以磷酸根甲基化之反義去氧核醣核酸探針藉由多孔性二氧化矽奈米粒子作為載體進行基因靜默調控之研究

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吳佳嶸(Chia-Jung Wu) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

以磷酸根甲基化之反義去氧核醣核酸探針藉由多孔性二氧化矽奈米粒子作為載體進行基因靜默調控之研究
(Studies of Methyl Phosphotriester Oligonucleotide as Anti-sense Oligo by Mesoporous Silica Nanoparticles for Gene Silencing)

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摘要(中)

在許多疾病包括癌症中，細胞內的蛋白質調控產生失衡，影響著蛋白質的合成與降解、訊號傳遞和代謝途徑等，甚至影響腫瘤的惡化及癌症發展。在這其 messenger RNA (mRNA) 在蛋白質轉譯形成及調節基因表達都發揮重要的作用。藥物的發展進程中，許多研究會使用反義寡核苷酸 (Antisense oligonucleotide, ASO) 做為探針，以達成基因調控的目標。ASO 與目標基因 (DNA,mRNAandmicroRNA) 進行鹼基配對，通過不同的生物機制對目標基因進行表達調控，因此從生物辨識工程的角度上反義寡核苷酸的專一性及親和力將成為一項關鍵因素。
為了實現此目標，本研究使用本實驗室所研發之磷酸根甲基化 DNA (Phosphate-methylated DNA) 作為反義寡核酸探針。nDNA 因磷酸骨幹經過特定位置的甲基化修飾，使帶電的磷酸根轉變成為三酯鍵而形成電中性 (Neutralized DNA, nDNA)，其可減少核酸雙股同時帶負電的靜電排斥效應，期待不會影響專一性外並能提升雜交穩定性。且經甲基化修飾的核酸探針，擁有抵抗核酸脢降解的能力，因此更具有成為反義核酸藥物的潛力。
為了使反義寡核酸探針進入細胞內，本研究使用多孔性二氧化矽奈米粒子(Mesoporous Silica Nanoparticle, MSN) 作為核酸類似物載體。並經 PEI 修飾，使表面帶正電，可透過靜電吸附力與核酸探針結合，再經由包吞作用進入細胞，進行基因抑制。
首先，為確保經 PEI 修飾之 MSN 不會造成細胞毒性，以 Cell Counting
Kit-8(CCK-8) 試劑進行細胞毒性檢測。結果表明，當奈米粒子濃度低於50 μg/mL 時不會引起細胞毒性，並且在 24、48 及 72 小時的共培養時間下， MSN-PEG/PEI 濃度低於 75 μg/mL 條件下，其測得生物活性皆高於 90%。並且由電泳實驗，探討 MSN-PEG/PEI 對 nDNA 吸附能力。結果顯示，使用 nDNA 與 MSN 重量百分比 1:128 為最佳吸附條件，且 MSN-PEG/PEI 在此條件下對 nDNA 與 DNA 吸附能力相當。
於 Human plasma-like medium 環境中，以 nDNA 作為反義寡核苷酸探針對 mRNA 進行抑制逆轉錄，其結果顯示，與 DNA 相比，nDNA 能更有效的抑制逆轉錄反應，其中又以修飾於序列片段中間部分的 N3-mid nDNA 效果最佳。
在基因靜默調控實驗中，本研究使用人類結腸癌細胞-綠螢光蛋白 (HCT116-GFP) 細胞，並以綠色螢光蛋白 (Greenfluorescentprotein,GFP) 作為報告基因 (Reporter gene) 。使用 nDNA 探針或 DNA 探針與 HCT116-GFP 細胞中 GFP mRNA 進行雜交，以抑制 mRNA 轉譯形成 GFP，並使用螢光顯微鏡及微量分光光度計對綠色螢光蛋白進行定量及定性分析。
結果所示，部分甲基化的反義 nDNA 探針，因擁有較佳的專一性及雜交能力，因此比 DNA 探針更有效地抑制綠色螢光蛋白之表達。且修飾於核酸
探針中間時，因提升中間雜交穩定性，因此不易與 mRNA 解旋，擁有更佳的抑制能力。同時比較市售轉染試劑 (TransIT-X2) 與 MSN-PEG/PEI 之傳遞核酸探針之能力，結果顯示在 30 nM 時，兩著對於核酸傳遞能力相當，但當濃度提高時，MSN-PEG/PEI 有較佳的傳遞能力。
本研究已經成功利用 MSN-PEG/PEI 作為 nDNA 載體，在細胞層次研究中利用 50 nM 之 N3-mid nDNA@MSN 與 HCT116-GFP 細胞經 24 小時共培養，可靜默 50% 綠螢光蛋白表達，並且可持續對綠螢光蛋白進行表達抑制達 48 小時，並期望在未來能夠以 nDNA 作為探針對人體內基因進行調控，以作為核酸藥物。

摘要(英)

Most diseases, including cancers, are diagnosed by protein dysregulation within cells, which impacts protein synthesis and degradation, signaling, metabolic pathways, and even tumor progression. In this context, messenger RNA (mRNA) plays a critical role in protein translation formation and the regulation of gene expression. Many therapeutic drug development studies use antisense oligonucleotides (ASO) to achieve gene regulation targets.
Our laboratory has developed phosphate-methylated DNA (designated as neutralized DNA or nDNA) antisense oligonucleotides by modifying the phosphate backbones with site-specific methyl groups to reduce the inter-strand repulsion force and manipulate the stability of nucleic acid duplexes without losing the duplex formation specificity.
This study exploited mesoporous silica nanoparticles (MSN) as a carrier to deliver ASO into cells by positively charging their surfaces using polyethyleneimine (PEI), enabling them to adsorb nucleic acid probes electrostatically and to penetrate cells via endocytosis and endosomal escape to suppress gene expression.
Initial trials ensured that the PEI-modified MSNs concentrations below 50 μg/mL has no cytotoxicity. While the electrophoresis measurements showed that a weight ratio of nDNA: MSN = 1:128 was optimal for nDNA to be adsorbed by MSNs.
Moreover, despite an equivalent loading efficiency at the dosage of 30 nM, MSNs were more capable in delivering nucleic acids than commercially available transfection reagents (TransIT-X2) at the higher dosages.
In the human plasma-like medium environment, using nDNA as antisense oligonucleotide probes to inhibit reverse transcription of GFP-mRNA showed that nDNA was more effective in inhibiting the reverse transcription reaction compared to DNA. As a result of the modifications in the middle portion of the sequence fragment, N3-mid nDNA exhibited the best effect.
Experimentally, the nDNA probes were compared with DNA probes silencing gene regulation by hybridizing with GFP mRNA in HCT116-GFP cells to inhibit mRNA translation to form GFP. Empirical results demonstrated profoundly that the nDNA probes were more potent than the DNA antisense in suppressing the expression of green fluorescent protein, owing to their enhanced specificity and hybridization stability, especially the ASOs with center- methylated positions. With different designs of the nDNA probes, we also observed different potency of the antisense.
In summary, MSN-PEG/PEI was successfully employed as an nDNA carrier, and 50 nM of N3-mid nDNA@MSN was co-cultured with HCT116-GFP cells for 24 hours, resulting in a 50% reduction in green fluorescent protein expression. The silencing effect persisted for 48 hours.

關鍵字(中)

★ 核酸類似物
★ 奈米粒子
★ 基因靜默

關鍵字(英)

論文目次

摘要 i
ABSTRACT iv
致謝 vi
目錄 viii
圖目錄 xii
表目錄 xv
一、緒論 1
二、文獻回顧 3
2.1 核酸分子介紹 3
2.1.1 核酸分子 3
2.1.2 去氧核醣核酸 4
2.1.3 核醣核酸 6
2.1.4 信使核糖核酸 7
2.2 核酸類似物 9
2.2.1 鎖核酸 9
2.2.2 肽核酸 11
2.2.3 中性去氧核醣核酸 13
2.3 反義寡核苷酸對基因的調控 19
2.4 奈米粒子 22
2.4.1 奈米粒子之內吞作用 23
2.4.2 多孔性二氧化矽奈米粒子 24
2.5 基因分子檢測 27
2.5.1 聚合酶連鎖反應 27
2.5.2 即時定量聚合酶連鎖反應 29
2.5.3反轉錄聚合酶連鎖反應 31
三、實驗方法與儀器設備 33
3.1 實驗藥品 33
3.1.1 細胞培養 33
3.1.2 細胞轉染 33
3.1.3 二氧化矽奈米粒子 34
3.1.4 細胞毒性分析 35
3.1.5 洋菜凝膠電泳 (Agarose gel) 35
3.1.6 模擬生物體內mRNA雜交 35
3.1.7 即時聚合酶鏈式反應 36
3.1.8 綠螢光蛋白表達抑制實驗 37
3.2 儀器設備 38
3.3實驗方法 39
3.3.1 細胞解凍 39
3.3.2 細胞冷凍保存40
3.3.3 細胞培養 41
3.3.4 MSN吸附 42
3.3.6 細胞轉染 44
3.3.7 細胞毒性分析47
3.3.8 洋菜凝膠電泳 (Agarose gel) 48
3.3.9 逆轉錄即時聚合酶鏈式反應 (RT-qPCR) 49
3.3.10 SYBR Green 螢光熔點量測 52
3.3.11 綠螢光蛋白表達抑制實驗 53
3.3.12 MSN的細胞攝取 55
四、實驗結果與討論 56
4.1以多孔性二氧化矽奈米粒子做為反義寡苷酸探針載體系統建立 56
4.2多孔性二氧化矽奈米粒子吸附能力之探討 60
4.2.1 不同MSN種類對於nDNA探針吸附能力之比較 60
4.2.2 MSN對於nDNA與DNA探針吸附能力之比較 64
4.3 細胞攝取 66
4.4 細胞增殖 (細胞毒性分析) 69
4.5 反義寡核苷酸探針在HUMAN PLASMA-LIKE MEDIUM中抑制能力 72
4.5.1不同nDNA探針位置設計對GFP-mRNA逆轉錄抑制能力 75
4.5.2不同nDNA探針數量設計對GFP-mRNA逆轉錄抑制能力 78
4.6 使用反義寡核苷酸探針對抑制GFP轉譯能力 81
4.6.1 nDNA 與 DNA 探針對抑制轉譯能力之比較 83
4.6.2不同nDNA探針設計對抑制轉譯結果之研究 88
4.6.3不同時間長度對抑制結果之研究 93
五、結論 97
六、未來展望 99
七、參考文獻 100

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指導教授

陳文逸

審核日期

2023-7-20

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