發(fā)布時(shí)間：2018-04-20 09:05

  本文選題：α-硫辛酸 + 支持細(xì)胞��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：探討α-硫辛酸對(duì)氧化應(yīng)激下大鼠睪丸支持細(xì)胞的氧化損傷是否具有保護(hù)作用，并通過細(xì)胞骨架蛋白表達(dá)角度研究α-硫辛酸對(duì)氧化應(yīng)激下大鼠睪丸支持細(xì)胞的保護(hù)作用途徑及相關(guān)的機(jī)制。 方法：取大鼠睪丸進(jìn)行支持細(xì)胞原代培養(yǎng)，，并通過細(xì)胞形態(tài)學(xué)進(jìn)行細(xì)胞鑒定。將各種濃度的H2O2(0,50,100,200,400,800,1600μmol/L)誘導(dǎo)體外培養(yǎng)的大鼠睪丸支持細(xì)胞氧化損傷，并通過CCK-8法檢測(cè)睪丸支持細(xì)胞的活性及DCFH-DA熒光探針法檢測(cè)細(xì)胞內(nèi)活性氧水平進(jìn)行睪丸支持細(xì)胞氧化損傷模型的鑒定。將原代培養(yǎng)大鼠睪丸支持細(xì)胞按以下四組進(jìn)行處理：對(duì)照組，即正常細(xì)胞培養(yǎng)，不加任何處理因素；α-LA組，僅加入含終濃度200μmol/Lα-LA的培養(yǎng)基；H2O2組，即加入含終濃度400μmol/LH2O2的培養(yǎng)基；α-LA+H2O2組，即預(yù)先加入含終濃度200μmol/Lα-LA孵育4h后再加入含終濃度400μmol/LH2O2的培養(yǎng)基。各組處理12h后進(jìn)行實(shí)驗(yàn)檢測(cè)。采用CCK-8法檢測(cè)各組睪丸支持細(xì)胞的活性及DCFH-DA熒光探針法檢測(cè)各組睪丸支持細(xì)胞內(nèi)活性氧水平；酶活性檢測(cè)試劑盒檢測(cè)抗氧化酶活性；免疫組織化學(xué)法、蛋白質(zhì)免疫印跡法檢測(cè)波形蛋白及微管蛋白的表達(dá)。 結(jié)果：大鼠睪丸支持細(xì)胞經(jīng)分離、接種后，細(xì)胞貼壁生長(zhǎng)，3至4天后細(xì)胞呈現(xiàn)單層融合狀態(tài)生長(zhǎng)。H2O2損傷組較正常對(duì)照組，細(xì)胞活力有所降低，并隨H2O2濃度的升高，細(xì)胞活力降低明顯，同時(shí)，細(xì)胞內(nèi)ROS水平上升，H2O2作用于細(xì)胞致氧化損傷的適合濃度為400μmol/L。與對(duì)照組比較，H2O2刺激后支持細(xì)胞存活率下降，具有統(tǒng)計(jì)學(xué)意義(P0.05)；細(xì)胞內(nèi)ROS水平上升(P0.05)；總抗氧化能力、超氧化物歧化酶、過氧化氫酶和谷胱甘肽還原酶的活性均降低（P0.05）；波形蛋白及微管蛋白的表達(dá)也明顯降低(P0.05)，而α-LA干預(yù)后可提高細(xì)胞存活率，降低細(xì)胞內(nèi)ROS水平，提高抗氧化酶的活性，并提高波形蛋白及微管蛋白的表達(dá)。 結(jié)論：氧化應(yīng)激可導(dǎo)致大鼠睪丸支持細(xì)胞波形蛋白及微管蛋白表達(dá)降低，使細(xì)胞存活率下降；α-LA通過清除細(xì)胞內(nèi)ROS，提高抗氧化酶的活性及波形蛋白和微管蛋白的表達(dá)而對(duì)氧化應(yīng)激的支持細(xì)胞起保護(hù)作用。
[Abstract]:Objective: to investigate the protective effect of 偽 -lipoic acid on oxidative injury of testicular Sertoli cells in rats under oxidative stress. The protective effects of 偽 -lipoic acid on rat testicular Sertoli cells under oxidative stress and its related mechanism were studied by cytoskeleton protein expression. Methods: Sertoli cells were cultured from rat testis and identified by cell morphology. Oxidative damage of rat testicular Sertoli cells was induced by different concentrations of H _ 2O _ 2: 50100200400800c1600 渭 mol / L in vitro. The activity of testicular Sertoli cells was detected by CCK-8 and the reactive oxygen species was detected by DCFH-DA fluorescence probe method to identify the oxidative injury model of testicular Sertoli cells. Primary cultured rat testicular Sertoli cells were treated in the following four groups: control group, that is, normal cell culture without any additional factors, 偽 -LA group, which was treated with H _ 2O _ 2 medium containing final concentration of 200 渭 mol/L 偽 -LA, that is, medium containing final concentration of 400 渭 mol/LH2O2, 偽 -LA H2O2 group, The medium containing final concentration of 200 渭 mol/L 偽 -LA was added for 4 h and then added to the medium containing final concentration of 400 渭 mol/LH2O2. After 12 hours of treatment, experimental examination was carried out in each group. The activity of testicular Sertoli cells in each group was detected by CCK-8 method and reactive oxygen species in Sertoli cells was detected by DCFH-DA fluorescence probe method, the antioxidant enzyme activity was detected by enzyme activity test kit, and the activity of antioxidant enzyme was detected by immunohistochemical method. The expression of vimentin and tubulin was detected by Western blot. Results: rat testicular Sertoli cells were isolated and inoculated. After 3 to 4 days of adherent growth, the cells showed monolayer fusion growth. H2O2 injury group was significantly lower than the normal control group, and the cell activity decreased with the increase of H2O2 concentration. At the same time, the level of ROS in cells was increased. The suitable concentration of H2O2 was 400 渭 mol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) for oxidative damage induced by H _ 2O _ 2. Compared with the control group, the survival rate of Sertoli cells decreased after stimulation with H _ 2O _ 2, which had statistical significance (P 0.05), the level of ROS in cells increased (P 0.05), the total antioxidant capacity, superoxide dismutase (SOD), total antioxidation ability, superoxide dismutase, The activities of catalase and glutathione reductase both decreased, vimentin and tubulin expression also decreased significantly. 偽 -LA increased cell survival rate, decreased intracellular ROS level, and increased antioxidant enzyme activity. The expression of vimentin and tubulin was increased. Conclusion: oxidative stress can decrease the expression of vimentin and tubulin in rat testicular Sertoli cells. 偽 -LA can protect the supporting cells from oxidative stress by scavenging intracellular ROSs and increasing the activity of antioxidant enzymes and the expression of vimentin and tubulin.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉作強(qiáng);陳捷;方小武;吳日然;陳昂;韋劍洪;;精子DNA損傷對(duì)試管嬰兒助孕結(jié)局的影響[J];蚌埠醫(yī)學(xué)院學(xué)報(bào);2013年05期

2 李廣裕;梁季鴻;梁世坤;韋國(guó)強(qiáng);宋衛(wèi)儒;張訊;朱春暉;;金水寶膠囊治療免疫性不育患者臨床觀察[J];湖南中醫(yī)藥大學(xué)學(xué)報(bào);2011年12期

3 李世葵;杜賢;鄭利平;;精漿微量元素含量與精液異常的相關(guān)性分析[J];臨床輸血與檢驗(yàn);2012年02期

4 蔣歡歡;賀小進(jìn);宋兵;曹云霞;;精子染色質(zhì)完整性對(duì)IVF/ICSI臨床結(jié)局的預(yù)測(cè)價(jià)值[J];中華男科學(xué)雜志;2011年12期

5 丁之德;;男性不育基礎(chǔ)研究的現(xiàn)狀及可能性途徑[J];中國(guó)男科學(xué)雜志;2010年03期

6 潘鋒;徐志鵬;石亮;何增;龔曹科;戴玉田;潘連軍;;精液常規(guī)參數(shù)初檢合格后不同時(shí)間點(diǎn)再分析結(jié)果及其與精子DNA損傷的關(guān)系[J];中華男科學(xué)雜志;2013年01期

7 鄧曉俊;郎根強(qiáng);章益峰;褚健;莊劍秋;曹建偉;;精索靜脈曲張程度與精液質(zhì)量的相關(guān)性研究[J];海軍醫(yī)學(xué)雜志;2013年01期

8 蘇春霞;王凌;白瑞萍;王耀榮;;精液結(jié)果異常與疾病關(guān)系的分析[J];內(nèi)蒙古醫(yī)學(xué)雜志;2013年04期

9 冷慧敏;周亮;錢衛(wèi)平;;胰島素樣生長(zhǎng)因子-2基因印跡模式異常與男性不育的關(guān)系[J];生殖與避孕;2012年04期

10 鄭九嘉;樓哲豐;鄭蔚虹;金建遠(yuǎn);倪吳花;李平;金龍金;;線粒體呼吸功能與精子活力、核DNA損傷的相關(guān)性分析[J];中國(guó)細(xì)胞生物學(xué)學(xué)報(bào);2012年01期

相關(guān)碩士學(xué)位論文 前2條

1 李世佳;ELSPBP1基因及Lyzl基因家族與生殖關(guān)系的初步研究[D];寧夏醫(yī)科大學(xué);2013年

2 楊超偉;中藥方劑—五子衍宗丸對(duì)腎虛精虧證大鼠睪丸支持細(xì)胞分泌功能的影響[D];北京中醫(yī)藥大學(xué);2014年

本文編號(hào)：1777138