本文關(guān)鍵詞：多肽淀粉樣纖維與細(xì)胞膜之間的相互作用　出處：《陜西師范大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胰島素 β淀粉樣肽 淀粉樣纖維 細(xì)胞毒性 人紅細(xì)胞

【摘要】： 蛋白質(zhì)淀粉樣纖維是造成多種人類疾病的因素之一,如Alzheimer's disease(AD)是由神經(jīng)元中β淀粉樣肽的沉積所致。目前已知淀粉樣纖維具有細(xì)胞毒性,可導(dǎo)致人紅細(xì)胞的溶血,但具體作用機制不清。為進(jìn)一步了解淀粉樣纖維的細(xì)胞毒性作用,探究其作用機制,本實驗以紅細(xì)胞作為細(xì)胞膜模型,研究了胰島素淀粉樣纖維及β淀粉樣肽的一段特征性序列Aβ_(25-35)與紅細(xì)胞之間的相互作用,分析淀粉樣纖維引起的細(xì)胞溶血,對細(xì)胞形態(tài),細(xì)胞膜骨架,以及細(xì)胞膜流動性的影響;以Aβ_(25-35)對紅細(xì)胞的損傷為模型,研究了一定劑量γ射線的照射對此損傷作用的影響。 實驗的主要方法和結(jié)果: 一、胰島素淀粉樣纖維對紅細(xì)胞的影響 1.胰島素淀粉樣纖維對紅細(xì)胞滲透脆性的影響 將一定量的胰島素纖維作用后的紅細(xì)胞置于滲透壓梯度的緩沖溶液中,采用分光光度計檢測溶血率,并繪制紅細(xì)胞膜滲透脆性曲線,發(fā)現(xiàn)胰島素淀粉樣纖維改變了細(xì)胞膜的滲透脆性。 2.胰島素纖維在孵育過程中巰基暴露趨勢與其細(xì)胞毒性的關(guān)系研究 在胰島素淀粉樣纖維的孵育過程中的不同時段取樣,同時檢測其溶血效果和巰基暴露的程度。結(jié)果發(fā)現(xiàn)胰島素纖維孵育3小時后溶血效應(yīng)開始增強,4.5至7.5小時之間溶血效應(yīng)最強,之后開始下降,孵育9小時后溶血效果不再隨時間變化。比較溶血與巰基暴露的時間曲線發(fā)現(xiàn),胰島素巰基開始暴露時的纖維其溶血效果最強,但之后巰基繼續(xù)暴露并不能增加其溶血效應(yīng)。 二、Aβ淀粉樣肽對紅細(xì)胞的影響 1.Aβ_(25-35)的溶血效應(yīng) 紅細(xì)胞經(jīng)孵育不同時間后的Aβ_(25-35)處理后,檢測溶血率變化趨勢,結(jié)果顯示在前40分鐘隨著Aβ_(25-35)孵育時間延長,溶血率上升;在40分鐘后則下降。紅細(xì)胞經(jīng)不同終濃度Aβ_(25-35)處理后檢測溶血率,發(fā)現(xiàn)溶血率隨著的Aβ_(25-35)濃度上升而提高。 2.Aβ_(25-35)對紅細(xì)胞膜形態(tài)及膜骨架蛋白的影響 紅細(xì)胞經(jīng)一定濃度的Aβ_(25-35)處理后,于掃描電鏡下觀察,可見紅細(xì)胞發(fā)生輕微聚集,并有部分紅細(xì)胞出現(xiàn)棘突,細(xì)胞形態(tài)發(fā)生改變。提取紅細(xì)胞血影并用一定濃度的Aβ_(25-35)處理后,使用原子力顯微鏡觀察。發(fā)現(xiàn)與對照組相比,Aβ_(25-35)作用后的紅細(xì)胞血影,規(guī)則的網(wǎng)格狀結(jié)構(gòu)丟失,表明膜表面骨架蛋白的分布受到影響。 3.Aβ_(25-35)對紅細(xì)胞流動性的影響 使用DPH作為熒光探針,插入細(xì)胞膜的磷脂雙層并穩(wěn)定后,通過探針的各相異性值檢測細(xì)胞膜的流動性變化。發(fā)現(xiàn)Aβ_(25-35)處理過后的紅細(xì)胞與對照組細(xì)胞膜的流動性并無明顯差別。結(jié)果顯示,Aβ_(25-35)對細(xì)胞膜的損傷不是膜脂流動性的變化所引發(fā)。 三、γ射線對Aβ_(25-35)誘導(dǎo)的紅細(xì)胞損傷的影響 本研究應(yīng)用兩種不同劑量(50 Gy,100 Gy)的γ射線,對Aβ_(25-35)與紅細(xì)胞的相互作用進(jìn)行了研究,結(jié)果發(fā)現(xiàn),兩種劑量的γ射線照射均對Aβ_(25-35)導(dǎo)致的紅細(xì)胞損傷具有保護(hù)作用。 1.溶血率測定 當(dāng)Aβ_(25-35)作用于經(jīng)不同劑量γ射線照射后的紅細(xì)胞時,溶血效應(yīng)與未經(jīng)照射的紅細(xì)胞相比明顯減弱,說明γ射線對Aβ_(25-35)誘導(dǎo)溶血作用具有抑制作用。 2.紅細(xì)胞的形態(tài)變化 γ射線照射后的紅細(xì)胞經(jīng)Aβ_(25-35)處理后,仍維持良好的細(xì)胞形態(tài),呈正常的雙凹圓盤形,表明γ射線有助于維持紅細(xì)胞的正常形態(tài)。 3.紅細(xì)胞滲透脆性的變化 將γ射線照射后的紅細(xì)胞置于滲透壓改變的梯度緩沖液中,采用分光光度計檢測溶血率發(fā)現(xiàn),γ射線照射后,紅細(xì)胞對低滲溶液的耐受性有所提高。 4.紅細(xì)胞膜脂流動性的改變 γ射線照射后的紅細(xì)胞加入一定劑量的Aβ_(25-35)后孵育,使用DPH作為熒光探針,檢測膜流動性。實驗發(fā)現(xiàn),γ射線照射后紅細(xì)胞的膜流動性增強。 結(jié)論:胰島素纖維與Aβ_(25-35)均會對紅細(xì)胞膜造成損傷,導(dǎo)致細(xì)胞溶血。在一定時間內(nèi),淀粉樣纖維隨著孵育時間的延長,溶血作用也得到增強,之后則呈現(xiàn)減弱,說明兩種淀粉樣纖維的細(xì)胞毒性作用均與其結(jié)構(gòu)變化有密切關(guān)系,并且Aβ_(25-35)更易形成淀粉樣纖維。胰島素纖孵育過程中觀察到自由巰基的暴露,胰島素纖維的自由巰基能夠和膜骨架相關(guān)蛋白的巰基交換,這可能與其溶血作用相關(guān);Aβ_(25-35)導(dǎo)致了膜下骨架蛋白的變化,而磷脂層的流動性則不受影響,表明Aβ_(25-35)對紅細(xì)胞的損傷涉及到Aβ_(25-35)與膜下骨架蛋白的相互作用。在本文的實驗條件下,γ射線對Aβ_(25-35)誘導(dǎo)的紅細(xì)胞損傷具有保護(hù)作用,并且增加了細(xì)胞膜的流動性。
[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.

【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 夏愛軍,張獻(xiàn)清,穆士杰,吳原茹,張清平;γ射線輻照對紅細(xì)胞抗氧化能力的影響[J];中國輸血雜志;2004年06期

2 張英,劉樹錚;低劑量輻射減輕化療對小鼠免疫功能的抑制作用[J];白求恩醫(yī)科大學(xué)學(xué)報;1997年05期

3 黎儒青;蔣天倫;肖瑞卿;任德會;曾杰;帥桂珍;;不同劑量γ射線對紅細(xì)胞制品中細(xì)胞因子含量的影響[J];第三軍醫(yī)大學(xué)學(xué)報;2006年24期

4 肖小煒;非小細(xì)胞肺癌患者自體血液照射回輸?shù)姆派浔Ｗo(hù)作用[J];湖北省衛(wèi)生職工醫(yī)學(xué)院學(xué)報;2001年01期

5 王俊玲;王衛(wèi)東;秦廣雍;;紅細(xì)胞膜輻射損傷的研究進(jìn)展[J];核農(nóng)學(xué)報;2007年05期

6 周劍濤;蛋白質(zhì)構(gòu)象病[J];生命的化學(xué);2001年04期

7 賀方學(xué),范士懷,葛來增,申源峰,呂相坤;自體血照射回輸提高食管癌患者抗輻射的作用[J];華人消化雜志;1998年10期

8 范士懷,胡相起,賀方學(xué),葛來增,申源峰,呂相坤;自體血低劑量輻射刺激療法防治癌癥血象降低的臨床研究[J];中華放射醫(yī)學(xué)與防護(hù)雜志;2003年01期

，

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