氧自由基对大鼠小肠上皮细胞的损伤及硫辛酸的干预研究

发布时间：2018-03-29 00:01

本文选题：大鼠　切入点：肠上皮细胞　出处：《上海交通大学》2011年硕士论文

【摘要】：目的:本试验通过体外培养大鼠小肠上皮细胞(IEC),建立黄嘌呤氧化酶/黄嘌呤(XO/X)和过氧化氢(H2O2)对IEC氧化损伤模型,研究不同氧自由基对小肠上皮细胞损伤与功能的关系,以及硫辛酸(LA)对XO/X与H2O2氧化胁迫下IEC的干预作用。方法:实验一:对原代培养大鼠IEC的分离方法做出改进与简化:联合运用300 U/mL胶原酶Ⅺ和100 mg/ml中性蛋白酶I分离新生大鼠小肠,可得到完整隐窝单位和少数单个小肠上皮细胞,在5%FBS-DMEM/F12生长培养基中,3-4d形成细胞集落,7-8d细胞汇合需要传代,用0.05% Trypsin-EDTA消化细胞,传代后细胞增殖能力较强,运用刮除法和相差消化相差贴壁法进行纯化,经免疫组化法鉴定能够得到纯度80%以上的小肠上皮细胞,为研究营养素对肠上皮细胞的作用提供了理想的体外模型。实验二:XO/X与H2O2损伤IEC模型的建立:以不同浓度的XO/X( 5、10、50、100、200U/L XO和10μmol/L X)和不同浓度的H2O2 (0.1μmol/L、1μmol/L、2.5μmol/L、10μmol/L、30μmol/L、100μmol/L和1mmol/LH2O2),分别作用IEC细胞3、12、24、48h,以四唑盐(MTT)比色法检测细胞活力氧化,确定XO/X和H2O2损伤模型适宜损伤浓度和损伤时间。实验三:XO/X和H2O2氧化胁迫模型下大鼠IEC损伤与修复及LA的干预作用:XO/X模型下,空白对照组:细胞正常培养24h; XO/X损伤组:细胞中加入50U/L XO,10μmol/L X后,培养24 h; XO/X+LA药物组:细胞与不同浓度的LA(0.lμg/ml、1μg/ml和10μg/ml)预孵3 h,再加入50U/L XO培养24h。H2O2模型下:空白对照组:细胞正常培养24h; H2O2损伤组:加入100μmol/L H2O2后,培养24 h; H2O2+LA药物组:细胞与不同浓度的LA(0.lμg/ml、1μg/ml和10μg/ml)预孵3 h,再加入100μmol/L H2O2培养24 h,分别测定LA对各组细胞活力,IEC抗氧化指标(SOD、GSH-pX和MDA)、功能酶(脂肪酶、淀粉酶)、肠道损伤特异性指标(DAO和LDH)的影响。结果:1. XO/X损伤IEC模型的适宜浓度为50U/L XO和10μmol/L X,适宜培养时间为24h; H_2O_2损伤IEC模型的适宜浓度为100μmol/ml H_2O_2,适宜培养时间为24h。 2. XO/X(50U/L XO/10μmol/L X)与H_2O_2(100μmol/ml)氧化胁迫下的IEC,细胞活力显著下降(P0.05),IEC的SOD、GSH-pX、脂肪酶、淀粉酶、二胺氧化酶活性显著下降(P0.05),细胞培养液中MDA含量显著上升(P0.05);且50U/L XO对IEC造成的氧化损伤相较100umol/ml的H_2O_2严重。 3. LA可以促进IEC的增殖,显著提高XO/X与H_2O_2两种模型氧化胁迫下IEC的SOD、GSH-pX活性,降低细胞培养液中MDA的含量(P0.05);同时LA提高了氧化胁迫下IEC分泌的淀粉酶(P0.05)和脂肪酶(P0.05)的活性;显著降低了氧化胁迫下IEC培养液中LDH的活性,提高了IEC裂解液和培养液中DAO的活性。
[Abstract]:Objective: to establish a model of IEC oxidative damage induced by xanthine oxidase / xanthine XO / XO / XO / H 2O 2 by cultured rat intestinal epithelial cells in vitro, and to study the relationship between injury and function of different oxygen free radicals on intestinal epithelial cells. Intervention of XO/X and H2O2 on IEC under oxidative stress. Methods: experiment 1: to improve and simplify the isolation method of primary cultured rat IEC: to separate the small intestine of newborn rats by using 300 U/mL collagenase XI and 100 mg/ml neutral protease I, respectively. The complete crypt units and a few single small intestinal epithelial cells were obtained. In the 5S-DMEM / F12 growth medium for 3 to 4 days, the colony forming cells needed to be subcultured for 7-8 days. The cells were digested with 0.05% Trypsin-EDTA, and the proliferation ability of the cells was stronger after passage. The method of scraping and phase contrast digestion was used to purify the intestinal epithelial cells. The purity of intestinal epithelial cells was over 80% by immunohistochemical method, which provided an ideal model for the study of the effect of nutrients on intestinal epithelial cells in vitro. Experiment 2: the IEC model of H2O2 damage induced by 10 渭 mol / L X / X and H2O2: using different concentrations of XO / X (5101050100U / L XO and 10 渭 mol / L XX) and different concentrations of H2O2 0.1 渭 mol / L ~ (-1) 渭 mol / L ~ (-1) 渭 mol / L ~ (10) 渭 mol / L ~ (10) 渭 mol / L ~ (10) 渭 mol / L ~ (30) 渭 mol / L ~ (100) mol/L and ~ (1) mol / L ~ (H _ 2O _ (2)) respectively, the cells were oxidized at 3122448 h by MTT colorimetry. The suitable damage concentration and time for XO/X and H2O2 damage models were determined. Experimental 3: XO / X and H2O2 oxidative stress Model Rats IEC damage and repair and the intervention of LA in the Model: control group: normal culture for 24 h, XO/X injury group: after adding 50U/L XO 10 渭 mol/L X to the cells, the control group was treated with 10 渭 mol/L X of 50U/L XO / X, and the control group was treated with 10 渭 mol/L X of 50U/L XO / X for 24 hours. Cultured for 24 h; XO/X LA drug group: cells were preincubated with LA(0.l 渭 g / ml 1 渭 g/ml and 10 渭 g / ml for 3 h, then added to 50U/L XO culture 24h.H2O2 model: blank control group: normal culture for 24 h, H2O2 injury group: after adding 100 渭 mol/L H2O2, The cells were preincubated with different concentrations of LA(0.l 渭 g / ml 1 渭 g/ml and 10 渭 g / ml for 3 h and then added 100 渭 mol/L H2O2 for 24 h. The effects of amylase, the specific index of intestinal injury, Dao and LDH. Results 1. The suitable concentration of XO/X damage IEC model was 50U/L XO and 10 渭 mol/L X, the suitable culture time was 24 h, and the suitable concentration of H_2O_2 injury IEC model was 100 渭 mol/ml H 2O 2 and the suitable culture time was 24 h. 2. Under oxidative stress of XO/X(50U/L XO/10 渭 mol/L X and H_2O_2(100 渭 mol / ml, the activity of IECs decreased significantly. The activity of diamine oxidase decreased significantly, and the content of MDA in cell culture medium increased significantly, and the oxidative damage of 50U/L XO to IEC was more serious than that of 100umol/ml H_2O_2. 3. La could promote the proliferation of IEC, increase the activity of SODH-pX in IEC under oxidative stress of XO/X and H_2O_2, decrease the content of MDA in cell culture medium (P0.05), and increase the activity of amylase (P0.05) and lipase (P0.05) secreted by IEC under oxidative stress. The activity of LDH in IEC medium was significantly decreased under oxidative stress, and the activity of DAO in IEC lysate and culture medium was increased.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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