臺灣博碩士論文加值系統

小分子褐藻醣膠是萃取自褐色海藻的低分子量含有豐富岩藻類多醣。人類研究，透過體外試驗和小鼠實驗已經證實小分子褐藻醣膠具有抗腫瘤效果與提升化療藥物的能力。臨床可用於改善直結腸癌患者疾病控制率，但在獸醫方面，則研究極少。在人醫，血管內皮生成因子、乙型轉化生長因子和尿液嗜中性白血球明膠酶被認為與臨床的腫瘤分期和疾病進展相關，而在獸醫較少研究。本實驗目的是研究小分子褐藻醣膠在犬貓惡性腫瘤之抗腫瘤和輔助化療的效用。犬貓患有惡性腫瘤且口服至少三個月小分子褐藻醣膠列入前瞻研究。依口服小分子褐藻醣膠的劑量分成三組，分別是一般劑量組、高劑量組和化療搭配一般劑量小分子褐藻醣膠組。使用商業化的酵素連結免疫吸附分析法套組分析患者血清血管內皮生成因子、乙型轉化生長因子的濃度；使用酵素連結免疫吸附分析法和西方墨點法分析尿液嗜中性球明膠酶的表現。納入研究之動物有20隻犬與8隻貓，患有16種不同腫瘤類別，列入此試驗。依據實驗結果得知血管內皮生成因子、乙型轉化生長因子的濃度會隨著腫瘤的進展而增加，尤其是在肺臟轉移的病患。在患有軟組織肉瘤的犬貓，血管內皮生成因子以及乙型轉化生長因子的濃度在口服褐藻醣膠後下降。在臨床高劑量小分子褐藻醣膠可以延長貓軟組織肉瘤無疾病進展期，及犬口腔惡性黑色素瘤病患的整體存活時間。服用高劑量小分子褐藻醣膠病患也具有較佳的疾病穩定及較高的存活率。尿液嗜中性球明膠酶的單體、三體和與第九型金屬基質形成之複合體會表現於乳腺腫瘤、轉移型腺癌、惡性賽托利細胞瘤、和惡性黑色素瘤之病患，且表現量隨著疾病進展而增加。透過分子分析與病患的臨床狀態比較，高劑量小分子褐藻醣具有部分抗腫瘤效果；且在軟組織肉瘤以及口腔惡性黑色素瘤具有臨床效益。

Oligo fucoidan is a low molecular weight fucose-rich polysaccharide which is extracted from brown seaweeds. In human studies, oligo fucoidan demonstrated anti-tumor effect and enhancement of chemotherapeutic activity both in vitro and in vivo. Clinically, oligo fucoidan can improve the tumoral disease control rate; however, it is rarely investigated in veterinary medicine. In human cancer, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and neutrophil gelatinase-associated lipocalin (NGAL) were considered to be associated with clinical status and tumor progression. The aim of this study was to investigate the antitumor and enhancement of adjuvant chemotherapeutic effects of oligo fucoidan in canine and feline patients. Dogs and cats with malignant tumors had been administered more than 3 months of oligo fucoidan were enrolled. Administration of oligo fucoidan was assigned to regular dose, high dose, and the combination of regular dose and chemotherapy. Twenty dogs and 8 cats with 16 types of malignant tumors were included. The serum samples were collected to analyze VEGF and TGF-β1 by commercial ELISA kit, and the urine samples were used for NGAL analysis by ELISA and western blot. The concentration of VEGF and TGF-β1 increased when the tumor progression especially when lung metastasis occurred. Moreover, VEGF and TGF-β1 concentration decreased after fucoidan treatment in patients with soft tissue sarcoma. Clinically, high dose oligo fucoidan prolonged the progression free survival in patients with feline fibrosarcoma, and prolong the overall survival time in dogs with oral malignant melanoma. The patients received high dose oligo fucoidan had more stable status and higher survival rate. In urine samples, monomer and trimer of NGAL and NGAL/MMP9 heterodimer increased when disease progressed in animals with mammary tumor, metastatic adenocarcinoma, malignant Sertoli cell tumor, and malignant melanoma. In conclusion, by molecular analysis, oligo fucoidan was shown to have some antitumor activity in animals with malignant tumors. In addition, it showed clinical benefit in patients with soft tissue sarcoma and oral malignant melnoma

摘要 i
Abstract ii
Contents iv
List of Tables vi
List of Figures viii
List of Abbreviations x
Chapter 1 Introduction 1
Chapter 2 Materials and methods 7
2.1 Animals 7
2.2 Oligo fucoidan and samples 8
2.2.1 Oligo fucoidan 8
2.2.2 Sample collection 8
2.3 Clinical monitoring and response evaluation 9
2.4 Serum VEGF and TGF-β1 immunoassay 10
2.4.1 VEGF 10
2.4.2 TGF-β1 11
2.5 Detection of NGAL in urine 12
2.5.1 Sandwich ELISA 12
2.5.2 Western blot analysis 13
2.5.2.1 Samples preparation 13
2.5.2.2 SDS-PAGE 13
2.5.2.3 Western blotting 13
2.6 Statistical analysis 15
Chapter 3 Results 16
3.1 Animals 16
3.2 Clinical reponse 17
3.3 Clinical outcome and survival analysis 20
3.3.1 Clinical detail during and after oligo fucoidan treatment 20
3.3.2 Survival analysis 26
3.4 VEGF-immunoassay 28
3.4.1 Precision data of VEGF-immunoassay 28
3.4.2 Statical analysis of VEGF-immunoassay 28
3.5 TGF-β1-immunoassay 33
3.5.1 Precision of TGF-β1-immunoassay 33
3.5.2 Statical analysis of TGF-β1-immunoassay 33
3.6 Comparison of molecular assay and clinical data 39
3.6.1 The serum VEGF and TGF-β1 baseline concentration among tumor types 39
3.6.2 The serum VEGF and TGF-β1 baseline concentration corresponding to the animals’ clinical status 39
3.6.3 Mean of progression free survival of primary disease and metastatic disease 39
3.6.4 Mean concentration of alteration when the disease progression in the primary disease and metastatic disease 40
3.6.5 Concentration of serum VEGF in various tumors in different disease status 40
3.6.6 Concentration of serum TGF-β1 in various tumors between two groups in different disease status 41
3.7 Detection of NGAL in urine sample 44
3.7.1 Sandwich ELISA for the detection of NGAL 44
3.7.2 Western blot analysis for the detection of NGAL 46
Chapter 4 Discussion 49
References 57

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