식물기반 치료용 항체생산

김영관,소양강,박다영,김현순,전재흥,추영국,고기성,Kim, Young-Kwan,So, Yang-Kang,Park, Da-Young,Kim, Hyun-Soon,Jeon, Jae-Heung,Choo, Young-Kug,Ko, Ki-Sung 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.3

Antibodies are powerful and versatile tools to play a critical role in the diagnosis and treatment of many diseases. Their application has been enhanced significantly with the advanced recombinant DNA and heterologonous expression technologies, allowing to produce immunotherapeutic proteins with improved biofunctional properties. However, with currently available technologies, mammalian cell-based therapeutic antibody production, as an alternative for production in humans and animals, is often not plentiful for passive immunotherapeutics in treatment of many diseases. Recently, plant expression systems for therapeutic antibodies have become well-established. Thus, plants have been considered to provide an attractive alternative production system for therapeutic antibodies, as plants have several advantages such as the lack of human pathogens, and low cost of upstream production and flexible scale-up of highly valuable recombinant glycoproteins. Recent advances in modification of posttranslational processing for human-like glycosylation in transgenic plants will make it possible that plant can become a suitable protein expression system over the animal cellbased current production system. This review will discuss recent advances in plant expression technology and issues for their application to therapeutic antibody production.

식물 및 곤충세포 발현시스템을 이용한 암 진단 및 치료용 항체 생산

이정환(Jeong-Hwan Lee),김득수(Deuk-Su Kim),이재혁(Jaehyouk Lee),명순철(Soon-Chul Myung),황경아(Kyung-A Hwang),추영국(Young-Kug Choo),고기성(Kisung Ko) 대한비뇨기종양학회 2012 대한비뇨기종양학회지 Vol.10 No.2

Therapeutic proteins can prevent or treat wide ranges of diseases from cancer and viral or bacterial infections. Production of the therapeutic proteins has been well established mainly in mammalian and bacterial cells including transgenic organisms by recombinant DNA techniques. Recent plant and insect biotechnology and advanced molecular immunology have established molecular biofarming system as an alternative way to produce recombinant pharmaceutical proteins such as immunotherapeutic monoclonal antibodies (mAb). The plants and insects as an expression system have several advantages, which include the lack of animal pathogenic contaminants, low cost of production, and ease of agricultural scale-up compared to other currently available systems. Thus, the paradigm of plant is being shifted as a food source to so-called plant bioreactor for production of therapeutic proteins. Currently, we have successfully developed a plant and insect cell expression system for production of anti-cancer and anti-virus monoclonal antibodies. The effective heterologous production system for recombinant therapeutics requires the appropriate expression machinery with optimal combination of transgene expression regulatory conditions such as control of transcriptional and post transcriptional events. In this paper, the possibility of targeting proteins to the ER and possibly storing them to the protein storage vacuoles with glycosylation modification are discussed in plant and insect cells.

Differential Expression Patterns of Gangliosides in the Liver and Heart of NIH-miniature Pigs

Jae-Sung Ryu(유재성),Kyu-Tae Chang(장규태),Ji-Su Kim(김지수),Dong Hoon Kwak(곽동훈),Young-Choon Lee(이영춘),Keon Bong Oh(오건봉),Young-Kug Choo(추영국) 한국생명과학회 2010 생명과학회지 Vol.20 No.4

갱글리오시드는 포유동물 세포막의 중요한 구성요소로서 세포와 세포 혹은 세포와 단백질간의 상호작용을 포함한 다양한 면역학적 역할을 수행하고 있다. 이 연구는 NIH-미니돼지의 간과 심장을 인간에게 이식할려고 할 때 예측되어지는 거부 반응과 관련된 구성성분들 중 시알산음 함유하고 있는 스핑고당지질인 갱글리오시드에 대해 조사하였다. 얇은 막크로마토그래피와 면역조직화학적분석을 실시한 결과 NIH-미니돼지의 간은 갱글리오시드의 발현이 심장보다 높게 나타났다. 갱글리오시드 GD3, GDla, GDlb, GTlb는 간과 심장의 두 기관에서 발견되었다. 그러나 GQlb는 간에서만 발견되었고 심장에서는 검출되지 않았다. 이러한 결과는 갱글리오시드의 발현양상은 간과 심장에서 조직특이적이라는 것을 의미한다. 한편, GM3를 포함한 다른 갱글리오 시리즈인 갱글리오시드들은 NIH-미니돼지의 간과 심장에서 검출되어지지 않았다. 이와 같은 연구결과로부터 갱글리오시드는 미니돼지의 장기중 특히, 간과 심장의 이종장기이식과 관련된 면역거부반응에서 어떤 역할을 수행하고 있다고 여겨진다. Gangliosides are a major component of the plasma membrane of mammalian cells, which are directly involved in a variety of immunological events, including cell-to cell or cell-to-protein interactions. In this study, we investigated whether gangliosides, sialic acid-containing glycosphingolipids, are related to rejection during the xenotransplantation of NIH-miniature pig livers and hearts to humans. Both high performance thin-layer chromatography and immunohistochemistry analyses revealed that the expression of gangliosides in the liver tissue of NIH-miniature pigs was higher than that in the heart. Gangliosides GD3, GD1a, GD1b, GT1b and GQ1b were observed in both the liver and heart, whereas GQ1b was detected only in the liver, indicating that the ganglioside expression profiles are tissue specific. Moreover, other ganglio-series gangliosides, including GM3, were not detected in the livers and hearts of NIH-miniature pigs. Taken together, these results suggest that gangliosides may play important roles in immune responses in clinical xenotransplants of pig livers and hearts.

Effect of Buan Mulberry on Metabolic Improvement in Streptozotocin-Induced Diabetic Rats

Ju Taek Lee(이주택),Jae-Sung Ryu(류재성),Dong Hoon Kwak(곽동훈),Yun Jum Park(박윤점),Seong Sun Kang(강성선),Pyoung Jun Kim(김병준),Kyung-A Hwang(황경아),Young-Kug Choo(추영국) 한국생명과학회 2010 생명과학회지 Vol.20 No.12

본 연구에서는 streptozotocin (STZ)에 의해 유도된 당뇨병 흰쥐를 이용하여 부안산 오디의 항 당뇨효과를 in vivo 실험을 중심으로 조사하였다. 기존의 연구는 주로 in vitro 실험을 통해 이루어 졌으며 일부 지역의 오디는 STZ에 의해 유도된 당뇨병 흰쥐에서 신진대사 증진, 항산화 및 체내 지방 저하 효과가 있음이 시사 되어졌다. 먼저, Sprague-Dawley 수컷 흰쥐를 난괴법을 통해 하나의 정상군(Normal)과 대조군(Diabetic), 인슐린 처리군(Insulin), 0.5% 오디 투여군(0.5% Mulberry), 1.0% 오디 투여군(1.0% Mulberry) 그리고 2.0% 오디 투여군(2.0% Mulberry) 등으로 분류 한 후 STZ로 당뇨병을 유도하였다. 부안산 오디를 STZ에 의한 당뇨병 흰쥐에 4주간 투여한 후 정상군과 비교 했을때 다양한 농도의 오디를 투여한 그룹에서 몸무게, 혈중 인슐린의 농도는 감소했지만, 신장무게, 혈당량, 요량 및 음수량은 증가하였다. 추가적으로 STZ에 의한 당뇨병 흰쥐와 이들 동물들에게 오디를 투여한 그룹을 비교한 결과 오디를 투여한 그룹은 신장 무게, 혈당, 요량 및 음수량이 확연히 감소하였다. 또한 오디를 투여한 그룹에서의 인슐린 농도는 대조군과 비교하여 유의적으로 증가하였다. 이와 같은 연구결과로부터 부안산 오디는 향후 당뇨 합병증을 억제하기 위한 기능성 식품의 원료로 사용 되어질 가능성을 시사한다. This study was designed to evaluate the anti-diabetic effects of Buan mulberries by using an insulin- dependent diabetes mellitus animal model. Several studies have shown that mulberries have metabolism- improving, antioxidant, and lipid-lowering properties in rats with streptozotocin (STZ)-induced diabetes. In this study, Sprague-Dawley male rats were randomly assigned to 1 normal control group and 5 STZ-induced diabetes groups: rats that had STZ-induced diabetes and did not receive any agents (diabetic group; negative control), rats that had STZ-induced diabetes and received insulin (insulin group; positive control), rats that had STZ-induced diabetes and received 0.5% mulberry extract (0.5% mulberry group), rats that had STZ-induced diabetes and received 1.0% mulberry extract (1.0% mulberry group), and rats that had STZ-induced diabetes and received 2.0% mulberry extract (2.0% mulberry group). Mulberry extracts were administered to the diabetic animals for 4 weeks. The rats that received mulberry extracts showed lower body weights and insulin levels, as well as higher kidney weights, blood glucose levels, urine quantities, and water intake in comparison with the normal controls. Further, the insulin concentrations in the mulberry-fed animals were higher than those in the diabetic group, and the kidney weights, blood glucose levels, urine quantities, and water intake in the mulberry-fed animals were lower than the corresponding values in the diabetic controls. These results suggest that mulberry may be an effective functional food to prevent diabetes-related complications.

주령별 쥐 대뇌피질 신경 조직의 초기 발달단계에서 UDP-GlcNAc 함량 및 단백질의 O-GlcNAc 수식 변화

이윤희 ( Yoon Hee Lee ),김성민 ( Sung Min Kim ),박제권 ( Jae Kweon Park ),김성욱 ( Sung Oog Kim ),도수일 ( Su Il Do ),추영국 ( Young Kug Choo ),박용일 ( Yong Il Park ) 한국키틴키토산학회 2009 한국키틴키토산학회지 Vol.14 No.4

체내 포도당 영양분 대사와 이로 인한 UDP-GlcNAc의 생성 대사와 단백질들에 대한 O-GlcNAc 수식 및 세포표면 당 단백질의 시알산 발현은 신경계 기능에 중요한 영향을 미치게 된다. 이러한 대사의 변화를 이해하는 것은 뇌의 비정상적인 발달에 의한 선천성 뇌신경계 질환 혹은 알츠하이머병과 같이 특징적인 뇌대사의 저하를 보이는 병의 진단 및 평가에 중요하나, 현재 이에 관련된 기작이 명확하게 밝혀져 있지 않다. 이를 위한 기초연구로서, 본 연구에서는 임신한지 18일 되는 태아쥐와, 생후 하루, 4주, 8주째의 신생쥐 및 청년기쥐로부터 대뇌피질 조직을 분리하여 초기 뇌조직 발달에 있어서 UDP-GlcNAc 함량 변화, 단백질의 O-GlcNAc 수식 변화 및 시알산 발현 변화를 조사하였다. 단백질의 O-GlcNAc의 양은 쥐의 나이가 증가함에 따라 점차 증가하는 양상을 보인 반면, 전구체가 되는 UDP-GlcNAc의 양은 감소하는 양상을 보였으나, O-GlcNAc과는 반대로, 역시 UDP-GlcNAc을 전구체로 하여 생합성 되는 시알산의 경우는 뇌조직이 발달해 감에 따라 UDP-GlcNAc의 감소와 함께 점차 감소하는 경향을 보였다. 이는 포도당 영양분의 공급 및 UDP-GlcNAc 대사와 단백질의 O-GlcNAc 수식화(O-GlcNAcylation)와 시알산 발현이 보다 복잡한 메카니즘에 의해 조절됨을 의미하며, 관련된 조절 단백질, 혹은 효소에 대한 더욱 많은 연구가 필요할 것으로 사료된다. 이들의 대사 관계 및 조절에 대한 기작이 밝혀 진다면 알츠하이머 등 노화에 따른 인지능력 저하나 청소년기 비정상적인 발달에 따른 기형아 등 선천성 신경계 질환을 예방하거나 치료할 수 있는 단서를 제시할 수도 있을 것으로 사료된다. The extrinsic glucose supply, the biosynthesis of UDP-GlcNAc, protein O-GlcNAcylation and sialylation of cell surface proteins are now increasingly important aspects for normal function of brain neuronal system. As a preliminary study, we investigated changes in protein O-GlcNAcylation, sialylation and the content of UDP-GlcNAc in the rat brain cortical tissues of different ages, embryonic(E18), new-born(P0), 4-week(P4W) and 8-week-old(P8W) rats. As the rats grow up to young adult(8-week-old), O-GlcNAcylation in total proteins and number of protein species gradually increased along with sharp decrease in UDP-GlcNAc content. In contrast to O-GlcNAcylation, total sialic acid content in total proteins of each tissue showed an increase from E18 to P0 and then gradually decreased. These results may suggest that UDP-GlcNAc metabolism and protein O-GlcNAc modification and sialic acid expression are controlled by rather complicate mechanism during the normal development of early post-natal brain tissues. Elucidating the mechanisms of the metabolic processes between these factors and their regulation would be beneficial for the prevention and treatment of neurodegenerative diseases such as hereditary and Alzheimer`s diseases.

생쥐난자의 체외수정과 배발달에서 다양한 정자농도의 효과에 관한 연구

추영국 圓光大學校 基礎自然科學硏究所 1996 基礎科學硏究誌 Vol.15 No.1

체외수정율에 영향을 미치는 생쥐난자의 체외수정시 체외수정용 배양액소적의 양과 정자농도가 체외수정율 및 배발달율에 미치는 영향을 조사하였으며, 그 결과는 아래와 같았다. 1. 25,100 및 400㎕의 체외수정용 배양액소적에서의 체외수정율은 각각 44.0,83,3 및 50,3%였다. 2. 1×10^4,1×10^6 및 1×10^8의 정자농도를 이용하여 체외수정을 유도한 결과 체외수정율은 각각 34.1,77.1 및 50.6%였다. 3. 1×10^4,1×10^6 및 1×10^8에서 체외수정된 수정란의 정상적인 배반포기까지의 배발달율은 각각 0,40.0 및 30.0%였다. This study was carried out investigate whether the insemination drop volume and sperm concentration has been influenced upon the rates of in vitro fertilization, and to elevate utilizating efficiency and improve the fertilization efficiency of mouse oocytes in vitro. The results obtained in these experiments were summarized as follows: 1. The percentage of in vitro fertilization on differented insemination drop volume for 25, 100, 400㎕ was 44.0,83.3,50.3%respectively. 2. In order to determine more accurately at what sperm concentration during in vitro fertilization of oocytes can be fertilization by spermatozoa the oocytes were collected at 14­16 hr times after hCG injection and inseminated for various sperm concentration. 3. The percentage of oocytes developed to 2­cell stage following sperm concentration for 1×10^4,1×10^6 833, 960, 80%, repectively.

消癌散의 항암효과 및 血管新生抑制에 미치는 영향

김용수,이성원,추영국,정규용,안성훈,정우열,우원홍 대한동의생리학회,대한동의병리학회 2003 동의생리병리학회지 Vol.17 No.1

Cancer, which is expressed in various forms, is one of the leading causes of human death. Soamsan (SAS) is composed of ten medicinal herb, the prescription was made according to the principles of Oriental traditional medicine based on the concept of synergic effects and interaction of among the components. SAS has been used for the cancer therapy, but the mechanism of it's effect is not well known. In the present study, the cytotoxic effect of the SAS water extract on cancer cell lines was investigated by the method of MTT in A549 cell lines and the anti-angiogenic effect was shown in the assay of chorioallantoic membrane (CAM) and in the cornea of rat administerd orally with SAS water extraction. The viability of A549 cell lines was not affected by the whole extract of SAS but the n-Hexan fraction of SAS water extract showed strong cytotoxicity which was not seemed to be done by the apoptotic mechanism. SAS water extract showed inhibition effects of angiogenesis induced in the cornea of rat and CAM assay. As the above results, it is suggested that SAS can be a candidate for new prescription for cancer therapy.

혈관신생 및 이식암세포증식 억제를 통한 가미소암산의 항암작용연구

윤성찬,안성훈,문연자,김진강,추영국,정규용,김영목,우원흥 대한동의생리학회,대한동의병리학회 2003 동의생리병리학회지 Vol.17 No.4

Gamisoamsan is a prescription originated in Soamsan which is known as an anti-cancer remedy in the traditional Korean Medicine. To enhance the synergic effects of anti-cancer activity of Soamsan, this study reconstituted the original components of Soamsan with a slight modification and produced a novel herbal remedy, namely Gamisoamsan. To investigate the effects of Gamisoamsan on anti-cancer reaction, I studied the effects of Gamisoamsan on angiogenesis via chorioallantoic membrane (CAM) assay, corneal neovascularization assay and the effects on expression of growth factor which are VEGF, TGF-β, bFGF and IMUP-1. Anti-cancer effects of Garnisoamsan was also abserved through hematological parameters, tumor volume and survival rate in mice. Gamisoamsan inhibited embryonic angiogenesis of blood vessels in CAM assay and inhibited neovascularization of rat cornea. Gamisoamsan reduced cell proliferation in HT1080 cells and IC50 was 2.18 mg/ml. Gamisoamsan reduced the expression of VEGF, TGF-β, bFGF and IMUP-1 which was known as vascular growth factor and this effects of Gamisoamsan was predominant than VP-16. The treatment of Gamisoamsan decreased the CT-26 cell inoculated-tumor volume in mice colon adenocarcinoma and increased mice survival which was inoculated CT-26 cells. The results of the present study suggest that Gamisoamsan extracts has a potential anti-tumor activity and may be an useful remedy to prevent and/or treat cancer.