模拟高原缺氧大鼠肠上皮细胞损伤自噬调控机制的研究

发布时间：2018-02-12 20:38

本文关键词： 自噬缺氧 3-甲基腺嘌呤肠上皮细胞 PI3K/Akt信号通路　出处：《甘肃中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探讨模拟高原缺氧大鼠肠上皮细胞自噬变化情况及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路调控自噬的机制,为进一步研究靶向防治高原相关肠道疾病提供新的理论依据。方法:以大鼠小肠隐窝上皮细胞(IEC-6)为体外实验对象,应用三气培养箱模拟高原缺氧(5%O2)构建IEC-6细胞缺氧损伤模型为实验组(Hypoxia),以正常含氧培养箱中的IEC-6细胞作为对照组(Control):(1)MTT法检测常氧条件下不同浓度的3-MA以及缺氧条件加入筛选出浓度的3-MA(3-MA+Hypoxia)对IEC-6细胞6 h、12 h、24 h、48 h各时间点的毒性作用,以及在对IEC-6细胞6 h、12 h、24 h、48 h各时间点的细胞存活率;(2)应用单丹磺酰戊二胺(MDC)染色法、透射电镜及蛋白免疫印迹(Western blot)法分别检测缺氧处理IEC-6细胞6 h、12 h、24 h、48 h各时间点自噬体和或自噬溶酶体点状荧光颗粒、自噬体双层膜结构及自噬相关蛋白LC3和Beclin-1的表达变化;(3)选择缺氧自噬最明显时间点加入自噬抑制剂3-MA后,用荧光显微镜观察MDC染色的自噬体和或自噬溶酶体点状荧光颗粒,透射电镜观察自噬体双层膜结构,Western blot法检测自噬相关蛋白LC3和Beclin-1以及PI3K/Akt信号通路相关蛋白Akt和磷酸化Akt(p-Akt)的表达变化。结果:(1)3-MA对IEC-6细胞毒性的MTT结果显示:随着3-MA浓度的增加及作用时间的延长,它对IEC-6细胞增殖的抑制率逐渐增强,呈现时间依赖性和浓度依赖性。当3-MA浓度为≤5mmol/L时,对IEC-6细胞增殖的抑制率及药物毒性较小,选择5mmol/L为3-MA的实验浓度;(2)缺氧对IEC-6细胞增殖的MTT结果:6 h、12 h细胞存活率出现了下降,但差异无统计学意义(P0.05),处理24 h、48 h后细胞存活率明显下降,差异有统计学意义(P0.05)。在缺氧加入自噬抑制剂3-MA同时培养后,从处理12h开始细胞存活率较单纯缺氧组显著下降,24 h、48 h下降更明显,差异有统计学意义(P0.05);(3)MDC染色结果显示:常氧组细胞内未见明显自噬体点状荧光颗粒,从缺氧6 h起细胞内开始出现点状荧光颗粒,细胞内点状荧光颗粒大多分部细胞核周围,在24 h时自噬点状荧光颗粒达到顶峰,作用48 h后细胞总数及细胞内点状荧光颗粒开始下降;(4)透射电镜结果显示:缺氧6 h组开始出现双层膜结构的自噬体,随着缺氧时间的延长,双膜自噬体的数量逐渐增加,其中以缺氧24 h组自噬体增多最为明显;缺氧48 h组细胞内仍可见大量自噬体囊泡,但细胞结构破坏,细胞核及细胞膜不程度碎裂;(5)Western blot法结果显示:缺氧6 h、12 h、24 h、48 h组自噬相关蛋白LC3及Beclin-1表达均显著高于常氧组,48 h后表达有所下降,差异有统计学意义(P0.05);(6)缺氧加入自噬抑制剂3-MA后,透射电镜及MDC染色结果均表明,IEC-6细胞核周围的自噬体较缺氧组显著减少;Western blot法检测结果显示,自噬相关蛋白LC3及Beclin-1表达较单纯缺氧组明显减少,差异有统计学意义(P0.05);单纯缺氧组细胞p-Akt蛋白表达水平明显高于常氧组,差异有统计学意义(P0.05),使用自噬抑制剂3-MA后,p-Akt蛋白表达量显著下降,差异有统计学意义(P0.05);Akt在缺氧IEC-6细胞损伤后无明显变化,差异无统计学意义(P0.05)。结论:(1)模拟高原缺氧IEC-6细胞损伤时可诱导自噬发生;(2)PI3K/Akt信号通路可能对自噬有调节作用。
[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.

【学位授予单位】：甘肃中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574

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