本文選題：大鼠　切入點：腸上皮細胞　出處：《上海交通大學》2011年碩士論文

【摘要】：目的:本試驗通過體外培養(yǎng)大鼠小腸上皮細胞(IEC),建立黃嘌呤氧化酶/黃嘌呤(XO/X)和過氧化氫(H2O2)對IEC氧化損傷模型,研究不同氧自由基對小腸上皮細胞損傷與功能的關系,以及硫辛酸(LA)對XO/X與H2O2氧化脅迫下IEC的干預作 用。方法:實驗一:對原代培養(yǎng)大鼠IEC的分離方法做出改進與簡化:聯(lián)合運用300 U/mL膠原酶Ⅺ和100 mg/ml中性蛋白酶I分離新生大鼠小腸,可得到完整隱窩單位和少數(shù)單個小腸上皮細胞,在5%FBS-DMEM/F12生長培養(yǎng)基中,3-4d形成細胞集落,7-8d細胞匯合需要傳代,用0.05% Trypsin-EDTA消化細胞,傳代后細胞增殖能力較強,運用刮除法和相差消化相差貼壁法進行純化,經免疫組化法鑒定能夠得到純度80%以上的小腸上皮細胞,為研究營養(yǎng)素對腸上皮細胞的作用提供了理想的體外模型。 實驗二:XO/X與H2O2損傷IEC模型的建立:以不同濃度的XO/X( 5、10、50、100、200U/L XO和10μmol/L X)和不同濃度的H2O2 (0.1μmol/L、1μmol/L、2.5μmol/L、10μmol/L、30μmol/L、100μmol/L和1mmol/LH2O2),分別作用IEC細胞3、12、24、48h,以四唑鹽(MTT)比色法檢測細胞活力氧化,確定XO/X和H2O2損傷模型適宜損傷濃度和損傷時間。 實驗三:XO/X和H2O2氧化脅迫模型下大鼠IEC損傷與修復及LA的干預作用:XO/X模型下,空白對照組:細胞正常培養(yǎng)24h; XO/X損傷組:細胞中加入50U/L XO,10μmol/L X后,培養(yǎng)24 h; XO/X+LA藥物組:細胞與不同濃度的LA(0.lμg/ml、1μg/ml和10μg/ml)預孵3 h,再加入50U/L XO培養(yǎng)24h。H2O2模型下:空白對照組:細胞正常培養(yǎng)24h; H2O2損傷組:加入100μmol/L H2O2后,培養(yǎng)24 h; H2O2+LA藥物組:細胞與不同濃度的LA(0.lμg/ml、1μg/ml和10μg/ml)預孵3 h,再加入100μmol/L H2O2培養(yǎng)24 h,分別測定LA對各組細胞活力,IEC抗氧化指標(SOD、GSH-pX和MDA)、功能酶(脂肪酶、淀粉酶)、腸道損傷特異性指標(DAO和LDH)的影響。 結果:1. XO/X損傷IEC模型的適宜濃度為50U/L XO和10μmol/L X,適宜培養(yǎng)時間為24h; H_2O_2損傷IEC模型的適宜濃度為100μmol/ml H_2O_2,適宜培養(yǎng)時間為24h。 2. XO/X(50U/L XO/10μmol/L X)與H_2O_2(100μmol/ml)氧化脅迫下的IEC,細胞活力顯著下降(P0.05),IEC的SOD、GSH-pX、脂肪酶、淀粉酶、二胺氧化酶活性顯著下降(P0.05),細胞培養(yǎng)液中MDA含量顯著上升(P0.05);且50U/L XO對IEC造成的氧化損傷相較100umol/ml的H_2O_2嚴重。 3. LA可以促進IEC的增殖,顯著提高XO/X與H_2O_2兩種模型氧化脅迫下IEC的SOD、GSH-pX活性,降低細胞培養(yǎng)液中MDA的含量(P0.05);同時LA提高了氧化脅迫下IEC分泌的淀粉酶(P0.05)和脂肪酶(P0.05)的活性;顯著降低了氧化脅迫下IEC培養(yǎng)液中LDH的活性,提高了IEC裂解液和培養(yǎng)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.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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