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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林文澧(Win-Li Lin) | |
| dc.contributor.author | Tian-Ning Tsao | en |
| dc.contributor.author | 曹恬寧 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:18:41Z | - |
| dc.date.available | 2021-11-05 | |
| dc.date.available | 2022-11-24T03:18:41Z | - |
| dc.date.copyright | 2021-11-05 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-29 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80841 | - |
| dc.description.abstract | "研究背景與目的 根據世界衛生組織的資料統計，大腸直腸癌是全世界發生率第三高之癌症，而死亡率則是排名第二，僅次於肺癌。若在癌症初期就介入治療，病患的五年存活率高達90%，但若造成了癌症的遠端轉移，五年存活率則大幅降到僅剩14%，因此控制癌症之轉移為大腸直腸癌病患首要需解決之問題。 根據先前研究顯示，超音波熱治療可刺激腫瘤大量釋放熱休克蛋白70（heat shock protein 70, HSP70），釋放到細胞外之HSP70可活化免疫系統。因此本研究建立雙側腫瘤動物模式下，利用脈衝式超音波熱治療（pulsed wave ultrasound hyperthermia, pUSHT）作為原位癌症疫苗，並輔以過繼性自然殺手細胞治療（adoptive NK cell therapy, ACT-NK），強化pUSHT誘發之免疫反應，期許在控制原位腫瘤的同時達到抑制遠端腫瘤生長，建構一套具系統性治療效果之合併治療手段。 實驗材料與方法 本研究主要分成三部分，依序為細胞實驗、動物實驗及腫瘤分析。細胞實驗使用CT26小鼠大腸直腸癌細胞株，利用細胞存活實驗探討43°C熱治療15、30、45及60分鐘對細胞株之生長抑制能力；細胞株於43°C 60分鐘治療參數下，第2、4、8及24小時之HSP70表現量；最後探討體外NK細胞對CT26細胞株之胞殺能力在有無熱療下之差異，其中本實驗用之NK細胞皆取自小鼠脾臟，並在體外進行11天之培養擴增。 動物實驗使用國家動物中心提供周齡為8-10週之BALB/c母鼠，在治療開始前7日於小鼠大腿右側及左側皮下種植CT26細胞以建立雙側腫瘤動物模型。pUSHT參數如下：探頭發射頻率1MHz、強度：1.44W/cm2、工作週期：50%、總施打時間為15分鐘；而ACT-NK以間隔六日施打一次、共二次。動物實驗之治療成效以腫瘤生長曲線及存活率呈現，並記錄小鼠體重變化作為治療對小鼠生存壓力影響之依據。 腫瘤樣本分為治療開始後第8日進行腫瘤淋巴細胞浸潤分析及治療結束後第6日進行蘇木精-伊紅染色（hematoxylin and eosin stain, H E stain）。每組各犧牲3隻小鼠，取下其超音波治療側及非超音波治療側之腫瘤進行分析。 實驗結果 由細胞實驗結果可知，CT26細胞在43°C熱水槽加熱下30分鐘，其存活率（48.6%）即能與Control組產生統計上之顯著差異（p < 0.001），而加熱45分鐘之存活率（38.5%）及60分鐘（17.7%）之存活率亦與Control組產生統計上之顯著差異（p < 0.0001）。後續選用43°C、60分鐘之加熱條件，探討CT26細胞在加熱後2、4、8、24小時之HSP70表現量，在8小時後與Control組達顯著性統計差異（p < 0.0001），且在24小時分析HSP70之表現量為Control組之19.95倍，較8小時之表現量17.55倍高，但兩者間並無顯著差異，因此本研究之動物實驗參考HSP70表現量之實驗，以pUSHT後24小時再進行ACT-NK。 動物實驗方面，由雙側腫瘤模型可得合併治療組無論是在超音波治療側或是非超音波治療側腫瘤，皆有最佳之腫瘤生長抑制結果，與Control組達到統計上之顯著差異（超音波治療側p < 0.0001；非超音波治療側p < 0.05）在治療開始後第15日，合併治療組超音波治療側腫瘤相較於Control組減少68.4%之生長，非超音波治療側腫瘤則減少22%之生長。 在治療結束後第6日進行腫瘤H E染色分析，由超音波治療側腫瘤可見pUSHT組及合併治療組之切片有物理性破壞之痕跡，而非超音波治療側腫瘤則較無明顯差異；腫瘤淋巴細胞浸潤分析結果雖然合併治療組無論在超音波治療側或是非超音波治療側腫瘤之淋巴細胞浸潤皆為四組最高，但四組並無達到統計上顯著差異。進一步分析腫瘤浸潤之CD4+T細胞、CD8+T細胞及NK細胞，合併治療組在超音波治療側浸潤之CD8+ T細胞及IFNγ+CD8+ T細胞皆高於其他三組，且達到統計上顯著差異；而非超音波治療側在四組間IFNγ+CD8+ T細胞、IFNγ+CD4+ T細胞及IFNγ+ NK細胞浸潤之總和雖無顯著差異，合併治療組卻呈現四組最高之趨勢，可見合併治療在非超音波治療側雖無明顯之作用，但還是有活化免疫細胞的趨勢存在。 結論 本研究於細胞實驗中證實熱治療確實有增加NK細胞對CT26細胞胞殺能力之效果，在動物模型中，pUSHT結合ACT-NK無論是在超音波治療側腫瘤及非超音波治療側腫瘤皆展現出最佳之抑制腫瘤生長的能力，且小鼠之存活率相較於其他三組也顯著提升，在治療期間亦無對小鼠造成過度之負擔。腫瘤樣本分析部分，由H E可觀察到超音波熱治療對腫瘤的確能產生除熱效應外之機械效應；淋巴細胞浸潤分析方面，合併治療組在超音波治療側有顯著之CD8+T細胞、IFNγ+CD8+T細胞浸潤效果，可見合併治療的確可以活化CD8+ T細胞並增加其浸潤；非超音波治療側之T細胞及NK細胞雖在合併治療組有相對活化之趨勢，但組間無統計上差異，可見合併治療在針對非超音波直接治療的腫瘤，還是無法突破其免疫抑制環境，使更多免疫細胞可浸潤到腫瘤中。 " | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:18:41Z (GMT). No. of bitstreams: 1 U0001-2909202118503600.pdf: 3679043 bytes, checksum: 3fab96a936a9e07338c01a8c8493ff8f (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 謝辭 i 中文摘要 ii Abstract v 圖目錄 x 表目錄 xi 縮寫與翻譯對照表 xii 第一章 緒論 1 1.1大腸直腸癌 1 1.2大腸直腸癌免疫療法 1 1.3超音波熱治療 1 1.3.1超音波熱效應 2 1.3.2超音波機械效應 3 1.3.3超音波誘發自然殺手細胞之免疫效應 3 1.4自然殺手細胞 4 1.4.1自然殺手細胞與癌症 4 1.4.2過繼性自然殺手細胞轉移療法 6 1.5研究目的 7 第二章 實驗材料與方法 8 2.1實驗材料 8 2.1.1細胞株及細胞培養 8 2.1.2 實驗動物 8 2.1.3 普留淨注射液 8 2.1.4 脈衝式超音波熱治療系統之裝置 9 2.2 實驗流程 10 2.3 實驗方法 11 2.3.1 NK細胞擴增與培養 11 2.3.3 NK細胞之胞殺性測試 12 2.3.4 西方墨點法 13 2.3.5 熱電耦溫度測試 15 2.3.6 脈衝式超音波熱治療參數設定 16 2.3.7動物實驗 16 2.4 腫瘤組織切片 18 2.4.1蘇木精-伊紅染色 18 2.5腫瘤淋巴細胞浸潤分析 18 2.6數值統計與分析 19 第三章 實驗結果 20 3.1 細胞實驗 20 3.1.1 MTT細胞存活分析─熱劑量反應 20 3.1.2 西方點墨法─熱休克蛋白測定 20 3.1.3 NK細胞擴增與培養 22 3.1.4 NK細胞之胞殺性測試 22 3.2 熱電耦溫度測試 23 3.3 動物實驗 24 3.3.1動物腫瘤大小分析 24 3.3.2 動物體重變化 27 3.3.3 動物存活分析 28 3.4組織切片 29 3.5腫瘤淋巴細胞浸潤分析 33 第四章 討論 38 4.1 細胞實驗證據 38 4.2動物實驗證據 39 4.3腫瘤分析證據 39 4.4研究限制 42 第五章 結論與未來展望 43 參考文獻 44 | |
| dc.language.iso | zh-TW | |
| dc.subject | 過繼性自然殺手細胞轉移療法 | zh_TW |
| dc.subject | 抗癌免疫反應 | zh_TW |
| dc.subject | 雙側腫瘤模型 | zh_TW |
| dc.subject | 癌症 | zh_TW |
| dc.subject | 超音波熱治療 | zh_TW |
| dc.subject | cancer | en |
| dc.subject | bilateral tumor model | en |
| dc.subject | antitumor immune response | en |
| dc.subject | adoptive natural killer cell transfer therapy | en |
| dc.subject | ultrasound hyperthermia | en |
| dc.title | 應用過繼性自然殺手細胞結合超音波熱治療強化全身性抗癌免疫反應 | zh_TW |
| dc.title | Adoptive Transfer of Natural Killer Cell in Combination with Ultrasound Hyperthermia Enhances Systemic Antitumor Immunity for Cancer Treatment | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 繆希椿(Shi-Chuen Miaw) | |
| dc.contributor.oralexamcommittee | 梁祥光(Hsin-Tsai Liu),(Chih-Yang Tseng) | |
| dc.subject.keyword | 超音波熱治療,過繼性自然殺手細胞轉移療法,抗癌免疫反應,雙側腫瘤模型,癌症, | zh_TW |
| dc.subject.keyword | ultrasound hyperthermia,adoptive natural killer cell transfer therapy,antitumor immune response,bilateral tumor model,cancer, | en |
| dc.relation.page | 47 | |
| dc.identifier.doi | 10.6342/NTU202103460 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-10-01 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
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