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|Other Titles: ||Novel atomic force microscopy applications for clinical detections|
|Authors: ||吳榮信;Wu, Jung-Hsin|
李世元;Lee, Adam Shih-Yuan
|Keywords: ||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|
|Issue Date: ||2012-06-21 06:38:01 (UTC+8)|
|Abstract: ||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 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.
|Appears in Collections:||[化學學系暨研究所] 學位論文|
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