本論文可分為三個部分，第一部分為使用氧化應激法 (oxidative stress) 處理肺癌細胞 (Lewis lung carcinoma, LLC)，使其細胞膜表面表現大量抗原 (antigen) 與損傷分子 (damage-associated molecular patterns, DAMPs)，再將細胞擠壓過濾製備奈米囊泡，最後使奈米囊泡回溶於細胞培養液，用於培養巨噬細胞 (reticulum cell sarcoma, J774A.1) 進行細胞體外實驗。第二部分為將LLC混合細胞基底質 (matrigel matrix) 注射於C57BL/6 (B6) 小鼠的皮下組織，並觀察腫瘤大小及量測小鼠體重，觀察奈米囊泡疫苗抑制腫瘤生長的效果。第三部分為使用靜電紡絲製備聚乙二醇 (polyethylene oxide, PEO) 奈米纖維，並將神經細胞 (neurona Schwann cell, RT4-D6P2T) 培養於PEO奈米纖維上，並使用MTT assay檢測細胞存活率，探討PEO奈米纖維作為神經導管的可能性。
癌症為台灣十大死因之首，為全球十大死因的第二名，僅次於心血管疾病，其中肺癌佔最大比例，由此可知癌症治療的重要性。本論文使用三種不同的氧化應激法處理肺癌細胞：(1) 使用含有濃度為30 μM H2O2的培養液 (dulbecco's high glucose modified eagles medium, DMEM)，培養LLC細胞，製備成H2O2疫苗；(2) 使用4 μg/ml 光敏劑 (photosensitizer) 維替泊芬 (verteporfin) 培養1小時，再使用光動力療法 (photodynamic therapy, PDT)，亦即於690 nm波長下強度為10 J/cm2的二極體雷射光束照射，收取上清液製備成Vs疫苗；過濾擠壓LLC製備成Ve疫苗(3) 使用大氣電漿 (atmospheric pressure plasma jet, APPJ) 處理，利用頻率為24 kHz、電壓為7 kV、氣體流率為1 L/min的參數，並固定電漿與樣品的距離為2 cm，處理LLC細胞60秒，製備成APPJ疫苗。利用上述的三種方式處理肺癌細胞，使肺癌細胞形成癌症免疫原性細胞 (immunogenic cancer cell)，再經由過濾擠壓法 (extrusion)，製備細胞外奈米囊泡 (extracellular nanovesicles, EVs)，探討其作為癌症疫苗的可能性。
將製備完成的奈米囊泡，用於活化J774A.1，並使其分化為M1型態並釋出一氧化氮 (nitric oxide, NO)，藉由格里斯試驗 (Griess assay) 檢測NO，藉此推測疫苗活化J774A.1的能力。由格里斯試驗法可以得知，使用H2O2疫苗活化J774A.1細胞，使其釋出較多的一氧化氮，濃度為16.87  2.46 μM。M1形態的巨噬細胞除了會釋出一氧化氮外，亦會抑制腫瘤細胞生長、或是殺死癌細胞，因此，本論文將被活化的J774A.1細胞與LLC癌細胞共培養，再使用流式細胞分選儀檢測共培養48小時後培養皿中的細胞比例，結果使用冷凍解凍法 (F/T) 製備的LLC細胞碎片，對活化J774A.1與LLC的LLC/J774A.1比例為3.48  0.64，而由H2O2、Vs、Ve、及APPJ疫苗活化的J774A.1與LLC的LLC/J774A.1比例分別為3.44  0.59、2.00  0.48、1.42  0.15與1.81  0.11，顯示使用PDT處理LLC、並使用過濾擠壓法製備的疫苗活化J774A.1，對抑制LLC效果最佳；由F/T收集的細胞碎片，用於活化J774A.1並抑制LLC的結果可以得知，結合氧化應激法與過濾擠壓法，製備奈米囊泡可有效的活化J774A.1，此種奈米囊泡應用於癌症疫苗有相當大的潛力。第二部分為將LLC細胞注射於小鼠皮下組織，並觀察腫瘤生長速率，觀察結果顯示腫瘤於16到28天時明顯增大，小鼠體重亦會增加約1到2克。
第三部分為使用靜電紡絲製備直徑為200至400 nm的PEO奈米纖維，並使用RT4-D6P2T神經細胞進行細胞活性分析實驗，結果顯示使用2 wt.%磷灰石混合10 wt.% PEO製備的奈米纖維可使RT4-D6P2T增生，MTT assay檢測結果顯示從培養第一天到第七天的呈色強度由0.082增加至2.26。因此，使用靜電紡絲製備奈米纖維應用於神經導管具有相當大的潛力。

The Ministry of Health and Welfare of Taiwan reported that, cancer was the first leading cause of death in Taiwan at 2017, which was also the second important cause of death globally. The most common types of cancers was lung cancer which caused 1.76 million deaths in 2018. Therefore, cancer therapies are of great importance. This study aims to prepare cancer vaccine by oxidative stress treatment and extrusion.
This study applied three different kinds of oxidative stress sources to treat Lewis lung carcinoma (LLC) cell: (i) 30 μM H2O2 (H2O2 vaccine), (ii) photo-dynamic therapy (PDT), keep the medium to collect nanovesicles as Vs vaccine, and extruded LLC after PDT treatment as Ve vaccine, and (iii) atmospheric pressure plasma jet (APPJ), which can get APPJ vaccine. The cancer cells became immunogenic cancer cells after exposure to oxidative stresses, and the cells were induced to apoptosis cells. Immunogenic cancer cells would then express damage-associated molecular patterns (DAMPs) and antigens on the surfaces of cell membranes followed by releasing spontaneous vesicles, which could activate the antigen presenting cells. In order to promote the yield of vesicles, the apoptosis cancer cells were extruded by with 80 psi dry air of filter extrusion, followed by centrifugation.
After H2O2 treatment and filter extrusion, the average diameter of vesicles was 207 nm and concentration of vesicles was 3.84  1010 NVs/2  107 cells, analyzed by nanoparticle tracking analysis (NTA). The efficiency of activating reticulum cell sarcoma (J774A.1) by different oxidative stress treated vesicles was determined by Griess assay. The results showed that the highest concentration of NO was 16.87  2.46 μM, by H2O2 treatment on vaccine for the activation of J774A1. The cytotoxicity of the J774A.1 primed with various vaccines towards LLC cells was evaluated in vitro by co-culturing LLC cells and J774A.1 to understand the potential of vesicles as cancer vaccine. The LLC/J774A.1 ratio by co-culture of LLC and J774A.1 activated by F/T, H2O2, Vs, Ve, and APPJ vaccine was 3.48  0.64, 3.44  0.59, 2.00  0.48, 1.42  0.15, and 1.81  0.11, respectively. The results showed that the incorporation of oxidative stress and extrusion method possessed good efficacy and reproducibility for the preparation of cancer vaccine
For the polyethylene oxide (PEO) electrospinning nanofibers, RT4-D6P2T cells were cultivated on the nanofibers as scaffolds to evaluate the potential for nerve repair. The effects of the applied voltages for electrospinning on the average diameter of the PEO nanofibers were evaluated by scanning electron microscopy (SEM). In order to prevent the dissolution of PEO nanofibers in aqueous solutions, 2.5 wt% of pentaerythritol triacrylate (PETA) was used as a chemical cross-linker. The correlations between the diameter of nanofibers and the cell responses were determined by MTT assay. The results of SEM observation indicated that the average diameter of the as-electrospun PEO nanofibers was 418 ± 134 nm, 286 ± 89 nm, and 328 ± 95 nm at the applied voltages of 10, 15, and 20 kV, respectively. To promote the biocompatibility of the electrospun PEO nanofibers, 2.0 wt% of apatite was incorporated into the polymer solution during electrospinning processes. With the addition of apatite, the average diameter of PEO nanofibers increased from 286 ± 89 to 426 ± 133 nm. MTT assay showed that PEO nanofibers containing 2 wt% apatite can significantly promote the cell growth comparing with the pristine PEO fibers, such that the cell density increased from 0.082 to 2.26 with the prolonged incubation time from 1 to 7 days. The results suggested that the as-prepared PEO nanofibers incorporated with 2.0 wt% of apatite displayed great potential for the applications in nerve tissue repair.

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