Measurement and Application of Particles Deposition Uniformity

常見基因轉殖的基因載入模式，包括︰基因槍、直接肌肉注射、複合基因、電穿孔、細菌或病毒感染等。其中以基因槍的基因載入模式，對細胞危害性低、所需的載入物質劑量低、可載入的DNA劑量大、以及可廣泛的應用在各種體內和體外的培養細胞等優點，使得基因槍成為生物技術上的新趨勢。 本研究針對影響基因槍之金微粒沈積均勻度與收集效率的因子，像是氦氣出口大小( 0.05, 0.1, 0.5, 5, 20 mm)、出口位置( 0, 15, 25 mm)、氦氣氣流壓力(100, 150, 250, 400, 600 psi)、錐狀體長度(5, 15, 35, 65, 105, 155 mm)、金微粒沉積面積的直徑(10, 15, 25, 30, 50 mm)、筒狀體長度(25, 35, 45, 65, 95, 155 mm)等，以探討對基因轉殖效率的影響，進而找出最佳化的轉殖效度：氦氣出口大小0.05 mm、出口位置0 mm、氦氣氣流壓力600 psi、錐狀體長度65 mm、金微粒沉積面積的直徑50 mm。改良後的基因槍收集效率增加為原先設計的5.2倍、均勻度提升了2.5倍、轉殖效度提升了17.3倍。The ways of gene deliver during gene transfection included gene gun, injection directly into the muscle, complex DNAs, electroporation, bacterial systems, and viral delivery systems. Among them, the advantages of the gene gun was below: low harmfulness for cell, only small amounts of DNA for requirement, but large DNA fragments may be transferred, applicable to both in vitro and in vivo transformation. So the gene gun will become a new trend in biological technology. In this research, we confer relationship between uniformity and slot width (0.05, 0.1, 0.5, 5, 20 mm), slot height (0, 15, 25 mm), helium pressure (100, 150, 250, 400, 600 psi), transition cone (5, 15, 35, 65, 105, 155 mm), diameter of target filter (10, 15, 25, 30, 50 mm), column height (25, 35, 45, 65, 95, 155 mm). The best transfection occurs when slot width is 0.05 mm, slot height is 0 mm, transition cone is 65 mm and diameter of target filter is 50 mm. Collection efficiency is 5.2 times than original design, uniformity is 2.5 times than original design and transfection is 17.3 times than original design.摘要…………………………………….…….………….……….….…………...….i Abstract………………………………………………………………….………….ii 目錄…………………………………………………………….…………….…..…iii 圖目錄……………………………………………………………………….….….vi 符號表……………………………………………………………….…………….viii 一、研究目的與背景…………………………………………………………………1 1.1研究目的…………………………………………………………...……….1 1.1.1提升基因槍射出金微粒的沈積均勻度……………………………….1 1.1.2降低基因轉殖的昂貴成本………………………………………...…..1 1.2背景………………………………………………………………………....1 1.2.1生物技術在醫療、食品及農業等應用科學的重要性……………….1 1.2.2常見的基因轉殖技術………………………………………………….3 1.2.3基因槍的優缺點……………………………………………………….6 1.2.4影響基因槍轉殖效率的原因……………………………………….....7 1.2.5基因轉殖常用的細胞………………………………………………...10 1.2.6電化噴霧……………………………………………………………...11 二、慣性衝擊的原理……………………………………………………………….12 2.1雷諾數……………………………………………………………………..12 2.2史托克常數………………………………………………………………..13 三、實驗方法………………………………………………………………………14 3.1儀器設備…………………………………………………………………..14 3.1.1基因槍選用…………………………………………………………...14 3.1.2微粒的選擇……………………………………………………….…..15 3.1.3子彈的製備…………………………………………………………..16 3.1.3.1清洗及乾燥尼龍管………………………………………………16 3.1.3.2子彈製備流程……………………………………………………16 3.1.4影像擷取系統………………………………………………………...18 3.1.4.1顯微鏡外接數位相機……………………………………………18 3.1.4.2視野校正……………………………………………...………….18 3.2視野選取方式…………………………………………………………......19 3.2.1等距離法……………………………………………………………...19 3.2.2等面積法…………………………………………………………...…19 3.3影像分析軟體……………………………………………………………..19 3.4覆蓋率與均勻度…………………………………………………………..22 3.4.1覆蓋率………………………………………………………………...22 3.4.2均勻度………………………………………………………………...23 3.5計數效率與收集效率…………………………………………………..…24 3.6基因轉殖效度的計算…………………………………………………..…25 3.7標準化…………………………………………………………………..…27 四、結果與討論……………………………………………………………………27 4.1等距離法與等面積法比較…………………………………………..……27 4.1.1等距離法……………………………………………………..….……27 4.1.2等面積法……………………………………………………….……..27 4.2均勻度與覆蓋率對轉殖效率的影響…………………………………..…27 4.2.1氦氣出口大小(SW)………………………………………………..…..28 4.2.2氦氣出口位置(SH)……………………………………………………29 4.2.3壓力………………………………………………………………...…30 4.2.4錐狀體的長(TC)……………………………………………................31 4.2.5筒狀體的長(CH)………………………………………………………32 4.2.6金微粒沉積面積的直徑(TD)…………………………………………33 4.2.6.1錐狀體長(TC) 5 mm……………………………………………...33 4.2.6.2錐狀體長(TC) 25 mm…………………………………………….34 4.2.6.3錐狀體長(TC) 65 mm………………………………………….…36 五、結論與建議…………………………………………………………………….37 參考文獻……………………………………………………………………………38 圖目錄 Figure 1：BHK-21纖維母細胞之單層細胞………………………………………43 Figure 2：B16-f10上皮細胞………………………………………………………..43 Figure 3：電化噴霧(Electrospray)…………………………………………………44 Figure 4：基因槍…………………………………………………………………...45 Figure 5：子彈製備系統(Tubing Preparation System)…………………………….46 Figure 6：切管器(Tubing Cutter)…………………………………………………...46 Figure 7：子彈匣及子彈(Cartridge Holder and Cartridges)………………….……47 Figure 8：顯微鏡外接數位相機……………………………………………………47 Figure 9：光柵(Light Railings)………………………….…………………………48 Figure 10：慣性衝擊示意圖…………………………………………………….…48 Figure 11：基因轉殖效度的計算……………………………………………….…49 Figure 12：灰階……………………………………………………………………..50 Figure 13：影像處理之雜訊濾除………………………………………………….51 Figure 14：影像處理之影像邊緣強化…………………………………………….52 Figure 15：影像處理之二值化.……………………………………………………53 Figure 16：影像處理之黑白互換………………………………………………….54 Figure 17：影像處理之濾除不要的微粒…………………………………………55 Figure 18：影像處理之分離疊合之微粒…………………………………………56 Figure 19：影像處理之微粒數目計數……………………………………………57 Figure 20：系統圖…………………………………………………………………58 Figure 21：等距離法與等面積法微粒分佈圖………………………………….…59 Figure 22：等距離法與等面積法計數效率……………………………………….60 Figure 23：轉殖效度模式……………………………………………………….….61 Figure 24：氦氣出口大小(SW)……………………………………………………...62 Figure 25：氦氣出口位置(SH)……………………………………………………...63 Figure 26：氦氣壓力………………………………………………………………..64 Figure 27：錐狀體高度(TC)……………………………………………………..…65 Figure 28：筒狀體高度(CH)……………………………………………………….66 Figure 29：標的面積直徑(TD)-錐狀體長(TC) 5 mm……………………………..67 Figure 30：標的面積直徑(TD)-錐狀體長(TC) 25 mm……………………………68 Figure 31：標的面積直徑(TD)-錐狀體長(TC) 65 mm……………………………69 Figure 32：最佳化的Spacer………………………………………………............71 Figure 33：原始設計與改良後Spacer比較……………………………………...72 Figure 34：等距離法……………………………………………………………….73 Figure 35：等面積法……………………………………………………………….7