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    題名: 原子力顯微術在臨床檢測上的新應用
    其他題名: Novel atomic force microscopy applications for clinical detections
    作者: 吳榮信;Wu, Jung-Hsin
    貢獻者: 淡江大學化學學系博士班
    李世元;Lee, Adam Shih-Yuan
    關鍵詞: 粘質沙雷氏菌;原子力顯微術;rssC基因;表面微結構;溶菌酶;病毒感染抑制效應;病毒崩;狗腎細胞;黏滯力;軟硬度;人類乳突病毒;聚合酶鏈鎖反應;西方點墨法;專一性抗體抗原作用力;子宮頸癌;S. marcescens;atomic force microscopy(AFM);rssC gene;surface ultrastructure(or nanostructure, topography);lysozyme;inhibitory effects;VirusBom;madin-darby canine kidney (MDCK) cell;adhesion forces;stiffness values;human papilloma virus(HPV);polymerase chain reaction(PCR);western blotting;Unbinding force;cervical cancer
    日期: 2012
    上傳時間: 2012-06-21 06:38:01 (UTC+8)
    摘要: Part 1:使用原子力顯微術針對黏質沙雷氏菌之野生株與rssC基因突變株做表面的量測驗證。此外,利用溶菌酶處理前後的野生、突變株,亦可在量測後得到其個別的表面微結構相關參數,諸如:峰頂至基底高、平均高、表面粗糙度與根均方高度等等。上述數據經統計比較後發現,突變株的表面構造比野生株較為粗糙且陡峭。除此之外,經溶菌酶處理後的野生株其表面型態無明顯變化。先前的研究指出,rssC基因主要作用在菌外層膜之脂肪酸的生合成,rssC基因突變會造成菌外層膜的缺陷。本論文直接藉由觀察菌外層膜之結構變化,成功區分出突變株與野生株種,未來更可以將AFM延伸應用到它種細菌表面蛋白的基因表現。
    Part 2:使用原子力顯微術量測A型流感病毒(H1N1)的表面奈米結構。根據病毒崩針對H1N1的病毒感染抑制效應之實驗結果,我們得到病毒崩可以在30~300ppm的濃度下對H1N1產生感染抑制的效果,而且須到達1000ppm濃度以上才會對狗腎細胞產生細胞毒性。所以利用300ppm的病毒崩,針對H1N1進行作用,再利用AFM做病毒粒子表面微結構與表面力學量測分析,量測後得到的表面微結構相關參數經統計比較後發現,H1N1的表面高度陡降(~90%↓),其表面力學性質,如黏滯力、硬度等,皆呈現下降趨勢。未來AFM可以較有效率的方式應用於抗病毒藥物的研究與開發。
    Part 3:先藉由聚合酶鏈鎖反應與西方點墨法確認帶有人類乳突病毒16型之患者檢體,再輔以AFM進行病毒粒子表面微結構與專一性抗體抗原作用力暨軟硬度分析。經純化後所得的人類乳突病毒粒子,以原子利顯微鏡量測後其大小約為50~55nm,此結果與電子顯微鏡相符合。實驗結果發現正常人與HPV患者間,兩者的專一性作用力與軟硬度分布,經統計處理後的高斯分布曲線可以完全分離不重疊。本研究的價值在於直接以HPV患者的檢體作為分析對象,實驗證實與非患者間可以做出有效鑑別。未來的研究可將AFM的應用推展到子宮頸癌臨床診斷上。
    Part 1:Atomic force microscopy (AFM) has been used to identify the surfaces of intact S. marcescens wild-type CH-1 cells and rssC mutant CH-1∆C cells. CH-1 and CH-1∆C cells were observed before and after treatment with lysozyme, and their topography-related parameters, e.g., a valley-to-peak distance, mean height, surface roughness, and surface root-mean-square values were defined and compared. The data obtained suggest that the cellular surface topography of mutant CH-1∆C becomes rougher and more precipitous than that of wild-type CH-1 cells. Moreover, it was found that, compared with native wild-type CH-1, the cellular surface topography of lysozyme-treated CH-1 was not changed profoundly. The rssC gene is thus predicted to be mainly responsible for fatty-acid biosynthesis of the S. marcescens outer membrane, and the cell membrane aberrance sites are assumed to be located in the lipid. This study represents the first direct observation of the structural changes in membranes of bacterial mutant cells and offers a new prospect for predicting gene expression in bacterial cells.
    Part 2:AFM has been used to probe the surface nanostructures of influenza viruses type A (H1N1). And according to inhibitory effects by VirusBom in H1N1 infection, we got that VirusBom could inhibit the native H1N1 infection in the concentration between 30~300 ppm, and it would be cytotoxicity to the madin-darby canine kidney (MDCK) cell if the concentration exceed 1000 ppm. H1N1 virions were observed before and after treatment with VirusBom by AFM, and their topography-related parameters and mechanical properties were defined and compared. Profiles displayed the height of H1N1 virions was from 56.66 ± 6.98 nm to 5.85 ± 0.83 nm (~90%↓) after treatment with 300ppm VirusBom, and the mechanical properties such as adhesion forces and stiffness values were also reduced. This new approach could be a useful technique for further investigating the antiviral drugs.
    Part 3:We identified oncogenic HPV type-16 virions by PCR analysis and Western blotting from specimen of normal (C2) and HPV patient (E12). And then we used an AFM to probe the surface ultrastructure and to measure their unbinding force and stiffness. The size of isolated single HPV virion was similar it’s SEM image (~50nm), and the histograms of unbinding forces were wider for the HPV patient than for the normal. The corresponding mean unbinding forces, obtained by fitting of the histogram to a Gaussian function, were 312.7±99.9 pN, and 53.7±24.6 pN at pulling velocities of 333.3 nm/s respectively. These novel findings showed the detailed surface mechanical properties, including stiffness and unbinding forces of HPV type-16 virions, which are essential to their infection potential and virulence. This new approach could be a useful technique for further investigating the potential role among subtypes of HPVs in the oncogenesis of human cervical cancer.
    顯示於類別:[化學學系暨研究所] 學位論文

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