本文關(guān)鍵詞： 自噬 缺氧 3-甲基腺嘌呤 腸上皮細(xì)胞 PI3K/Akt信號通路　出處：《甘肅中醫(yī)藥大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討模擬高原缺氧大鼠腸上皮細(xì)胞自噬變化情況及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信號通路調(diào)控自噬的機制,為進(jìn)一步研究靶向防治高原相關(guān)腸道疾病提供新的理論依據(jù)。方法:以大鼠小腸隱窩上皮細(xì)胞(IEC-6)為體外實驗對象,應(yīng)用三氣培養(yǎng)箱模擬高原缺氧(5%O2)構(gòu)建IEC-6細(xì)胞缺氧損傷模型為實驗組(Hypoxia),以正常含氧培養(yǎng)箱中的IEC-6細(xì)胞作為對照組(Control):(1)MTT法檢測常氧條件下不同濃度的3-MA以及缺氧條件加入篩選出濃度的3-MA(3-MA+Hypoxia)對IEC-6細(xì)胞6 h、12 h、24 h、48 h各時間點的毒性作用,以及在對IEC-6細(xì)胞6 h、12 h、24 h、48 h各時間點的細(xì)胞存活率;(2)應(yīng)用單丹磺酰戊二胺(MDC)染色法、透射電鏡及蛋白免疫印跡(Western blot)法分別檢測缺氧處理IEC-6細(xì)胞6 h、12 h、24 h、48 h各時間點自噬體和或自噬溶酶體點狀熒光顆粒、自噬體雙層膜結(jié)構(gòu)及自噬相關(guān)蛋白LC3和Beclin-1的表達(dá)變化;(3)選擇缺氧自噬最明顯時間點加入自噬抑制劑3-MA后,用熒光顯微鏡觀察MDC染色的自噬體和或自噬溶酶體點狀熒光顆粒,透射電鏡觀察自噬體雙層膜結(jié)構(gòu),Western blot法檢測自噬相關(guān)蛋白LC3和Beclin-1以及PI3K/Akt信號通路相關(guān)蛋白Akt和磷酸化Akt(p-Akt)的表達(dá)變化。結(jié)果:(1)3-MA對IEC-6細(xì)胞毒性的MTT結(jié)果顯示:隨著3-MA濃度的增加及作用時間的延長,它對IEC-6細(xì)胞增殖的抑制率逐漸增強,呈現(xiàn)時間依賴性和濃度依賴性。當(dāng)3-MA濃度為≤5mmol/L時,對IEC-6細(xì)胞增殖的抑制率及藥物毒性較小,選擇5mmol/L為3-MA的實驗濃度;(2)缺氧對IEC-6細(xì)胞增殖的MTT結(jié)果:6 h、12 h細(xì)胞存活率出現(xiàn)了下降,但差異無統(tǒng)計學(xué)意義(P0.05),處理24 h、48 h后細(xì)胞存活率明顯下降,差異有統(tǒng)計學(xué)意義(P0.05)。在缺氧加入自噬抑制劑3-MA同時培養(yǎng)后,從處理12h開始細(xì)胞存活率較單純?nèi)毖踅M顯著下降,24 h、48 h下降更明顯,差異有統(tǒng)計學(xué)意義(P0.05);(3)MDC染色結(jié)果顯示:常氧組細(xì)胞內(nèi)未見明顯自噬體點狀熒光顆粒,從缺氧6 h起細(xì)胞內(nèi)開始出現(xiàn)點狀熒光顆粒,細(xì)胞內(nèi)點狀熒光顆粒大多分部細(xì)胞核周圍,在24 h時自噬點狀熒光顆粒達(dá)到頂峰,作用48 h后細(xì)胞總數(shù)及細(xì)胞內(nèi)點狀熒光顆粒開始下降;(4)透射電鏡結(jié)果顯示:缺氧6 h組開始出現(xiàn)雙層膜結(jié)構(gòu)的自噬體,隨著缺氧時間的延長,雙膜自噬體的數(shù)量逐漸增加,其中以缺氧24 h組自噬體增多最為明顯;缺氧48 h組細(xì)胞內(nèi)仍可見大量自噬體囊泡,但細(xì)胞結(jié)構(gòu)破壞,細(xì)胞核及細(xì)胞膜不程度碎裂;(5)Western blot法結(jié)果顯示:缺氧6 h、12 h、24 h、48 h組自噬相關(guān)蛋白LC3及Beclin-1表達(dá)均顯著高于常氧組,48 h后表達(dá)有所下降,差異有統(tǒng)計學(xué)意義(P0.05);(6)缺氧加入自噬抑制劑3-MA后,透射電鏡及MDC染色結(jié)果均表明,IEC-6細(xì)胞核周圍的自噬體較缺氧組顯著減少;Western blot法檢測結(jié)果顯示,自噬相關(guān)蛋白LC3及Beclin-1表達(dá)較單純?nèi)毖踅M明顯減少,差異有統(tǒng)計學(xué)意義(P0.05);單純?nèi)毖踅M細(xì)胞p-Akt蛋白表達(dá)水平明顯高于常氧組,差異有統(tǒng)計學(xué)意義(P0.05),使用自噬抑制劑3-MA后,p-Akt蛋白表達(dá)量顯著下降,差異有統(tǒng)計學(xué)意義(P0.05);Akt在缺氧IEC-6細(xì)胞損傷后無明顯變化,差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:(1)模擬高原缺氧IEC-6細(xì)胞損傷時可誘導(dǎo)自噬發(fā)生;(2)PI3K/Akt信號通路可能對自噬有調(diào)節(jié)作用。
[Abstract]:Objective: To study the simulation of autophagy plateau hypoxia rat intestinal epithelial cells and changes of phosphatidylinositol 3- kinase (PI3K) / protein kinase B (Akt) signal pathway regulation mechanism of autophagy, provide a new theoretical basis for the further study of targeted prevention and treatment of high altitude related intestinal diseases. Methods: rat intestinal crypt epithelial cells (IEC-6) in vitro experiment object, application of three gas incubator simulated high altitude hypoxia (5%O2) to construct IEC-6 cell hypoxia model for the experimental group (Hypoxia), with normal oxygen incubator in IEC-6 cells as the control group (Control): (1) different concentrations of 3-MA and hypoxia MTT assay under normoxic conditions join the selected concentration of 3-MA (3-MA+Hypoxia) on IEC-6 cells in 6 h, 12 h, 24 h, 48 h toxicity at each time point, and in IEC-6 cells for 6 h, 12 h, 24 h, 48 h each time point, the cell survival rate; (2) using single dansyl amyl two amine (MDC) Staining, transmission electron microscopy and Western blotting (Western blot) were detected in hypoxia treated IEC-6 cells for 6 h, 12 h, 24 h, 48 h each time point of autophagosome and autolysosome or punctate fluorescent particles, autophagic double membrane structure and autophagy related protein LC3 and Beclin-1 expression (3); the most obvious choice of hypoxic time points adding autophagy autophagy inhibitors 3-MA, MDC staining and observation of autophagosomes or autolysosome punctate fluorescent particles by fluorescence microscopy, observe autophagic double membrane structure TEM, Western blot method was used to detect autophagy related protein LC3 and Beclin-1 and PI3K/Akt signaling pathway related protein Akt and phosphorylated Akt (p-Akt) the expression of 3-MA. Results: (1) the cytotoxicity of IEC-6 MTT results showed that: with the increase of 3-MA concentration and treatment time, it inhibited the proliferation of IEC-6 cells was gradually increased in a time dependent manner And concentration dependent. When the 3-MA concentration is less than 5mmol/L, the inhibition rate and low toxicity on the proliferation of IEC-6 cells, 5mmol/L is selected as the experimental concentration of 3-MA; (2) hypoxia on the proliferation of IEC-6 cells MTT results: 6 h, 12 h cell survival rate decreased, but the difference was not statistically significant (P0.05), 24 h, 48 h after the cell survival rate decreased significantly, the difference was statistically significant (P0.05). At the same time adding autophagy inhibitor 3-MA in hypoxia cultured from 12h start cell survival was significantly decreased in hypoxia group, 24 h, 48 h decreased more significantly, the difference was statistically significant (P0.05); (3) MDC staining showed: normoxic cells had no obvious autophagosomes punctate fluorescent particles from hypoxia 6 h cells appeared punctate fluorescent particles around intracellular punctate fluorescence particles are 24 h in the division of the nucleus, punctate fluorescent particles reach the peak of autophagy, as Drop by 48 h after the total number of cells and intracellular punctate fluorescent particles; (4) transmission electron microscopy showed that autophagy hypoxia 6 h group began to appear double membrane structure, with the duration of hypoxia, the number of double membrane autophagosomes increased gradually, among which 24 h hypoxia group autophagosomes increased obviously; hypoxia 48 h group cells still showed a large number of autophagosomes, but the damage of cell nucleus and cell membrane, degree of fragmentation; (5) Western blot results showed that: hypoxia 6 h, 12 h, 24 h, 48 h group of autophagy related protein LC3 and Beclin-1 expression were significantly higher than that in normoxia group. 48 the expression of H decreased, the difference was statistically significant (P0.05); (6) adding autophagy inhibitor 3-MA after hypoxia, transmission electron microscope and MDC staining showed that autophagy IEC-6 around the nucleus was significantly higher than that of hypoxia group reduced; the detection result of Western blot showed that autophagy related protein LC3 and Becli N-1 expression in hypoxia group was significantly reduced, the difference was statistically significant (P0.05); hypoxia group, the protein expressions of p-Akt were significantly higher than normal oxygen group, the difference was statistically significant (P0.05), the use of autophagy inhibitor 3-MA, p-Akt protein expression was significantly decreased, the difference was statistically significant (P0.05); no significant Akt changes in hypoxic IEC-6 cells after injury, there was no statistically significant difference (P0.05). Conclusion: (1) to induce autophagy in IEC-6 cells can be simulated high altitude hypoxia injury; (2) the PI3K/Akt signaling pathway may have a regulatory role for autophagy.

【學(xué)位授予單位】：甘肅中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R574

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王彬彬;武承鳳;張方信;馬強;;自噬介導(dǎo)腫瘤多藥耐藥的研究進(jìn)展[J];中國腫瘤臨床;2015年08期

2 王路喬;程曉曙;黃茶花;黃波;梁茜;;Rapamycin Protects Cardiomyocytes against Anoxia/Reoxygenation Injury by Inducing Autophagy through the PI3k/Akt Pathway[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2015年01期

3 向鏡芬;楊祥;龔劍鋒;雷偉健;鄧艷瓊;牟丹;鐘國權(quán);孟啟勇;;PI3K/Akt信號通路在膿毒癥腎臟損傷誘導(dǎo)的自噬中的調(diào)節(jié)作用[J];中國病理生理雜志;2014年06期

4 Xiao-Zhong Huang;Li-Bin Zhu;Zhong-Rong Li;Jing Lin;;Bacterial colonization and intestinal mucosal barrier development[J];World Journal of Clinical Pediatrics;2013年04期

5 李素芝;楊定周;鄭必海;劉厚東;陳有;吳前進(jìn);陳薇薇;;海拔5374m高原實地急性缺氧暴露下家兔腸黏膜屏障功能損傷觀察[J];華南國防醫(yī)學(xué)雜志;2011年04期

6 ;Over-starvation aggravates intestinal injury and promotes bacterial and endotoxin translocation under high-altitude hypoxic environment[J];World Journal of Gastroenterology;2011年12期

7 吳文明;張方信;張盼;鄧芝云;楊文翠;馬強;陳嘉嶼;康生朝;楊玉捷;;高原缺氧條件對大鼠腸黏膜組織及缺氧誘導(dǎo)因子-1α、誘導(dǎo)型一氧化氮合酶表達(dá)的影響[J];解放軍醫(yī)學(xué)雜志;2010年05期

8 張方信;吳文明;鄧芝云;楊文翠;于曉輝;汪泳;吳漢平;;谷氨酰胺對高原缺氧早期條件下大鼠腸黏膜組織形態(tài)學(xué)及誘導(dǎo)型一氧化氮合酶表達(dá)的影響[J];中華消化雜志;2010年03期

9 費洪榮;王鳳澤;;PI3K/Akt信號通路的調(diào)控與腫瘤血管生成[J];生命的化學(xué);2010年01期

10 周波;楊定周;周其全;;模擬高原低氧環(huán)境暴露下家兔小腸黏膜掃描電鏡觀察[J];胃腸病學(xué)和肝病學(xué)雜志;2009年08期

，

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