探討抗菌胜肽攜帶干擾核糖核酸於細胞抑制基因效率

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姓名

蔡勝凱(Sheng-Kai Tsai) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

探討抗菌胜肽攜帶干擾核糖核酸於細胞抑制基因效率
(Delivery of short interfering RNA using antimicrobial peptides)

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摘要(中)

Small interfering RNA (siRNA)具有專一性有效抑制特定基因表現的功能，然而它本身帶負電的緣故，無法有效接近帶負電的細胞膜、進去細胞質發揮正常功能。所以需要一個合適的載體把siRNA包覆，防止siRNA被降解和進入細胞。本研究利用Indolicidin(IL)以及其衍生物ILF89、PEI(M.W=25k da)-ILC、PEI(M.W=25k da)-CIL、PEI(M.W=750k da)-ILC以及PEI(M.W=750k da)-CIL作為基因載體。檢測出IL-siRNA和ILF89-siRNA粒徑大小分別為大約800-1000nm和300-600nm而在表面電位的部分IL-siRNA complex在小於N/P=20是呈現中性和負電，大於N/P=30以上則是18mV以上; ILF89其表面電位都呈現負電。以相同的配製方法對HEK293T這株細胞的基因-GAPDH(每個動物細胞都含有此基因)去做抑制實驗，IL、ILF89利用QPCR分析出來的實驗結果大約是30-40％和幾乎0％的mRNA抑制效果比預期的來的低。針對另一株細胞HT1080的基因-EGFP，以Flow cytometry分析螢光螢光蛋白質的表現，IL、ILF89當成載體攜帶siRNA其螢光幾乎沒有下降。原因是EGFP的半生期過長，長達26小時，蛋白質很穩定以致於抑制效果非常的不明顯。
以PEI(M.W=25k da)-ILC、PEI(M.W=25k da)-CIL、PEI(M.W=750k da)-ILC以及PEI(M.W=750k da)-CIL作為基因載體，分析的結果PEI接上IL的抑制效果都比PEI好; PEI(M.W=25k da)-IL的抑制效果則是比PEI(M.W=750 da)好。
IL、ILF89作為基因載體抑制效果沒有預期的高，然而PEI以共價鍵和IL相接，效果比PEI好，所以IL有發揮提高轉染的效果。

摘要(英)

Small interfering RNA (siRNA) can specifically inhibit certain gene expression, but siRNA cannot approach to negatively-charged cell membrane and not even be functionalized into cytoplasm. To overcome the above problems, a suitable carrier to cover siRNA from degradation and to deliver it into cell is required.
In this study, we selected Indolicidin(IL), its derivative ILF89, PEI(M.W=25k da)-ILC、PEI(M.W=25k da)-CIL、PEI(M.W=750k da)-ILC以及PEI(M.W=750k da)-CIL as the gene carriers. Indolicidin may assemble into a complex with siRNA by electrostatic interaction at different amine over phosphate molar ratio (N/P ratio). First, we covered siRNA with IL and ILF89, and found that the sizes of siRNA-peptide complex are 800nm to 1000nm and 300nm to 600nm, respectively. On the other hand, the zeta potential of siRNA-IL complex at N/P ratio less than 20 is neutral and negatively charged while that of N/P ratio higher than 30 is above 18mV. In contrast to siRNA-IL complex, zeta potential of siRNA-ILF89 complex is completely negatively charged. We applied the above method to the inhibitory examination on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of HEK293T cell line. From the real-time polymerase chain reaction (QPCR) analysis, we found that the inhibitory efficiencies of siRNA-IL and siRNA-ILF89 complex are 30% to 40% of mRNA and 0％ of mRNA, respectively, which is relatively lower than the expected result.
To another gene, enhanced green fluorescent protein (EGFP), expressed from HT1080 cell line, there is little decrease in the fluorescence intensity from the green fluorescent protein expression of both siRNA-IL and siRNA-ILF89 complex, which was analyzed by QPCR. We attributed the above results to the long half-life of EGFP (26 hour) that makes the protein stable resulting in low inhibitory efficiency.
From results of PEI-IL, we found that the inhibitory efficiencies of PEI-IL are higher than PEI and the inhibitory efficiencies of PEI(M.W=25kda)-IL are higher than PEI(M.W=750kda)-IL. Thus, IL shows enhanced transfection ability.

關鍵字(中)

★ 干擾核糖核酸
★ 抗菌胜肽

關鍵字(英)

★ siRNA
★ antimicrobial peptide

論文目次

目錄
摘要 IV
Abstract V
目錄 VII
圖目錄 X
表目錄 XII
第一章緒論 1
研究動機 1
研究目的 2
第二章文獻回顧 3
2.1.基因治療 3
2.1.1病毒型載體(Viral Vector) 3
2.1.2非病毒型載體(Non-Viral Vector) 4
2.2 CPP和Indolicidin進入細胞途徑 15
(1) Barrel-stave mechanism 16
(2) Carpet mechanism 16
(3) Toroidal pore mechanism 17
(4) Molecular electroporation model 18
(5) Sinking raft model 18
2.3胞吞作用(endocytosis) 20
2.4 siRNA (Small interfering RNA) 21
2.4.1 siRNA的發展和研究 21
2.4.2 siRNA的抑制機制 23
2.4.3 Off-target effect of siRNA 25
2.5 CPP和siRNA之間的鍵結 26
2.5.1 非共價作用力(靜電吸引力) 26
2.5.2 共價作用力 26
第三章實驗材料與方法 28
3. 1試藥與原料 28
3. 1. 1 siRNA 28
3.1.2細胞 28
3.1.3胜肽 29
3.1.4藥品 29
3.2儀器 31
3.3試藥配製 31
3.4 HEK293T和HT1080細胞培養 31
繼代 (Passage) 31
冷凍細胞 32
解凍細胞 33
3.5 PEI、IL轉染 34
3.5.1細胞轉染 34
3.5.2 萃取mRNA 35
3.5.3 mRNA轉成cDNA 37
3.5.4 QPCR 38
3.5.5 Flow Cytometry 38
3.6奈米粒子製備及物理化學性質鑑定 39
3.6.1 奈米粒子製備 39
3.6.2雷射粒徑分佈儀(dynamic light scattering, DLS)分析 39
第四章結果與討論 40
4-1奈米粒子物理性質鑑定 40
4.1.1 粒徑大小(DLS測量) 40
4.1.2表面電位(DLS測定) 40
4.2 穿膜胜肽對轉染效率影響 43
4.3 PEI結合穿膜胜肽對轉染效率影響 51
第五章結論 54
第六章參考文獻 55

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2013-8-29

推文