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標題: | 幾丁聚醣奈米粒子於疾病診斷與基因轉殖之應用評估 Application of Chitosan-Based Nano-particles for Disease Diagnosis and Gene Transfection |
作者: | Shu-Jyuan Yang 楊淑娟 |
指導教授: | 謝銘鈞 |
關鍵字: | 大腸直腸癌、幾丁聚醣,五胺基酮戊酸,吡,喀紫質,基因轉殖,超音波, Colorectal cancer,chitosan,5-aminolaevulinic acid,protoporphyrin IX, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 根據衛生署統計指出:2003 年台灣地區大腸直腸癌為國人罹患十大癌症排行榜為第三名,而直至目前為止,大腸直腸癌的成因仍不清楚。此外,大腸直腸癌的早期症狀並不明顯,且特異性也不高,易被病患及醫師所忽略,以致延誤了早期診斷及治療的時機。根據研究指出,大腸直腸癌病患如能於發病早期就被診斷出並加以治療,其五年存活率可高達88%。而近年來用以診斷腸胃的內視鏡主要是以巨觀的角度來觀察腸胃黏膜形態的變化以判斷其是否產生病變,其需借助技
術精良的內視鏡技術人員加以區分微小變異黏膜與正常黏膜組織間的差異。此外,為了增加診斷的準確性,常會需要輔以組織切片與顯微鏡觀察,然而組織切片的製備時間往往會增加內視鏡診斷大腸病變的時間,也增加造成併發症的風險。感光劑前驅物五胺基酮戊酸(5-aminolaevulinic acid)可被正常細胞與癌細胞經由原血紅素合成路徑(heme biosynthetic pathway) 代謝為螢光物質吡喀紫質(protoporphyrin IX)。然而,因癌細胞內原血紅素合成路徑中的酵素ferrochelatase活性降低,造成癌細胞中吡喀紫質大量的累積。本研究即是利用幾丁聚醣奈米粒子做為感光劑前驅物五胺基酮戊酸的藥物載體,其可被大腸直腸癌細胞吞噬,使包覆的藥物於細胞內被順利的代謝為螢光物質吡喀紫質,並累積於大腸直腸癌細胞內,以利於螢光內視鏡對早期大腸直腸癌的診斷。而幾丁聚醣的抗菌性可避免包覆於內的五胺基酮戊酸被腸道內的大腸桿菌吞噬,並可降低大腸桿菌內的吡喀紫質螢光於螢光內視鏡診斷所造成的干擾。此外,將幾丁聚醣分子表面鍵結葉酸分子,可增加奈米粒子對大腸直腸癌細胞的親和性,並經由葉酸受體媒介之胞噬作用大量地將帶有五胺基酮戊酸的奈米粒子吞噬於癌細胞內,進一步使吡喀紫質大量地累積於細胞內。最後,順利地將帶有負電荷的天然高分子海藻酸鹽分子與幾丁聚醣摻合以做為五胺基酮戊酸的藥物載體。因海藻酸鹽分子於溶小體的弱酸 環境中會帶有負電荷,會與帶有正電荷的幾丁聚醣形成離子性鍵結,使的大量鍵結於幾丁聚醣上的五胺基酮戊酸被釋放出來,進一步使吡喀紫質大量的累積於大腸直腸癌細胞內。而動物實驗也發現,將五胺基酮戊酸包覆於奈米粒子中可有效的被運送至腸道中並被腸道內的腸腫瘤吞噬,最後在腫瘤細胞內代謝為吡喀紫質螢光物質。因此,將五胺基酮戊酸包覆於鍵結有葉酸並摻有海藻酸鹽之幾丁聚醣奈米粒子中,可有效的應用於早期大腸直腸癌螢光內視鏡診斷。 而近年來,基因轉殖技術也被用於疾病的治療,但目前現有的基因轉殖技術往往受限於載體的安全性與低轉殖效率。因此,本研究將帶有綠色螢光蛋白基因質體包覆於幾丁聚醣奈米粒子,並轉殖子宮頸癌細胞。由實驗結果可知包覆於幾丁聚醣奈米粒子的綠色螢光蛋白質基因可順利的表現於細胞內,如再加上超音波的照射,於癌細胞內的轉殖效率可大大的被提升。因此,結合做為基因載體之幾丁聚醣奈米粒子與超音波可有效的應用於疾病的基因治療上。 Colorectal cancer is one of the leading causes of malignant death in Taiwan because it often remains undetected until later stages of the disease. Improved methods of detecting dysplasia and tumors during colonoscopy will improve mortality. In this study, a chitosan based nano-particle was successfully prepared and loaded with 5-aminolaevulinic acid (5-ALA) that could be taken up by colorectal cancer cells (HT29 and Caco-2) but escape from being engulfed by E. coli. that might lead to misinterpretation of endoscopic examination in clinical. The z-average diameter of the chitosan based nano-particles was about 100 nm, and the zeta-potential was higher than 20 mV that provided enough zeta-potential to prevent aggregation. Furthermore, folic acid can be covalently conjugated to chitosan molecules via its γ-carboxyl moiety and thus retain a high affinity for colorectal cancer cells bearing the folate receptor over-expression. The chitosan nano-particle conjugated with folic acid could be significantly taken up by HT29 and Caco-2 cell lines, most likely via receptor-mediated endocytosis, and enhance the protoporphyrin IX (PpIX) accumulation in cytoplasma after a short-term uptake period. When alginate was physically incorporated into the chitosan based nano-particles and then fed to colorectal cancer cells, the fluorescent intensity of PpIX in cells could be remarkably improved by competing with 5-ALA for chitosan in cellular lysosomes, resulting in a higher amount of 5-ALA release and PpIX accumulation. Therefore, the chitosan nano-particle conjugated with folic acid and incorporated with alginate appears to be an ideal vector for colorectal-specific drug delivery of 5-ALA for fluorescent endoscopic detection of colorectal cancer. Moreover, gene therapy has been used to treat a variety of health problems. However, inefficiency of transfection and the lack of safe gene vectors have limited progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapies. In this study, we also demonstrated the successful preparation of chitosan based nano-particles that were complexed with the pAcGFP1-C1 plasmid. The prepared nano-particles protected the DNA from enzymatic digestion and improved the efficiency of gene transfection of HeLa cells and 293T cells. The efficiency of gene transfection was further enhanced by exposure to an ultrasound (US) regimen either for in vitro or in vivo tests. Although US reduced cell viability, the simultaneous use of gene transfection and tumor destruction by a focused US may have advantages for the gene therapy of cancer. We conclude that the combined use of DNA-complexed chitosan based nano-particles and US provides a high efficiency gene transfection system for human cancer cells and shows promise for use in future clinical gene therapy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44938 |
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顯示於系所單位: | 醫學工程學研究所 |
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