초록 ▼

본 연구는 인체 말초혈액의 T 임파구를 polyclonal activation하여 proliferation을 유도하였을 때, G1 phase에서 특이적으로 그 활성이 증가하는 새로운 autophosphorylating protein kinase (MW ∼21 kDa, p21-autokinase)의 특성을 조사하여 인체 T 임파구의 activation과 cell cycle regulation에 있어서의 그 기능을 규명하는 것을 목표로 한다. $\rtrif$ Preparative 2D-electrophoresis를 이용한 p21-aut

본 연구는 인체 말초혈액의 T 임파구를 polyclonal activation하여 proliferation을 유도하였을 때, G1 phase에서 특이적으로 그 활성이 증가하는 새로운 autophosphorylating protein kinase (MW ∼21 kDa, p21-autokinase)의 특성을 조사하여 인체 T 임파구의 activation과 cell cycle regulation에 있어서의 그 기능을 규명하는 것을 목표로 한다. $\rtrif$ Preparative 2D-electrophoresis를 이용한 p21-autokinase의 정제 $\rtrif$ p21-autokinase의 polyclonal antibody의 제조 $\rtrif$ Phosphoaminoacid analysis에 의한 p21-autokinase의 autophosphorylating site의 규명 $\rtrif$ Human Jurkat T cells의 cell cycle진행을, hydroxy urea 와 nocadazole 등으로 blocking한 후 이어서 각 blocking으로부터 release하는 방법으로 동조시키고 이때 확보되는 cell cycle의 각 단계별 cells을 이용하여 p21-autokinase의 세포주기별 발현양상 및 autophosphorylating kinase의 활성변화를 검토 $\rtrif$ 본 연구에서 제조되는 p21-autokinase의 polyclonal antibody를 이용하여 Jurkat cDNA library로부터 p21-autokinase cDNA의 cloning 및 sequencing $\rtrif$ Yeast Two Hybrid System등을 이용한 p21-autokinase의 기질탐색 $\rtrif$ Western 및 Northern blot analysis에 의한 p21-autokinase의 tissue-specific expression 검토 $\rtrif$ p21-autokinase의 human leukemia 및 cancer cell lines에서의 발현양상 검토 $\rtrif$ p21-autokinase의 T cell activation에 있어서의 기능 규명 $\rtrif$ p21-autokinase의 기능 규명을 통하여, 인체 T 임파구의 activation 및 cell cycle regulation 기전에 대한 이해증진 $\rtrif$ p21 autokinase의 암 관련성 규명가능 $\rtrif$ p21-autokinase의 활성이 T cell activation에 있어서 G1/S transitoin에 essential 한 것으로 규명되면 p21-autokinase가 T 임파구 관련 면역기능의 억제제 개발의 새로운 target으로 활용가능. $\rtrif$ 분자량이 ∼21 kDa인 새로운 autophosphorylating protein kinase (p21-autokinase)의 특허등록

Abstract ▼

To characterize the functional role of an autophosphorylating protein kinase (MW ∼ 21 kDa, p21-autokinase) that is identified by kinase renaturation assay and is dominantly expressed in G1 phase of human peripheral T cells after polyclonal activation, the autokinase has been purified by preparative

To characterize the functional role of an autophosphorylating protein kinase (MW ∼ 21 kDa, p21-autokinase) that is identified by kinase renaturation assay and is dominantly expressed in G1 phase of human peripheral T cells after polyclonal activation, the autokinase has been purified by preparative 2D-electrophosresis system. MALDI-TOF mass spectrometric analysis of the p21-autokinase hase been done. In addition, kinase renaturation assay as well as Western blot analysis employing rat polyclonal antobody raised against the purified p21-autokinase was perfomed to understand the alteration of the protein level and kinase activity during T cell activation and cell cycle regulation. $\rtrif$ Determination of the functional role of p21-autokinase in T cell activation and cell cycle progression $\rtrif$ Purification of p21-autokinase from Jukart T cell lysate using preparative 2D-electrophoresis $\rtrif$ Preparation of polyclonal anti-p21-autokinase $\rtrif$ Determination of autophosphorylating amino acid residues of p21-autokinase by phosphoaminoacid analysis $\rtrif$ Synchronization of the cell cycle progression of Jurkat T cells by blocking using hydroxy urea and nocadazole, and by releasing from the individual blockings. Investigation of the cell cycle-related change in the level of p21-autokinase protein and its kinase activity under these synchronization conditions $\rtrif$ Expand our understanding of the mechanism of T cell activation and cell cycle regulation $\rtrif$ Characterization of the possible role of p21 autokinase involved in tumorigenesis $\rtrif$ When the requirement of p21-autokinase activity for the transition of G1/S transition during T cell activation is elucidated, p21-autokinase can be applicable as a target for the design of immunosuppresive drug. $\rtrif$ Western blot analysis employing rat polyclonal antobody raised against the purified p21-autokinase demonstrated that the protein level of p21-autokinase remained constant during trhe cell cycle progression of Jurkat T cells..

목차 Contents

• Ⅰ. 연구계획 요약문...3
• 1. 국문요약문...3
• Ⅱ. 연구결과 요약문...4
• 1. 국문요약문...4
• 2. 영문요약문...5
• Ⅲ. 연구내용...6
• 1. 서론...6
• 2. 연구방법 및 이론...8
• 3. 결과 및 고찰...12
• 4. 결론...21
• 5. 인용문헌...22