多肽淀粉样纤维与细胞膜之间的相互作用

发布时间：2018-01-15 15:22

本文关键词：多肽淀粉样纤维与细胞膜之间的相互作用　出处：《陕西师范大学》2008年硕士论文　论文类型：学位论文

【摘要】： 蛋白质淀粉样纤维是造成多种人类疾病的因素之一,如Alzheimer's disease(AD)是由神经元中β淀粉样肽的沉积所致。目前已知淀粉样纤维具有细胞毒性,可导致人红细胞的溶血,但具体作用机制不清。为进一步了解淀粉样纤维的细胞毒性作用,探究其作用机制,本实验以红细胞作为细胞膜模型,研究了胰岛素淀粉样纤维及β淀粉样肽的一段特征性序列Aβ_(25-35)与红细胞之间的相互作用,分析淀粉样纤维引起的细胞溶血,对细胞形态,细胞膜骨架,以及细胞膜流动性的影响;以Aβ_(25-35)对红细胞的损伤为模型,研究了一定剂量γ射线的照射对此损伤作用的影响。实验的主要方法和结果: 一、胰岛素淀粉样纤维对红细胞的影响 1.胰岛素淀粉样纤维对红细胞渗透脆性的影响将一定量的胰岛素纤维作用后的红细胞置于渗透压梯度的缓冲溶液中,采用分光光度计检测溶血率,并绘制红细胞膜渗透脆性曲线,发现胰岛素淀粉样纤维改变了细胞膜的渗透脆性。 2.胰岛素纤维在孵育过程中巯基暴露趋势与其细胞毒性的关系研究在胰岛素淀粉样纤维的孵育过程中的不同时段取样,同时检测其溶血效果和巯基暴露的程度。结果发现胰岛素纤维孵育3小时后溶血效应开始增强,4.5至7.5小时之间溶血效应最强,之后开始下降,孵育9小时后溶血效果不再随时间变化。比较溶血与巯基暴露的时间曲线发现,胰岛素巯基开始暴露时的纤维其溶血效果最强,但之后巯基继续暴露并不能增加其溶血效应。二、Aβ淀粉样肽对红细胞的影响 1.Aβ_(25-35)的溶血效应红细胞经孵育不同时间后的Aβ_(25-35)处理后,检测溶血率变化趋势,结果显示在前40分钟随着Aβ_(25-35)孵育时间延长,溶血率上升;在40分钟后则下降。红细胞经不同终浓度Aβ_(25-35)处理后检测溶血率,发现溶血率随着的Aβ_(25-35)浓度上升而提高。 2.Aβ_(25-35)对红细胞膜形态及膜骨架蛋白的影响红细胞经一定浓度的Aβ_(25-35)处理后,于扫描电镜下观察,可见红细胞发生轻微聚集,并有部分红细胞出现棘突,细胞形态发生改变。提取红细胞血影并用一定浓度的Aβ_(25-35)处理后,使用原子力显微镜观察。发现与对照组相比,Aβ_(25-35)作用后的红细胞血影,规则的网格状结构丢失,表明膜表面骨架蛋白的分布受到影响。 3.Aβ_(25-35)对红细胞流动性的影响使用DPH作为荧光探针,插入细胞膜的磷脂双层并稳定后,通过探针的各相异性值检测细胞膜的流动性变化。发现Aβ_(25-35)处理过后的红细胞与对照组细胞膜的流动性并无明显差别。结果显示,Aβ_(25-35)对细胞膜的损伤不是膜脂流动性的变化所引发。三、γ射线对Aβ_(25-35)诱导的红细胞损伤的影响本研究应用两种不同剂量(50 Gy,100 Gy)的γ射线,对Aβ_(25-35)与红细胞的相互作用进行了研究,结果发现,两种剂量的γ射线照射均对Aβ_(25-35)导致的红细胞损伤具有保护作用。 1.溶血率测定当Aβ_(25-35)作用于经不同剂量γ射线照射后的红细胞时,溶血效应与未经照射的红细胞相比明显减弱,说明γ射线对Aβ_(25-35)诱导溶血作用具有抑制作用。 2.红细胞的形态变化 γ射线照射后的红细胞经Aβ_(25-35)处理后,仍维持良好的细胞形态,呈正常的双凹圆盘形,表明γ射线有助于维持红细胞的正常形态。 3.红细胞渗透脆性的变化将γ射线照射后的红细胞置于渗透压改变的梯度缓冲液中,采用分光光度计检测溶血率发现,γ射线照射后,红细胞对低渗溶液的耐受性有所提高。 4.红细胞膜脂流动性的改变 γ射线照射后的红细胞加入一定剂量的Aβ_(25-35)后孵育,使用DPH作为荧光探针,检测膜流动性。实验发现,γ射线照射后红细胞的膜流动性增强。结论:胰岛素纤维与Aβ_(25-35)均会对红细胞膜造成损伤,导致细胞溶血。在一定时间内,淀粉样纤维随着孵育时间的延长,溶血作用也得到增强,之后则呈现减弱,说明两种淀粉样纤维的细胞毒性作用均与其结构变化有密切关系,并且Aβ_(25-35)更易形成淀粉样纤维。胰岛素纤孵育过程中观察到自由巯基的暴露,胰岛素纤维的自由巯基能够和膜骨架相关蛋白的巯基交换,这可能与其溶血作用相关;Aβ_(25-35)导致了膜下骨架蛋白的变化,而磷脂层的流动性则不受影响,表明Aβ_(25-35)对红细胞的损伤涉及到Aβ_(25-35)与膜下骨架蛋白的相互作用。在本文的实验条件下,γ射线对Aβ_(25-35)诱导的红细胞损伤具有保护作用,并且增加了细胞膜的流动性。
[Abstract]:Amyloid protein fiber is one of the factors causing many human diseases, such as Alzheimer's disease (AD) is composed of neurons in beta amyloid deposition induced by amyloid fibrils. Currently known toxicity, can cause hemolysis of human red blood cells, but the mechanism is not clear. In order to further understand the cytotoxicity of amyloid fibrils the exploration of the mechanism, the experiment on red blood cells as a cell membrane model of insulin amyloid fibrils and amyloid beta peptide a characteristic sequence of A beta _ (25-35) and the interaction between the red blood cells, analysis of amyloid fibrils caused by hemolysis, on cell morphology, cell membrane the skeleton, the impact of liquidity and cell membrane; with A beta _ (25-35) on red blood cell injury model, studied the effect of a certain dose of gamma ray irradiation on this injury.
The main methods and results of the experiment are:
The effect of insulin amyloid fiber on red blood cells
The effect of 1. insulin amyloid fiber on osmotic fragility of red blood cells
A certain amount of insulin after the action of red blood cells was placed in osmotic pressure gradient buffer solution. The hemolysis rate was detected by spectrophotometer and the osmotic fragility curve of erythrocyte membrane was drawn. It was found that insulin amyloid fiber changed the osmotic fragility of cell membrane.
Study on the relationship between the exposure trend of sulfhydryl group and its cytotoxicity during the incubation of 2. insulin fibers
In the insulin amyloid fibrils were incubated for different periods in the sampling process, and detect its hemolytic effect and sulfhydryl exposure. Results showed that insulin fibrils after incubation for 3 hours hemolytic effect began to increase from 4.5 to 7.5 hours between the strongest hemolytic effect, then started to decline, incubation effect of hemolysis after 9 hours incubation no change over time time curve of hemolysis and sulfhydryl exposure showed that insulin began when exposed to the thiol fiber hemolytic effect is strongest, but after continued exposure and thiol cannot increase its hemolytic effect.
Two, the effect of A beta amyloid peptide on red blood cells
1.A (25-35) _ beta hemolytic effect
A beta _ red cells after incubated for different time (25-35) after treatment, detection of hemolytic rate trends, results show that in the first 40 minutes with A beta _ (25-35) incubation time prolonged, the hemolysis rate increased; in 40 minutes decreased. Different concentrations of A beta _ by red blood cells (25-35 the detection rate of hemolysis) after treatment, found that the hemolysis rate of A beta _ (25-35) with the concentration increased.
2.A beta _ (25-35) influence on the morphology and membrane cytoskeleton protein
A beta _ concentration of red blood cells (25-35) treated by scanning electron microscope, visible red blood cells had mild aggregation, and some red cell spinous process, changes in cell morphology. The extraction of erythrocyte ghosts and A beta _ with certain concentration (25-35) after the treatment, the use of atomic microscope. Found. Compared with the control group, A _ beta (25-35) erythrocyte ghosts after the loss of grid structure rules, show that the distribution of membrane surface skeleton protein was affected.
3.A beta _ (25-35) effects on red cell mobility
Using DPH as a fluorescent probe is inserted into the cell membrane phospholipid bilayer and is stable, the liquidity of the anisotropic value detection of cell membrane probes. A beta _ (25-35) after the treatment of red blood cells with the control group, cell membrane fluidity and there is no obvious difference. The results show that A beta _ (25-35) caused by the damage of cell membrane is membrane fluidity changes.
Three, gamma ray of A beta _ (25-35) effects of red cell injury induced by the
This study used two different doses (50 Gy, 100 Gy) gamma rays of A beta _ (25-35) was studied, and the interaction of red blood cells and found that gamma ray irradiation of two doses of A beta _ (25-35) has a protective effect of red blood cells induced by injury.
Determination of 1. hemolysis
When the A beta _ (25-35) in the different dose of red blood cells, hemolytic effect compared with non irradiated red blood cells decreased significantly, indicating the A beta gamma ray _ (25-35) induced hemolysis has inhibitory effect.
Morphological changes of 2. red cells
After gamma ray irradiation of red blood cells by A beta _ (25-35) after treatment, still maintain good cell morphology was normal discocytes, shows that gamma ray helps to maintain the normal morphology of red blood cells.
Changes in osmotic fragility of 3. red cells
The red blood cells irradiated by gamma rays were placed in gradient buffer solution with osmotic pressure, and the hemolysis rate was detected by spectrophotometer. It was found that the tolerance of erythrocytes to hypotonic solution increased after gamma irradiation.
Changes in lipid fluidity of 4. erythrocyte membrane
A _ beta gamma ray irradiation after adding a certain dose of red blood cells (25-35) after incubation, using DPH as a fluorescent probe detection. The experimental results showed that membrane fluidity and membrane fluidity of erythrocytes increased after irradiation.
Conclusion: insulin and A beta _ fiber (25-35) can cause damage to the membrane of red blood cells, leading to cell hemolysis. In a certain period of time, amyloid fibrils with prolonging of incubation time, hemolysis was also enhanced, then decreased, that there is a close relationship between the cytotoxicity of amyloid fibrils were two with structural changes, and A beta _ (25-35) to form amyloid fibrils. Observe the exposure of the free thiol fiber to insulin during incubation, insulin free sulfhydryl fiber to thiol and membrane skeleton proteins related to the exchange, which might be relevant to hemolysis; A beta _ (25-35) leads to changes in cytoskeletal protein in the film, and the liquidity of the phospholipid layer is not affected, show that the A beta _ (25-35) damage to red blood cells involves A beta _ (25-35) interaction with the membrane skeleton protein. In this experimental condition of A _ beta gamma ray (25-35) induced by red fine Cell damage has a protective effect and increases the fluidity of the cell membrane.

【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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