);并且HQ在40～150μmol/L范圍內(nèi),隨著HQ濃度增高,V79細(xì)胞存活率下降,呈劑量-效應(yīng)關(guān)系。 二、LA對(duì)HQ所致V79細(xì)胞存活率下降的干預(yù)作用 與對(duì)照組比較,LA為50和100μmol/L時(shí),對(duì)細(xì)胞存活率沒(méi)有影響,但此濃度下的LA可以明顯拮抗50和100μmol/L HQ所引起的V79細(xì)胞存活率的下降,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 三、LA對(duì)HQ所致V79細(xì)胞線粒體膜電位影響的干預(yù)作用 對(duì)照組V79細(xì)胞線粒體膜電位為1.12,當(dāng)50、100μmol/L的HQ作用于V79細(xì)胞時(shí)線粒體膜電位明顯降低,分別為0.22和0.03;50μmol/L的LA單獨(dú)作用于V79細(xì)胞的線粒體膜電位為0.95;與單純HQ組比較,50μmol/L的LA能明顯拮抗50、100μmol/L的HQ對(duì)V79細(xì)胞線粒體膜電位造成的損傷作用,使其線粒體膜電位分別上升至0.79和0.63。 結(jié)論 1、對(duì)線粒體的損傷可能是HQ引起V79細(xì)胞毒性的重要途徑之一。 2、一定濃度的LA對(duì)HQ引起的V79細(xì)胞毒性和線粒體損傷有保護(hù)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.
【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R363

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