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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻(Tai-Horng Young) | |
dc.contributor.author | Yong-Chong Lin | en |
dc.contributor.author | 林泳冲 | zh_TW |
dc.date.accessioned | 2021-06-16T05:14:03Z | - |
dc.date.available | 2014-08-21 | |
dc.date.copyright | 2014-08-21 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-18 | |
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The international journal of biochemistry & cell biology 2011; 43(4): 622-31. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56051 | - |
dc.description.abstract | 現今對於唾液腺癌之治療主要還是以放射線與化療為主,然而在治療的過程中經常會伴隨著許多的副作用產生。其中唾液線組織的功能性喪失即為主要副作用之一,其會造成乾口症進而影響病患的生活品質。因此若能經由組織工程建立一個具有自動分泌唾液功能的裝置,勢必能改善此類病患的症狀。於先前的研究成果中,我們已成功建立一套穩定的唾液腺初代細胞培養技術,然而此細胞乃為高度分化之細胞族群,不易增殖並有效率的進行組織工程之相關研究。於此篇研究中,我們將結合基因調控與生醫材料來改善唾液腺細胞的功能。此外,開發DNA疫苗用以預防唾液腺癌的復發亦為此篇研究的一大課題。
首先,我們成功的建立帶有LMP1致癌基因的細胞株,其細胞生長速度與遷徙能力皆大幅提升,並用以做為測試DNA疫苗效力的腫瘤動物模式。實驗結果證實,我們所構築的DNA疫苗-pcDNA3.1/CRT/LMP1,可有效的用來預防腫瘤的生成,但對於已發生腫瘤的動物則顯示較差的治療效果。為了強化此治療效果,我們未來將搭配樹突細胞共同進行免疫治療,於唾液腺癌的治療上勢必有極佳的效果。樹突細胞於癌症之免疫治療的應用上,最重要的步驟是誘導單核球細胞進行分化作用。與傳統的培養方式相比,本篇研究證實幾丁聚醣培養系統可獲得較多的樹突細胞,並且受到免疫原的刺激後,可分泌較多的免疫調節激素IL-12,此激素於腫瘤的免疫治療上扮演相當重要的角色。本研究結果亦顯示,幾丁聚醣培養系統可有效的增加毒殺型T細胞的活性並增強抗腫瘤的效力。 先前的研究主要致力於針對術後唾液腺癌的病患,開發一有效的DNA疫苗並搭配樹突細胞免疫治療,用以預防唾液腺癌的復發。最後我們將結合基因調控與生醫材料來改善唾液腺細胞的功能,期許未來能夠改善病患的生活品質。傳統上對於唾液腺細胞培養的研究,皆是將生醫材料進行化學或物理性改質,進而改善唾液腺細胞的貼附、增殖與功能。而本篇研究則係進行唾液腺細胞的N-乙醯葡萄醣胺轉移酶-V基因調控,使其增強細胞表面的N-乙醯葡萄糖胺,進而提高其與生醫材料-幾丁聚醣的交互作用。本研究成功的結合基因調控與生醫材料的應用,經基因調控後的唾液腺細胞,其增殖與分化能力皆有提升,且貼附於幾丁聚醣的速率也顯著性的提高,最重要的是對於唾液分泌的功能有促進的效果。 | zh_TW |
dc.description.abstract | Currently, the mainstay for the treatment of salivary gland carcinoma is radiation and chemotherapy. However, all these treatments are associated with severe short- and long-term side effects. One of the major side effect is that may cause dysfunction of the salivary glands and lead in the desiccant oral environment. Therefore, a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Previous studies demonstrated that we had set up a stable protocol for primary cultivation of human salivary gland cells from surgery specimens. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. In this study, we combine the gene regulation and biomaterials to address whether the combination have a well effect on the salivary gland cells. In addition, creating a DNA vaccine for salivary gland carcinoma to prevent the tumor regrowth after surgical operation is also a topic in this study.
In the first work, we successfully established the tumor cell line, which carrying the oncogene-LMP1, to mimic the salivary gland tumor cells. The established tumor cell line exhibited the significantly higher proliferation rate and higher migration capability. In this work, we have demonstrated that pcDNA3.1/CRT/LMP1 DNA vaccinia was effective in preventing but less effective for treating LMP1-associated tumors in the tumor animal model. Because dendritic cells (DCs) can be engineered with various kinds of tumor-associated antigens to elicit robust immune response against cancer cells, DC vaccine can be combined with DNA vaccine to enhance the immunotherapy for salivary gland carcinoma. The induction of monocyte differentiation into dendritic cells (DCs) is the essential protocol for the DC-mediated cancer immunotherapy. In the second work, monocytes isolated from mouse bone marrow were cultured on chitosan substrate to evaluate the effect of the chitosan culture system on the induction and tumor protection of DCs. Compared to tissue culture polystyrene (TCPS), the chitosan culture system could enhance monocyte aggregation and detachment with increased MTT reduction activity and expression of DC marker CD11c and LPS co-receptor CD14. Moreover, compared to TCPS, chitosan could enhance lipopolysaccharides (LPS)-stimulated DCs to secrete higher amount of IL-12. More importantly, vaccination of tumor lysate-pulsed DCs harvested from chitosan could increase cytotoxic T-lymphocyte (CTL) activity and showed significantly enhanced anti-tumor effect than those from TCPS. Therefore, the current study demonstrated that a protocol to culture DCs on a less-adherent chitosan substrate followed by treatment with tumor lysate has the potential in future DC-based vaccine application. The final work is to combine the gene regulation and biomaterial to improve the salivary acinar cell regeneration. Traditionally, biomaterials can be physically or chemically modified for improving cell attachment, proliferation and function. Conversely, this work tried to modify human parotid gland acinar cells (ACs), not biomaterials, with enhanced expression of N-acetylglucosamine on the cell surface by delivering N-acetylglucosaminyl transferase V (GnT-V) gene into the cells. We hypothesized that upregulation of N-acetylglucosamine expression on the AC surface could enhance the AC-chitosan interaction due to chitosan consisting of the similar structure, D-glucosamine and N-acetylglucosamine. Compared to primary ACs, the GnT-V-transfected ACs could greatly enhance N-acetylglucosamine expression and was more effective in attachment onto chitosan surface. Moreover, upregulation of GnT-V expression could function in the proliferation of ACs on chitosan and the replicative capacity of human ACs was maintained after prolonged culture. More importantly, over-expression of GnT-V facilitated the secretion of α-amylase, the most abundant enzyme in human saliva, in ACs cultivated on chitosan, while primary cells could not exhibit such behavior. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:14:03Z (GMT). No. of bitstreams: 1 ntu-103-D96548008-1.pdf: 3254865 bytes, checksum: 35d9b5339508c4a34ef6a47e4aaab76f (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員會審定書 ..................................................................... i
致 謝 .................................................................................. ii 摘 要 ................................................................................. iii Abstract ...................................................................................... v Chapter 1. DNA Vaccine for EBV/LMP1-associated tumor animal model ........................................................................... 14 1.1 Background ........................................................................................ 14 1.2 Materials and methods ....................................................................... 16 1.2.1 Construction of pLenti6/UbC/V5-DEST /LMP1 vector ... 16 1.2.2 Construction of CRT-LMP1 DNA vaccine ....................... 17 1.2.3 Generation of TC-1/LMP1 tumor cells ............................. 17 1.2.4 RT-PCR assay .................................................................... 18 1.2.5 Immunocytochemistry ....................................................... 19 1.2.6 Western blot analysis ......................................................... 19 1.2.7 Flow cytometric analysis ................................................... 20 1.2.8 MTT assay ......................................................................... 21 1.2.9 In vivo tumor growth kinetics ........................................... 21 1.2.10 In vivo tumor protection experiments ............................. 22 1.2.11 In vivo tumor regression experiments ............................. 22 1.2.12 Cell wound-migration assays .......................................... 22 1.2.13 Statistical analysis ........................................................... 23 1.3 Results ................................................................................................ 23 1.3.1 TC-1/LMP1 tumor cell line ............................................... 23 ix 1.3.2 TC-1/LMP1 tumor animal model ...................................... 24 1.3.3 Process design for the effects of DNA vaccine on LMP1-associated tumor animal model ................................ 25 1.4 Discussion .......................................................................................... 26 Chapter 2. Chitosan substrate has the potential to be applied future DC-based vaccine in immunotherapy. ....................... 30 2.1 Background ........................................................................................ 30 2.2 Materials and methods ....................................................................... 32 2.2.1 Preparation of chitosan-coated wells ................................. 32 2.2.2 Generation of DCs ............................................................. 33 2.2.3 MTT assay ......................................................................... 33 2.2.4 Cell surface marker expression ......................................... 34 2.2.5 Quantification of IL-12 and IFN-γ synthesis .................... 34 2.2.6 Preparation of tumor cell lysate ........................................ 35 2.2.7 Cytotoxic T-lymphocyte (CTL) assay ............................... 35 2.2.8 Tumor challenge and DC vaccination ............................... 36 2.2.9 Statistical analysis ............................................................. 37 2.3 Results ................................................................................................ 37 2.3.1 Formation of monocyte spheroids on chitosan ................. 37 2.3.2 The effect of chitosan on DC maturation .......................... 38 2.3.3 Activation of LPS-stimulated DCs on chitosan. ............... 39 2.3.4 Antitumor effect of chitosan culture system ..................... 40 2.4 Discussion .......................................................................................... 42 Chapter 3. Combining of gene regulation, GnT-V, and chitosan, to improve the salivary acinar cell regeneration. . 48 3.1 Background ........................................................................................ 48 3.2 Materials and methods ....................................................................... 50 3.2.1 Isolation and culture of human parotid gland ACs ........... 50 3.2.2 Preparation of GnT-V-transfected ACs.............................. 51 3.2.3 RT-PCR assay .................................................................... 52 3.2.4 Western blot analysis ......................................................... 53 3.2.5 Immunofluorescence Microscopy ..................................... 54 3.2.6 Chitosan substrate preparation .......................................... 54 3.2.7 Cell attachment assays ....................................................... 55 3.2.8 Cell growth and population doublings .............................. 55 3.2.9 Statistical analysis ............................................................. 56 3.3 Results ................................................................................................ 56 3.3.1 Characterization of GnT-V-transfected ACs ..................... 56 3.3.2 Proliferation and differentiation of GnT-V-transfected ACs 58 3.3.3 Attachment of GnT-V-transfected ACs on chitosan .......... 59 3.3.4 α-amylase expression of GnT-V-transfected ACs on chitosan 60 3.4 Discussion .......................................................................................... 61 Chapter 4. Conclusion ............................................................ 66 References ................................................................................ 67 | |
dc.language.iso | en | |
dc.title | 幾丁聚醣應用於唾液腺癌之免疫治療與唾液腺細胞之功能性修復 | zh_TW |
dc.title | The application of chitosan on the immunotherapy for salivary gland carcinoma and functional regeneration of salivary gland acinar cell | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 婁培人(Pei-Jen Lou) | |
dc.contributor.oralexamcommittee | 林佳靜(Chai-Ching Lin),陳羿貞(Yi-Jane Chen),楊銘乾(Ming-Chien Yang),鄭廖平(Liao-Ping Cheng),洪智煌(Chih-Huang Hung) | |
dc.subject.keyword | 唾液腺癌,DNA疫苗,幾丁聚醣,樹突細胞,唾液腺細胞,N-乙醯葡萄醣胺,N-乙醯葡萄醣胺轉移?-V, | zh_TW |
dc.subject.keyword | salivary gland carcinoma,DNA vaccine,chitosan,dendritic cell,acinar cells,N-acetylglucosamine,N-acetylglucosaminyl transferase V, | en |
dc.relation.page | 104 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-18 | |
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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