초록 ▼

본 연구에서는 한국산 겨우살이 추출물 및 lectin의 항암효과를 알아보기 위해 실험적 간 발암모델을 이용하여 전암성 병변과 생화학적 변화에 미치는 영향 등을 알아보았으며, asialofetuin-column을 이용하여 한 종류의 lectin(VCA)이 분리하였다. 또한, 분리된 한국산 겨우살이 lectin의 구조 및 활성을 검색하고 겨우살이제제의 VCA의 역할을 밝히고자 하였다. 이를 위하여 VCA의 물리화학적 특성을 확인하고, 항체를 개발하여 정량법을 개발하였다. 또한, 분자량별로 추출물을 분획하여 각 분획의 항암활성을 검색하여

본 연구에서는 한국산 겨우살이 추출물 및 lectin의 항암효과를 알아보기 위해 실험적 간 발암모델을 이용하여 전암성 병변과 생화학적 변화에 미치는 영향 등을 알아보았으며, asialofetuin-column을 이용하여 한 종류의 lectin(VCA)이 분리하였다. 또한, 분리된 한국산 겨우살이 lectin의 구조 및 활성을 검색하고 겨우살이제제의 VCA의 역할을 밝히고자 하였다. 이를 위하여 VCA의 물리화학적 특성을 확인하고, 항체를 개발하여 정량법을 개발하였다. 또한, 분자량별로 추출물을 분획하여 각 분획의 항암활성을 검색하여 VCA의 역할을 규명하였다. 또한 VCA의 HL-60 세포에 대한 apoptosis 유도 여부를 검색하고, caspase-3, PARP 등의 marker를 사용하여 apoptosis 작용기전을 규명하였다. 또한 용액상 효소 결합법에 의한 lectin의 정량법의 개발을 위한 연구를 하였다.

Abstract ▼

The extracts form European mistletoe have been widely used as unconventional cancer therapy, and the yellow berry Korean mistletoe, a subspecies of European mistletoes, has been used traditionally as medicinal herbs for a long time. This study was performed to investigate the effects of Korean misle

The extracts form European mistletoe have been widely used as unconventional cancer therapy, and the yellow berry Korean mistletoe, a subspecies of European mistletoes, has been used traditionally as medicinal herbs for a long time. This study was performed to investigate the effects of Korean misletoe and lectin on $GST-P^{+}$ foci, serum GOT, GPT, α-fucosidase activity, and phase I, II enzymatic system in experimental rat hepatocarcinogenesis. Although mistletoe and lectin were not showed the effects on serum GOT, GPT, α-fucosidase activity and phase I, II enzymatic system, $GST-P^{+}$ foci number and area were significantly decreased by mistletoe and lectin. Especially, this effect was the highest in mistletoe 100㎍/㎏ BW injection group. Also in this study, the Viscum album var. coloratum agglutinin(VCA) was more efficiently isolated than the Viscum album, agglutinin (VAA) by asialofetuin-Sepharose 4B. To investigate the synergistic effects of the components in mistletoes, the water extract was divided into different molecular weight fractions. The fractions with lectin of both plants showed lower cytotoxicity than whole extract against Molt-4 cells, demonstrating that the lower molecular weight components may be involved in the synergistic effect. The fractions without lectin of European mistletoe showed no cytotoxicity while those of Korean mistletoe showed slightly higher cytotoxicity than European one. These results indicate that although the lectins of both plants are main components for exerting anticaner activity, the synergistic effect of lower molecular weight components may be more strongly involved in the activity of Korean mistletoes, We also investigated the apoptotic mechanism of lectin on the HL-60 cells. In HL-60 cells, addition of VCA resulted in a dose- and time- dependent growth suppression, morphological changes of apoptotic nuclei, and DNA fragmentation characteristics of apoptosis. To investigate that caspase-3 activation during VCA-induced apoptosis induces cleavages of PARP, the expression of PARP and the pattern of caspase-3 activation in HL-60 cells were investigated. The native and processed PARP forms typically seen in apoptotic cells were observed, and a decrease in expression of 32kDa form of caspase-3 was observed in dose-dependent manner. At last in this study, a simple and rapid homogeneous enzyme-linked binding assay method are developed for the determination of the Mistletoe lectin(ML). The major component of them is ML with galactose group sepecificity. For developing homogeneous assay for ML, the Alkaline phosphatase(AKP)-biotin and avidin-D-galactose conjugates aresynthesized and utilized. The activity of the AKP-biotin conjugate is inhibited in the presence of the avidin-galactose conjugate, and the observed inhibition is reversed in an amount dependent on the concentration of galactose binding protein, ML, added. AKP-biotin4 as the signal generator is shown to be inhibited up to 60% by the binding of the excess avidin. Avidin-gal4 was chosen as the signal modulator, since it gives a high inhibitory effect(up to 84%) and reverses the observed inhibition (up to 56%) with an excess ML. The dose-response curve for ML with fixed amounts of AKP-biotin4 and avidin-gal4 shows linearity over a narrow concentration range (30-50g), indicating that the assay can be used for the sensitive determination of the ML. These results could contribute to the scientific elucidation of mechanism for the cancer treatment of Korean mistletoe.

목차 Contents

• 제 1 장 서 론...15
• 제 1 절 연구개발의 필요성...15
• 제 2 절 연구목적(연차별)...16
• 제 3 절 연차별 연구내용 및 범위...17
• 제 2 장 국내외 기술개발 현황...18
• 제 1 절 국내외 기술 현황...18
• 제 2 절 현 기술상태의 취약성...20
• 제 3 장 연구개발수행 내용 및 결과...22
• 제 1 절 실험적 간암모델에서 겨우살이( Mistletoe) 추출물의 발암 억제효과 탐색 및 작용기전 연구...22
• 1. 연구방법...22
• 2. 연구결과...26
• 3. 요약 및 결론...31
• 제 2 절 VCA-1 분리, 항암활성, 역할 검색 및 lectin 정량...33
• 1.연구방법...33
• 2.연구수행 결과 및 고찰...35
• 제 3 절 생리활성물질에 대한 Avidin-Biotin System을 이용한 용액상 효소결합분석법...39
• 1.연구방법...39
• 2.연구결과...41
• 제 4 장 연구개발목표 달성도 및 대외기여도...46
• 제 5 장 연구개발결과의 활용계획...47
• 제 6 장 참고문헌...49