마우스 배아줄기세포에서 정상 및 비정상 간장실질세포 증식기전 연구 Establishment of mouse embryonic stem cells from TIS21 knock out mouse and detection of altered hepatocytes among regenerating liver cells원문보기
연구배경: 암세포는 최종분화 과정을 거치지 못한 미분화 또는 탈분화 상태의 세포라는 병리학적 성질에 근거하여 간암발생기전 연구의 일환으로써 배아줄기세포주 (MES) 확립과 간장세포로의 분화 유도를 통한 간장기능회복을 목적으로 시도되었기에 다음과 같은 목표를 설정하였다. 단계별 목표: 1단계- antiproliferative gene family 에 속하는 TIS21-knock out mice 제조 및 미분화도 측정. 2단계-MES의 간장세포로의 분화 유도, 간장 progenitor cell study, TIS21 및 andro
연구배경: 암세포는 최종분화 과정을 거치지 못한 미분화 또는 탈분화 상태의 세포라는 병리학적 성질에 근거하여 간암발생기전 연구의 일환으로써 배아줄기세포주 (MES) 확립과 간장세포로의 분화 유도를 통한 간장기능회복을 목적으로 시도되었기에 다음과 같은 목표를 설정하였다. 단계별 목표: 1단계- antiproliferative gene family 에 속하는 TIS21-knock out mice 제조 및 미분화도 측정. 2단계-MES의 간장세포로의 분화 유도, 간장 progenitor cell study, TIS21 및 androgen receptor에 의한 암발생기전연구 시도 연구내용: I-1) TIS21KO 마우스 제조를 시도하였던 $C57BL/6{\times}129svJ$ 마우스 MES 10 lines, C57BL/6 in-bread MES 4 lines, TOBKO-MES 12 lines, TIS21KO-MES 3 lines를 확립하여 미분화도 측정 (SSEA-1, c-kt 발현 및 CD45, CD34 미발현 측정). 최종적으로 ES cell을 SCID mouse 에 주사 후 teratoma 형성을 조사하여 stemness 확립여부 조사함. I-2) 분화능 조사위하여 embryoid body 형성하여 특히 간장세포기능 가진 세포의 존재를 확인함 (간장세포마커 RT-PCR, indocyanine green uptake, 배설능 조사) II-1) TIS21 같이 antiproliferative gene 에 속하는 TOB-KO mouse 도입하여 간장실질세포 분리 후 1차 배양계에서 liver progenitor cell 특성 규명함. II-2) TOBKO-MES 에서 간장실질세포로 분화 시도함. II-3) TISKO mouse 제조하였으며, TIS21 결함으로 늑골 형성에 이상 오는 표현형 확인 및 bone morphogenic protein activator 임을 확인함. II-4) TIS21KO mice 비장과 골수 progenitor cell 분리 후 FACS 분석으로 c-kit, Sca-1 발현이 정상대조군에 비하여 작지만 의미있게 증가되어 있음을 확임. 면역계의 이상이 시사되었음. III. in vitro TIS21 기능 연구하여 TIS21에 의한 증식억제 기전을 규명함: TIS21-cyclin B1 결합으로 G2/M phase 억제 및 TIS21-Pin-1 결합 후 mitochondrial depolarization 유도함을 밝힘. IV. 마우스 간발암 실험을 시도하여 남성호르몬이 간암발생에 미치는 연구 진행하였음. IV-1) 간장실질세포 증식억제 및 간암세포증식을 촉진하는 rat TGF-b1 promoter DNA cloning 하여 보고하였으며, DHT-AR complex 가 TRG-b1 발현 촉진함을 규명함 IV-2) AR 는 b-catenin 과 결합하여 b-catenin nuclear translocation을 통한 간발암작용 촉진함을 규명하면서, protein phosphatase 1/2A 억제제인 nodularin 에 의해 b-catenin threonine 잔기 인산화를 규명하여 보고 중임. IV-3) AR 과발현된 간암세포는 오히려 DHT 존재 시에 간암세포 증식을 억제하며, 호르몬 부재 시에 암세포증식을 촉진함을 발견하여 연구 중임.
Abstract▼
I. Purpose and aims of proposal: Based on the undifferentiation or dedifferentiation properties of cancer cells, we planned to establish mouse embryonic stem (MES) cells using the TIS21KO-and TobKO-mice as well as their control mice for restoration of liver function. The specific aims of this projec
I. Purpose and aims of proposal: Based on the undifferentiation or dedifferentiation properties of cancer cells, we planned to establish mouse embryonic stem (MES) cells using the TIS21KO-and TobKO-mice as well as their control mice for restoration of liver function. The specific aims of this project include 1) preparation of MES cells and characterization of their toti-potent properties for cell therapy trial 2) preparation of TIS21KO mice and study on liver progenitor cells in the KO mice 3) in vivo hepato-carcinogenesis study focused on the role of androgen receptor-testosterone complex, and 4) characterization of TIS21, a tumor suppressor protein. II. Content and scope of proposal: 1. Preparation and characterization of MES cells isolated from TIS21(Tob)-KO mice as well as their counterpart control mice. 2. Study on liver progenitor cells in the primary culture of hepatocytes isolated from the livers of KO and control mice. 3. Study on carcinogenesis; 3-1) in vivo animal study focused on the potential role of male sex hormone and its receptor, AR. 3-2) in vitro characterization of TIS21 protein and a potential mechanism of cell death induced by overexpression of TIS21 in tumor cells. III. Results of proposal: 1. MES cell preparation: ES cells were established from the TIS21(Tob)-KO and their control mice, and their stem cell properties were then characterized by FACS analyses using anti SSEA-1 and c-kit antibodies. The established MES cells were able to differentiate into hepatoblasts on the 18th day of embryoid body formation (table 1). 2. Study on adult liver progenitor cells: Primary culture of hepatic parenchymal cells isolated from the KO and control mice revealed that the expression of cyclin D1 and 3H-thymidine incorporation were significantly increased in the TobKo mice as compared with that in the control. At the same time, c-kit and Sca-1 expressions were also increased in the KO cells. The cells isolated from the spleen and BM of the TIS21KO mice also revealed increased progenitor cell population, when examined by FACS analysis with anti-c-kit and anti-Sca-1 antibodies. 3. Carcinogenesis study; 3-1) To investigate a potential role of male sex hormone and its receptor (AR), we isolated rat TGF-I promoter (-4784 to +68), based on our previous report (GenBank AF249327), and examined the regulation of its promoter activity by dihydrotestosterone (OHT) in Huh7 cells. Putative androgen response sequence half site (5'-TGTCCT-3') was identified to be located within -1932 to -1927, proved by mutant (S'-AGACCT-3') analysis and chromatin immunoprecipitation (ChIP) assay. Up-regulation ofTGF-1 expression mediated by AR was confirmed by hepatocellular carcinoma developed in nude mice with AR-overexpressed Huh7-cells. 3-2) In vitro characterization of TIS21 protein was carried out. Furthermore, the potential mechanism of cell death induced by overexpression of TIS21 in tumor cells was found to be through its binding to cyelin B I/cdc2 complex and Pin-I at the specific site of TIS2 I protein, which could regulate G21M phase of tumor cells, independent of p53 and caspase activation. IV. Future application of the proposal: 1. MES cell lines; When we establish the method of differentiation of ES cells to hepatocytes, the cells can be used for cell therapy on mice with acute and chronic liver failure. 2. Liver progenitor study; The data obtained from the liver progenitor cells of Tob(TIS2 I )KO mice will be employed to discriminate liver regeneration from carcinogenic proliferation. 3. Carcinogenesis study; As mentioned above, overexpression of AR induced cellular senescence rather than tumor cell proliferation in the presence of testosterone. Therefore, we plan to convert hepatoma cells to senescent cells. At the same time, we will attempt to induce cell death of TlS21-overexpressed cells, especially in hepatoma cells.
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