생쥐에 있어서 사람 성장호르몬 유전자의 발현

이경광(Kyung Kwang Lee),한용만(Yong Mahn Han),남궁욱(Uk Namgung),이철상(Chul Sang Lee),김용주(Yong Joo Kim),김재만(Jae Man Kim),김지영(Ji Young Kim),한문희(Moon Hi Han) 한국축산학회 1989 한국축산학회지 Vol.31 No.3

The structural gene of human growth hormone was fused with mouse MT-1 promoter and the fusion plasmid was designated as pMThGH. The 2.6Kb BamHI/Bst EⅡ DNA fragment of pMThGH was used as a source of human growth hormone fusion gene in these studies. For microinjection, DNA fragments were diluted with TE buffer in the range of 2-8ng/ul. Fertilized one-cell eggs were flushed from the oviducts on 22-24 hours after HCG injection. To remove cumulus cells, cumulus cell masses were treated with hyaluronidase (300units/ml) and then washed 3 times in fresh medium. The obscure pronuclei of eggs due to hybrid(C57BL/6x CBA) mouse were visible after centrifugation for 3 min. at 15,000rpm. Visualization of nuclear structures was aided by the use of a differential interference contrast microscope and microinjection was carried out under x200 magnification using a Leitz micromanipulator. The microinjected eggs were surgically transferred to the oviducts of synchronized recipients. The litters developed from microinjected eggs were weaned about 4-6 weeks after birth and a piece of tail from each mouse was cut off about 1-1.5cm to analyze foreign DNA. The genomic DNAs isolated from each tail were analyzed by dot hybridization and Southern blot to determine which mouse carried MThGH genes. The results obtained in these experiments were summarized as follows; I. Of 106 microinjected eggs cultured for 96 hours in vitro, 53 eggs(50.0%) were developed to normal blastocyst stage. 2. When microinjection was carried out in the medium with or without Cytochalasin B(5 ug/ml), survival rate of eggs was 72.9% and 50.2%, respectively. 3. 382 microinjected eggs were transferred to the oviducts of 23 recipients and 140 mice consequently developed from these eggs. 4. Of 77 mice analyzed by dot hybridization and Southern blot, 6 mice were positive for MThGH genes and each carried at least one copy of hGH gene. 5. Transgenic mice had very high levels of hGH in their serum, 450 - 6, 100mg/ml, and body weights of them were heavier 1.5-2.0 times than those of controls.

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

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.

한국재래흑염소에서 발정 및 과배란 유도와 외래유전자 주입에 적합한 1세포기 수정란의 채취

신상태,이두환,김명철,이운규,이철상,한용만,이경광 충남대학교 수의과대학 동물의과학연구소 1998 動物醫科學硏究誌 Vol.6 No.-

Three different treatments for induction of estrus in Korean native black goats were compared: follicle stimulating hormone(FSH, FSH-p^TM), FSH combined with MAP(intravaginal impregnated sponges, Veramix^ⓡ, containing 60 ㎎ medroxy progesterone acetate for 14 days), and FSH combined with progesterone(Ovaron^ⓡ, 10 ㎎ IM for 10 days) and PGF_2α(Lutalyse^ⓡ, 3 ㎎ IM at first FSH injection). FSH for inducing estrus and superovulation was given a total 20 ㎎ intramuscularilly in decreasing dosage injections twice daily over 4 days. The MAPs were withdrawn at the 3rd day of FSH injection. Estrus observations were conducted every 6 hours from last FSH injection for 24 hours by placing the does with fertile male goats. Estrus and superovulation were more successfully induced with treatment of MAP + FSH than other treatments(FSH only, or progesterone + PGF_2α + FSH) (estrus induction; 100 vs 42.8 and 71.4%, ovulation points; 11.4 vs 5.4 and 4.4, respectively). The effect of gonadotropin releasing hormone(GnRH) on the ovulation rate was also examined. However, no difference was observed for inducing ovulation with treatment or dosage(100 ㎍ 200 ㎍) of GnRH. Low midline laparotomies were performed, and then ovarian responses (ovulations and follicular development) were examined by exteriorization of the reproductive tracts. Ova were recovered from oviducts by retrograde flushing 60-146 hours after MAP removal, and were classified the developmental stages. Overall 66.1% (236/357) of recovery rate was obtained from 30 superovulated does. The optimal recovery time of microinjectable 1-cell zygotes was approximately 72-76 hours after MAP removal.