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피부발암원성 평가를 위한 HPV 16 E6/E7 유전자 형질전환 마우스
류재웅(Zae Young Ryoo),김길수(Kil-Soo Kim) 한국실험동물학회 2005 Laboratory Animal Research Vol.21 No.2
사람 유두종바이러스 16형(HPV16)은 자궁경부암 발생의 주요한 원인으로 알려져 있다. HPV16의 생체내 활성을 편평상피세포에서 발현시켜 알아보기 위하여 HPV16 E6/E7형질전환마우스를 개발하였다. 전체적으로 사람 keratinocyte 14(hK14) 프로모터에 HPV16 E6/E7 유전자가 삽입된 형질전환마우스는 신생자에서 모발의 출현이전에 나타나는 주름진 피부(wrinkled skin)를 포함하여 성숙개체에서의 비후화된 귀(thickened ears)와 탈모 등과 같이 다양한 표현형을 보인다. 심한 주름진 피부와 발모지연을 나타내는 형질전환 마우스에서 동물은 3-5주령에서 사망하였다. 조직학적인 분석에서 E6/E7 유전자는 형질전환마우스에서 상피증식(epidermal hyperplasia)을 일으키는 것을 확인하였다. 이러한 상피증식은 구획의 증식과 각화세포층의 확장을 특징으로 hyperkeratosis와 관련되어 있다. 피부의 증식도 관찰되었다. glutathione peroxidase(GPx)와 Cu/Zn superoxide dismutase(Cu/ZnSOD)의 효소활성도가 형질전환 마우스에서 감지되었고 이는 피부에서 keratinocyte와 관련되어 있다. 그러한 활성도는 정상 마우스에 비하여 형질전환 마우스에서 유의성 있게 높았다. 그리하여 이들 형질전환마우스는 hyperkeratosis에서 항산화효소의 표현에서 유용할 것으로 보이며, 여러 종류의 피부암의 처치에서 유전자치료의 적용을 히는 데에도 유용할 것으로 판단된다. Human papillomavirus type 16 (HPV16) has been known to the major factor for the development of uterine cervical carcinomas. We have extended these studies to investigate the in vivo activities of HPV16 E6/E7 when expressed in squamous epithelia of transgenic mice. Grossly, hK14 HPV16 E6/E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In the transgenic mice, the wrinkled skin phenotype on the body and legs died at the age of 3-4 weeks. Histological analysis of demonstrated that E6/E7 causes epidermal hyperplasia in multiple transgeniclineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratinocyte and was associated with hyperkeratosis. These transgenic mice expressed E6/E7 transgene mainly in skin, heart, pancreas and kidney. Hyperplasia was found at the skin. The enzyme activities of glutathione reductase (GR), glutathione peroxidase (GPx), and Cu/ZnSOD were measured from the transgene cause keratinocyte at the skin. The specific enzyme activities were significantly higher in transgenic mice skin compared to the normal mice skin. Thus, these transgenic mice may be useful for the expression of antioxidant enzymes in hyperkeratosis or other therapies for HPV-associated skin cancer.
Dong Hoon Yu,Zae Young Ryoo(류재웅) 한국실험동물학회 2008 한국실험동물학회 학술발표대회 논문집 Vol.2008 No.-
The circling (cir/cir) mouse is one of the murine models for human non-syndromicdeafness DFNB6. The mice have abnormal circling behavior, suggesting a balanced disorder and profound deafness. The causative gene was trans membrane inner ear (tmie) gene of which the mutation is a 40-kilobase genomic deletion including tmie gene itself. In this study, tmie-over expression trasngenic mice were established. Individuals with germline transmission have been mated with circling homozygous mutantmice (cir/cir) in order to produce the transgenic mutant mice (cir/cir-tg) as agenetherapy. After the genotyping, phenotypic analyses were performed so that the insertion of the new gene might compensate for the diseases such as hearingloss, circling behavior, or swimming inability. Some individuals exhibited complete recovery in their behavior and hearing but the others did not show any amelioration in behavioror hearing. Individual mice had very different levels of tmie transgene expression in the cochlea. These results clearly indicate that tmie protein plays an important role when the appropriate expression level of tmie was expressed in the innerear. The protein levels were variable in each individual and these are thought to induce the differences in disease amelioration levels.
Hei Jung Kim(김혜정),IngNyol Jin(진익렬),Zae Young Ryoo(류재웅) 한국생명과학회 2008 생명과학회지 Vol.18 No.5
류마티스 관절염은 활액막 내의 염증성 세포와 cytokine의 침적으로 인한 만성적인 염증이 계속 진행되어 연골과 뼈의 파괴 등이 발생하는 특징을 가진 심각한 자가면역질환의 일종이다. Calcineurin은 면역 세포의 활성화를 책임지는 대표적인 세포 내 신호전달 단백질이다. Calcineurin의 억제제로 알려진 Cabin-1을 과발현시키는 유전자 치료를 통해 류마티스 관절염의 새로운 치료법을 시도하게 되었다. 본 연구에서는 EL-4 세포에 calcineurin 저해제인 Cabin-1을 과발현시켜 배양된 세포에서 유전자의 발현정도와 발현의 지속성을 측정하고자 하였다. 또한 형질전환된 류마티스 모델 동물의 간, 림프절, 비장 등의 조직에서 Cabin-1 단백질 발현여부를 조사하고자 하였다. 그 결과 calcineurin이 EL-4 세포에서 발현함을 확인하였으며 calcineurin의 생리학적 억제제인 Cabin-1유전자를 transfection했을 경우 calcineurin phophatase activity가 감소됨을 확인하였다. 또한 Cabin-1의 생체 내 기능을 해석하기 위해 형질전환 생쥐를 생산하였으며 간, 비장, 폐, 말초혈액단핵세포 및 림프절에서 Cabin-1의 발현을 검증하였다. 그러나 Cabin-1 유전자가 T 세포의 발생과 활성에 영향을 끼쳐 심한 면역결핍증상으로 인해 Cabin-1 형질전환 생쥐의 계통을 유지할 수가 없었다. 결론적으로 calcineurin을 억제하는 Cabin-1유전자의 발현벡터를 구축하였으며 이는 in vivo에서 Cabin-1유전자와 calcineuirn사이의 기작 및 Cabin-1의 기능을 해석하는데 중요한 매개가 될 수 있다. 또한 류마티스 관절염을 포함하여 루푸스, 다발성 경화증, 당뇨병 등과 같은 다양한 면역질환의 발병 원인을 밝혀내고 치료에 응용될 수 있다. Rheumatoid arthritis (RA) is characterized by a tumor-like expansion of the synovium, which is composed of proliferation, infiltrating leukocytes and angiogenesis. Calcineurin is expressed and plays a critical role in inflammatory arthritis. Calcineruin binding protein (Cabin)-1 associates with calcineurin, leading to a non-competitive inhibitor of calcineurin activity. We investigated the expression pattern of calcineurin in inflammatory arthritis. The overexpression of Cabin-1 inhibited calcineurin phosphatase activity in in vitro. Moreover, we produced the T cell-specific Cabin-1 transgenic mice but they died without transmitting the transgene to offspring. The Cabin-1 transgenic mice have a dominant lymph node expression and a wide tissue distribution. It is postulated that transgenic mice died from the critical effects of the transgene on T cell development or activation. Data suggests that the inhibition of calcineurin by the overexpression of Cabin-1 may be an effective strategy for the suppression of inflammatory arthritis.
Dong Hoon Yu,이준규,Hyung Soo Yuh,Seo Jin Park,Hei Jung Kim,Ki Beom Bae,Young Rae Ji,Na Ri Kim,박시준,Do Hyung Kim,김성현,Myoung Ok Kim,이정웅,Zae Young Ryoo,류재웅 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.9
Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular-superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal odels of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC- SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1β, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.
Vasopressin-SV40T Antigen Expression in Transgenic Mice Induces Brain Tumor and Lymphoma
Ryoo, Zae-Young 가톨릭중앙의료원 가톨릭암센터 2002 암심포지움 Vol.- No.2
Little is known about the regulatory mechanisms of vasopressin(VP) gene expression. To understand the importance of various cis-acting elements in the regulation of VP gene expression, we have produced the transgenic mice regulated by VP constructs containing 3.8 kb of the 5'flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to a SV40 T antigen(Tag). The two VP-transgene constructs differed by the lengths of their VP gene 3'flanking regions(2.1 versus 3.6 kb). In pVPSV.IGR3.6 construct, all six of founder transgenic mice were died at the age of 2∼6 weeks. Among three transgenic mice in pVPSV.IGR2.1 construct, one transgenic line expressing high levels of SV40 Tag was propagated. The founder and all transgene positive adult mice have appeared with shorten mortality or apparent phenotypic abnormalities and 21% mice showed brain tumor at 5 week and 100% mice showed brain tumor after 24 week. Histological analysis of transgenic mice showed that tumor developed in brain is similar to primitive neuroectodermal tumors (PNET) and tumor in spleen and lymph node is similar to lymphoma in human. The phenotype differences the two transgenic mice, therefore, suggest that the tissue-specific expression might be regulated by cis-acting elements in 1.5 kb 3' flanking region. Which is not contained in pVPSV.IGR2.1. Therefore, the pVPSV.IGR2.1 mice will provide a valuable mouse model system for studying PNETs tumorigenesis in brain and for studying tumorigenesis in lymph node and spleen.