유전자재조합(형질전환체)과 윤리성

Dae-Yeul Yu(유대열) 한국실험동물학회 2003 한국실험동물학회 학술발표대회 논문집 Vol.2003 No.-

Studies on Aging Using Oxidative Stress Mouse Models

Dae-Yeul Yu(유대열) 한국실험동물학회 2010 한국실험동물학회 학술발표대회 논문집 Vol.2010 No.2

Aging is a multifactorial phenomenon characterized by a time-dependent decline in physiological function. The process of aging is one of the most complex and intriguing biological phenomenons. Through the years, hundreds (and perhaps more) of hypotheses have been proposed as potential reasons organisms age. One of the most studied and accepted hypotheses for the molecular basis of aging has been the oxidative stress theory of aging, which was first conceptualized by Denham Harman as the free radical theory of aging and has been modified to the Oxidative Stress Theory of Aging. The basis of this theory is that a chronic state of oxidative stress exists in all cells of aerobic organisms even under normal physiological conditions because of an imbalance between pro-oxidants and antioxidants, suggesting that antioxidants play an important role in protection of aging in mammalian cells. Several groups have genetically altered various components of the antioxidant defense system in mice to study the Oxidative Stress Theory of Aging. We already generated peroxiredoxin (Prx) Ⅰ and Ⅱ knockout mice to understand the roles of their proteins in vivo and are studying the mice to know whether Prxs are involved in protection of cellular senescence and organism aging. Underlying research results indicate that Prx Ⅰ and Ⅱ play a role in protection of cellular senescence in mice. In this symposium, I would like to introduce the results of transgenic/ knockout mice for antioxidant enzymes.

락토페린이 국내분리 유아 로타바이러스의 MA 104세포 감염에 미치는 영향

차광종,유대열,이종기,유제현,Cha, Kwang-Jong,Yu, Dae-Yeul,Lee, Chong-Kee,Yu, Jae-Hyeun 대한미생물학회 1999 Journal of Bacteriology and Virology Vol.28 No.1

It has long been known that lactoferrin prevents human beings from infection of virus. To prove this activity of lactoferrin, we evaluated the activities of different lactoferrins to an isolate human rotavirus K-21. Bovine lactoferrin inhibited infection of K-21 to MA-104 cell at the concentration of $25.9\;{\mu}M$ whereas bovine hydrolysed lactoferrin prevented rotavirus infection at $103.8\;{\mu}M$. However human lactoferrin prevented infection of K-21 at the concentration of $217.5\;{\mu}M$. These data suggested that lactoferrin activity may be unaffected by the intestinal digestive enzymes and bovine lactoferrin is more active than human lactoferrin with respect to prevention of rotavirus infection.

국내분리 유아 로타바이러스의 혈청형과 염기서열 분석

차광종,송진욱,조홍찬,김용휘,유대열,이중복,이종기,곡구효보,유제현,Cha, Kwang-Jong,Song, Jin-Ook,Cho, Hong-Chan,Kim, Yong-Hee,Yu, Dae-Yeul,Lee, Joong-Bok,Lee, Chong-Kee,Koki, Taniguchi,Yu, Jae-Hyeun 대한미생물학회 1999 Journal of Bacteriology and Virology Vol.28 No.1

Rotaviruses belong to Reoviridae causes diarrhea in human beings as well as domestic animals. This study was conducted to see what type of human rotaviruses are distributed in Seoul and Kyung-gi province. Twenty two of 81 patients showed rotavirus positive with diagnostic kit and RNA electropherosis. We isolated all of rotaviruses from the patients. Electropherotypes of 22 isolates showed 4:2:3:2 pattern whereas those migration patterns were long type. All of those isolates belonged to group A. Twenty out of 22 isolates reacted with monoclonal antibodies specific to G1, P1A and subgroup II, whereas rest of them, A-29 and K-30 reacted with subgroup I specific monoclonal antibody. The nucleotide sequence of an isolate K-21 showed $98{\sim}100%$ and $90{\sim}96%$ homologies with those of Wa and KU strain, respectively.

A Potential Demerit of the Pronuclear Microinjection Technique

왕애국,김선욱,문형배,현병화,남기환,서준교,김남순,유대열,이동석,Wang, Ai-Guo,Kim, Sun-Uk,Moon, Hyung-Bae,Hyun, Byung-Hwa,Nam, Ki-Hoan,Suh, Jun-Gyo,Kim, Nam-Soon,Yu, Dae-Yeul,Lee, Dong-Seok Korean Society of Life Science 2006 생명과학회지 Vol.16 No.4

Pronuclear microinjection (PMI) is a primary method for producing transgenic mice and offers a powerful tool for investigating gene function in vivo. The method has several reported advantages and disadvantages. Here, we report another potential shortcoming. The survival rate of fertilized one cell-stage embryos was significantly reduced after PMI procedure (65.4% (1202/1838)). In addition, the proportion of embryos developing to full-term was also significantly lower than that of embryos not undergoing PMI (26.5% (319/1202) vs 41.9% (57/136)). Moreover, 3 out of 21 (14.3%) founder control mice which were non-transgene-carrying littermates of transgenic founders showed histopathological changes in their liver, which was comparable to that in of transgenic lineages (4 out of 27 (14.8%)). In conclusion, the mechanical damages in chromosomes occurring during PMI procedure may be a potential factor influencing phenotypes of transgenic mice. 현재, 유전자의 in vivo 기능을 연구하기 위해 가장 많이 이용되고 있는 transgenic mice를 생산하기 위한 기본적으로 이용되고 있는 방법이 one cell-stage embryo에 pronuclear microinjection (PMI)이다. 그러나, 이 PMI 후에 one cell-stage embryo들의 생존율은 현저히 감소 (65.4%)할 뿐만 아니라 PMI 후의 embryo의 출생률(26.4%)이 PMI 처리를 하지 않은 것 (41.9%) 보다 현저히 낮다. 더욱이, PMI 방법에 의해 태어난 transgenic founder들의 간 조직에 병리학적 변화가 14.8% 정도에 대해서 같은 한배의 새끼 non-transgenic founder들의 경우도 간 조직에 병리학적 변화가 14.3%로 나타났다. 결론적으로, 이 PMI 방법에 의한 염색체에 물리적 손상은 형질전환 마우스의 생산 및 표현형에 영향을 미치는 잠재적 요소로 생각된다.

PCNA와 Cytokeratin 단클론 항체를 이용한 2중 면역조직화학 염색방법에 관한 연구

문형배 ( Hyung Bae Moon ),노종섭 ( Jong Sup Roh ),곽효일 ( Hyo Il Kwak ),유대열 ( Dae Yeul Yu ),한용만 ( Yong Mahn Han ),이경광 ( Kyung Kwang Lee ) 대한임상검사과학회 1996 대한임상검사과학회지(KJCLS) Vol.28 No.1

락토페린 구조 기능 및 생산에 관한 연구

유대열 한국유가공기술과학회 1997 Journal of Dairy Science and Biotechnology (JMSB) Vol.15 No.2

Lactoferrin is an 80 kDa, iron-binding glycoprotein present in milk and, to a lesser extent, in exocrine fluids such as bile and tears. It consists of a single-chain polypeptide with two globular lobes and is relatively resistant to proteolysis. Owing to its iron-binding properties, lactoferrin has been proposed to play a role in iron uptake by the intestinal mucosa and to act as a bacteriostatic agent by withholding iron from iron-requiring bacteria. Beside this, the functions proposed for lactoferrin are diverse and include immunomodulatory activity, regulation of myelopoiesis, cell growth promotion and differentiation, and antioxidant effects. Therefore lactoferrin has been a good target for commercial production. Until now lactoferrin has been purified from human, bovine, sheep, goat and horse milk. Complete amino acid sequences of the lactoferrin from human, murine, bovine, porcine, and caprine have been determined, either directly at the protein level or deduced from the nucleotide sequence and shown to display a high level of similarity. We, researchers in my laboratory, have been studied lactoferrin for mass production by using the techniques of transgenic animal, transgenic plant and microorganism. Here we present the data on structure, function and production of lactoferrin.

Pathologic and Immunohistochemical Studies on the Hepatocellular Carcinoma in the HBx Transgenic Mice

Moon, Hyung-Bae,Yu, Dae-Yeul,Lee, Kyung-KWang,Han, Yong-Mahn,Yun, Ki-Jung,Han, Won-Cheol,Kim, Bo-Yong,Chung. Yung-Jin,Shin, Dae-Kyun 圓光大學校 醫科學硏究所 1998 圓光醫科學 Vol.14 No.2

B형 간염바이러스(HBV) 감염은 만성간염, 간경화증, 및 간암의 발생과 밀접한 관계를 가지고 있으며, HBV에 존재하는 x항원(HBx)은 HBV 유전자의 발현이나 HBV 증식에 관여할 뿐 아니라 간세포암의 발생에 중요한 역할을 한다고 알려져 있다. 한편 분자생물학적으로 클론된 유전인자들을 포유동물의 germline에 이식하여 이식된 유전인자가 다음세대로 유전되게 하여 인체 질환을 실험동물에 유발시키는 형질전환동물 기법이 개발된 후, 인체질환의 연구에 형질전환동물이 많이 이용되고 있다. 이에 따라 HBx와 간암의 발생관계를 연구하기 위하여 HBx 형질전환 마우스를 개발한 결과 간세포암이 발생하여 이들에 대한 병리학적 및 면역조직화학적 연구를 시행한 결과는 다음과 같다. 1. 4개월부터 HBx 형질전환 마우스 모든 예에서 간세포의 공포성 변화 및 다양한 크기의 핵을 가진 변형세포 군집(Altered foci)이 관찰되었으며, 6개월부터는 육안적으로 종양은 발견되지 않지만 조직학적으로 간세포암의 특성을 보인 소결절성 병변(small nodular lesions)이 75%의 동물에서 관찰되었으며, 11개월부터 육안으로 확인 가능한 간세포암이 60%의 실험동물에서 관찰되었다. 2. 소결절성 병변 및 간세포암의 조직학적 특징은 다양한 형태 및 과염성 핵을 가지고, 핵과 세포질의 비는 감소하였으며, 간혹 비정상적인 핵분열이 관찰되었지만, 종양주위의 섬유화는 관찰되지 않았고, 이들 병변에서 증식세포핵항원 (Proliferating cell nuclear antigen)은 현저히 증가하였다. 3. HBx 단백에 대한 면역조직화학 염색 결과 변형세포군집, 작은 결절성 병변 및 간세포암 모두에서 HBx 양성소견을 나타냈으며 이의 분포양상은 비특이적이었다. 4. 유식세포분석기를 이용한 DNA ploidy 검사에서 변형세포군집 및 소결절성 병변에서는 DNA 2배수성 또는 4배수성이 관찰되었으며, 간세포암 3예 중 2예에서는 비배수성을 나타냈다. 이상의 결과로서 HBx 형질전환마우스에서 간세포암의 발생이 확인되었으며, HBV 감염에 의해 간암이 발생되는 과정에서 HBx 단백이 중요한 역할을 함을 알 수 있었고, HBx 형질전환 마우스는 간암의 연구에 중요하게 이용될 수 있을 것으로 사료되었다. Chronic infection with hepatitis B virus (HBV) is associated with a high incidence of liver disease, including chronic hepatitis, liver cirrhosis and HCC (HCC). The hepatitis B virus-encoded X antigen (HBx) stimulates virus gene expression and replication, which may be important for the establishment and maintenance of the chronic carrier state. The HBx protein, acting as a transcriptional transactivator of viral genes, may alter host gene expression and lead to the development of HCC. It has been reported by Kim et al in 1991 that HBx transgenic mice can have HCC. However Lee et al could not find HCC in their transgenic mouse system using the HBx gene. To confirm whether HBx could cause HCC in transgenic mice and present a useful model system for defining the molecular events for the transformation of HCC. We recently have generated transgenic mice by introducing HBx gene of the HBV into the mice. This study was carried out to examine the histopathologic and immunohistochemical characteristics of the liver in newly developed HBx transgenic mice. The incidence of HCC after 11 months was 60% (3/5). HCC were identified by gross examination and that indicates pleomorphic or hyperchromatic nuclei and partially infiltrative growing without surrounding fibrosis and that have DNA aneuploidy by flow cytometry in two of three cases of HCC. The incidence of small nodular lesions after 6 months was 75%(6/8) in the HBx transgenic mice, but small nodular lesions were not identified by gross examination and had same histological characteristics to HCC. Altered foci which revealed vacuolar changes in the cytoplasm of hepatocytes were found in all HBx transgenic mice from 4 months. DNA diploidy or tetraploidy were found in the tissue of the altered foci and small nodular lesions. The proliferation cell nuclear antigen (PCNA) markedly increased in small nodular lesions and HCCs in the transgenic mice. The HBx protein was expressed in the cytoplasm of all lesions including altered foci, small nodular lesions and HCC. These result indicated HCC can develop in the HBx transgenic mice and suggested that HBx protein can make the hepatocytes more susceptible to secondary events in hepatocarcinogenesis after HBV infection in human beings.

결핵성 육아종에서 Thioredoxin peroxidase-2 의 발현

박근호,유형륜,정영진,윤기중,한원철,유대열,문형배 圓光大學校 醫科學硏究所 1999 圓光醫科學 Vol.15 No.2

Background: Thioredoxin peroxidase(TPX) is a kind of recently discovered antioxidant enzyme which react as rapid hydrogen ion donor for the removal of hydroperoxide. The action and distribution of the TPX was poorly understood in the human diseases. This experiments were designed for the study about the distribution of the TPX in the chronic granulomatous inflammation and about the correlation between the expression of TPX and the site of inflammation, histological activities of tuberculous inflammation or existence of mycobacterium in the inflammatory foci. Methods: The immunohistochemical stains were performed for the localization of the TPX-2 in the epithelioid cells, giant cells and lymphocytes in the chronic granulomatous inflammation. The tissue sections were obtained from the paraffin blocks of the 54 cases of tuberculosis (lung 21 cases, lymph node 12 cases, bone and soft tissue 12 cases, kidney 9 cases; active 33 cases, inactive 21 cases by the histologic classification; presence of mycobacterium 15 cases, no mycobacterium 39 cases by PCR reaction). Results: The expression of TPX-2 was 16.7% in the giant cells, 27.8% in the epithelioid cells and 100% in the lymphocytes of tuberculous inflammations. The expression of TPX-2 in the giant cells and epithelioid cells of the tuberculosis were 28.6% and 57.1% of the pulmonary tuberculosis; 33.3% in each cells of the renal tuberculosis; 0% in each cells of the lymph node or bone and soft tissue tuberculosis. The expression of TPX-2 in the giant cells and epithelioid cells were 9.1% in each cells of the active tuberculosis and were 28.6% and 57.1% in each cells of the inactive tuberculosis by histologic classification. The expression of TPX-2 in the giant cells and epithelioid cells was 40% in each cells of tuberculosis which mycobacteria were detected and the expression of TPX-2 was 7.7% and 23.1% in each cells which mycobacteria were not detected by PCR reaction in the paraffin embedded tissue. Conclusions: The above results were summarized that the TPX-2 in the giant cells and epithelioid cells were more frequently expressed in the inactive tuberculosis than in the active tuberculosis. These results suggest that the TPX-2 is a kind of regulating or suppressing factors in the activity of the tuberculosis.

HBx형질전환 생쥐에서 발생한 간세포암종에서 H-ras 및 c-myc의 발현에 관한 연구

문형배,소병준,김학철,윤기중,한원철,조향정,유대열,정영진 圓光大學校 醫科學硏究所 2002 圓光醫科學 Vol.17 No.2

<연구목적> HBx형질전환 생쥐에서 발생한 간세포암종의 발암과정에 종양유전자(H-rgs, c-myc)의 발현 정도를 조사하고자 하였다. <연구방법> 정상생쥐 12마리(4-18개월) 및 HBx 형질전환 생쥐 44마리(4-18개월)를 대상으로 포르말린에 고정하고 파라핀에 포매한 간 조직을 이용하여 면역조직화학적염색을 실시하였다. 실험군은 정상 부위, 이형성 부위 및 종양 부위로 구분하였으며, 종양 부위는 소결절성병변 부위와 간세 포암종 부위로 구분하였고, 이형성병변 부위는 이형성병변만 발견되는 부위, 소결절성병변과 동반된 이형성병변 부위 및 간세포암종과 동반된 이형성병변 부위로 구분하였다. <연구결과> H-rgs의 발현은 정상 간조직에 비하여 이형성병변 부위(P<0.05) 및 종양 부위(P<0.01)에서 증가하였으며, 소결절성병변 부위과 간세포암종 부위 사이에서는 간세포암종 부위에서 증가된 경향이었으나 통계학적으로 유의한 차이가 없었으며, 각 이형성병변 부위 사이에서도 유의한 차이는 없었다. c-myc의 발현은 정상 간조직 및 이형성병변 부위에 비해 종양 부위에서 증가하였으며(P<0.001), 소결절성병변 부위와 간세포암종 부위에서는 비슷하였고, 각 이형성병변부위 사이에서도 비슷하였다. <결론> HBx형질전환 생쥐에서 발생하는 간세포암종의 발생에 H-rgs는 이형성 변화를 일으키는 시기에 관여하며, c-myc은 이형성병변에서 암으로 이행하는 시기에 관여할 것으로 생각한다. Background: This experiment was designed for the expression of H-ras and c-myc in hepatocarcinogenesis of the HBx transgenic mice. Methods: Immunohistochemical stains in the paraffin embedded tissue of the liver were used for the detection of H-ras and c-myc in the 12 normal mice and 44 HBx transgenic mice of the 4-18 month old. Results: Expression of the H-ras was significantly increased in the dysplastic area (P<0.05) and tumor area (P<0.01) than in the normal liver. But there were no differences of H-ras expression between areas of microscopically identified hepatocellular carcinoma (MI-HCC) and grossly identified hepatocellular carcinoma (GI-HCC) and dysplastic areas among the only dysplastic areas, dysplastic areas with MI-HCC and GI-HCC. Expression of the c-myc was significantly increased in the tumor area (P<0.001) than in the normal liver and dysplastic area. But there were no differences of c-myc expression between areas of MI-HCC and GI-HCC, and dysplastic areas among only dysplastic areas, dysplastic areas with MI-HCC and GI-HCC. Conclusions: Our study suggests that H-ras is related to the dysplastic change and c-myc is related to the neoplastic change in the hepatocarcinogenesis of the HBx transgenic mice.