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National Chung Hsing University Institutional Repository - NCHUIR > 理學院 > 生物物理學研究所 > 依資料類型分類 > 碩博士論文 >  利用原子力顯微鏡量測正常人與第二型糖尿病 患紅血球的奈米機械特性

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/97737

標題: 利用原子力顯微鏡量測正常人與第二型糖尿病 患紅血球的奈米機械特性
Nanomechanical Measurements of Normal and Type 2 Diabetes Erythrocytes with Atomic Force Microscopy
作者: 趙佑叡
Chao, Yu-Jui
Contributors: 林明忠;蘇維彬;張嘉升
Min-Jon Lin;Wei-Bin Su;Chia-Seng Chang
何孟書
Mon-Shu Ho
中興大學
關鍵字: Type 2 Diabetes;spectrin;Atomic Force Microscopy
第二型糖尿病;細胞骨架蛋白;原子力顯微術
日期: 2012
Issue Date: 2012-09-04 09:17:14 (UTC+8)
Publisher: 生物物理學研究所
摘要: 第二型糖尿病(Type 2 Diabetes)是現代文明病也是國人常見的慢性病之一,根據2009年衛生署統計糖尿病佔十大死因中第五名。然而在已往研究發現,第二型糖尿病患者的紅血球外觀有異常的形貌,本實驗主要針對第二型糖尿病患者,依照糖化血色素(Glycosylated haemoglobin, HbA1c)數值,將樣品區分為六的區段進行分析研究,希望從中了解不同嚴重程度糖化血色素對紅血球外貌,細胞骨架蛋白(spectrin),以及奈米機械特性的影響。
第一部份對於紅血球外貌的探討,我們利用掃描式電子顯微鏡(Scanning Electron Microscopy)觀測紅血球,從我們實驗結果得知隨著糖化血色素數值超過7.0%,發現不同變異性的紅血球隨之增加,發現棘狀紅血球(echinocyte)以及波峰形紅血球(“crest” erythrocyte),甚至扁平形紅血球(planocyte)的存在,而在糖化血色素數值超過10.0%的樣本中,發現不正常所佔的比例遠高於正常人,猜測當糖化血色素數值超過10.0%更容易造成其他併發症產生,而這些變異的紅血球主要是由於細胞骨架蛋白的缺陷所致。
第二部份針對紅血球細胞骨架蛋白做螢光免疫染色法(Immunofluorescence)檢測,也發現隨著糖化血色素數值增高,而骨架蛋白有缺陷甚至重新排列的現象,但是在糖化血色素數值介於6.0~6.9%之間,並無發現明顯異樣,因為在這數值間屬於糖尿病的模糊地段,於是我們利用原子力顯微術(Atomic Force Microscopy)做奈米機械特性量測,希望能從中得到進一步結果。
第三部份,我們利用原子力顯微鏡於等張環境中進行奈米量測,從結果中得知紅血球內外徑大小隨著糖化血色素數值升高成反比,進而量測紅血球的黏滯力大小發現紅血球隨著糖化血色素數值增高越來越沒彈性,代表紅血球在通過微血管時更不容易形變,導致血拴或是造成其他糖尿病病發症產生。然而在糖化血色素數值介於6.0~6.9%的樣品發現,黏滯力小於正常人,或許在未來可將原子力顯微鏡列入檢測方向,將這段模糊區間做更細微的研究分析,讓病患得到提早發現,及時治療的目標。
由於紅血球細胞骨架蛋白與細胞膜之間主要藉由架橋蛋白(Band 3)相聯結,而架橋蛋白在紅血球的生理結構上扮演著一的很重要的角色,細胞骨架蛋白的缺失是否會間接影響架橋蛋白的生理功能,未來可針對這一方面進行更詳細研究,以便在未來治療糖尿病上有另ㄧ項判斷的依據。
Type 2 Diabetes is one of the civilization diseases as it is at present. According to the world statistics made by Department of Health in 2009, diabetes is among the leading causes of death in people. Previous studies have found that erythrocytes from patients type 2 diabetes appear abnormal morphology. In this study, we investigate type 2 diabetes erythrocytes with 6 different glycosylated hemoglobin values. The studies of glycosylated hemoglobin values along with morphology, cytoskeleton protein(spectrin), and nanomechanical characteristics of erythrocytes are useful in understanding the property of type 2 diabetes which are which are helpful in further treatment.
First erythrocytes morphology were taken by scanning electron microscopy (SEM). When glycosylated hemoglobin exceeds 8.0%, variability and abnormality of erythrocytes increases. Echinocyte , “crest” erythrocyte, and even planocyte were present. When glycosylated hemoglobin value exceeds 10.0%, found abnormal erythrocytes/ normal erythrocytes ratio increase to 1/4 due toa higher possibility of other complications. Also these variability and abnormality of erythrocytes somehow crash of cytoskeleton protein.
Second, immunofluorescence test of erythrocytes cytoskeleton were taken. With the increase of glycosylated hemoglobin value, protein defectiveness and rearrangement of cytoskeleton of erythrocytes took place. In the range of glycosylated hemoglobin value 6.0 - 6.9%, there is no much divergence which is used to categorize to be “pre-diabetes” or “at risk of diabetes” patients. The nanomechanical measurements of erythrocyte membranes were investigated by atomic force microscopy(AFM). The information will be helpful to understand the abnormality of cytoskeleton.
Third, erythrocytes were measured by atomic force microscopy in phosphate buffer saline solution. Erythrocytes diameter decreased with the increase of glycosylated hemoglobin value. From the measurement of AFM force curves which is referred to membrane adhesion. The flexibility of erythrocytes is found to be decreased with the increase of glycosylated hemoglobin value. It implied the ability of erythrocytes deformation decreased through capillaries. The thrombosis and other complications would occur. The adhesion also can be investigated less than normal in the region of glycosylated hemoglobin value at 6.0 to 6.9%. It would be a good method to study “pre-diabetes” or “at risk of diabetes” patients by AFM in detection and treatment.
Finally, erythrocytes membrane and cytoskeleton is linked by Band 3 proteins. And Band 3 protein play an important role in erythrocytes. In the future, we can do more detail study in this area. Facilitate the future treatment of type 2 diabetes on the basis of another judgment.
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