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dc.description.abstractPlatelets are produced from megakaryocytes through complex events, megakaryopoiesis which includes increased nucleus numbers through the endomitosis (up to 128N) and cytoplasmic maturation leading to the production of proplatelets and release of platelets. A new substance analogously related to the compounds in deer antler, phosphocholine derivative, was synthesized. I investigated the effect of phosphocholine derivative on the signal pathways in megakaryopoiesis of human leukemic cell lines K562 and HEL cells. To enhance the megakaryopoiesis by phosphocholine derivative, we used co-culture system using OP9 stromal cells. Treatment of phosphocholine derivative in K562 and HEL cells induced morphological and biochemical changes, which are characteristic for megakaryocytes. Phosphocholine derivative-treated cells resulted in increased megakaryocyte markers, CD41, CD61, CD42a and CD42b compared to control cells. The cell size and nucleus number were increased in phosphocholine derivative-treated cells. To elucidate mechanism underlying megakaryopoiesis, I performed two inhibitory experiments. In phosphocholine derivative-treated K562 cells were inhibited PI3K inhibitor, LY294002 and mTOR inhibitor, rapamycin. In phosphocholine derivative-treated HEL cells were inhibited PI3K inhibitor, LY294002. These results suggest that megakaryopoiesis in phosphocholine derivative-treated K562 and HEL cells went through PI3K and mTOR signaling pathway. To understand the genetic events during megakaryopoiesis, I carried out Real-time PCR analysis in differentiated K562 cells differentiated. In previous studies, 10 genes were chosen as genes whore expression change more than 10 fold by a gene chip analysis. These genes, geneD, geneE, geneG, geneI, geneK, geneL, geneP2, geneP3, geneT and geneU were shown similar pattern between a gene chip data and Real-time PCR data. Among these genes, geneE, geneL, geneP3, geneU were chosen for silencing experiments. Megakaryocytic development, such as polyploidization and expressions of megakaryocytic specific markers, CD41, were suppressed when geneL and geneP3 genes were knock-downed by shRNA. To investigate gene overexpression in megakaryopoiesis, geneL gene was overexpressed by overexpression DNA. ;혈소판은 megakaryopoiesis라는 복잡한 과정을 통해 거핵구에 의해서 생성된다. Megakaryopoiesis는 핵 내 유사분열을 통한 핵의 수 증가, 전-혈소판의 형성을 위한 세포질의 성숙으로 이루어진다. 녹용으로부터 추출된 물질의 유도체인 새로운 합성 화합물 phosphocholine 유도체로 인간 백혈구 세포주인 K562, HEL 세포에서의 거핵구 발달 작용을 밝히고자 하였다. 거핵구로의 분화와 성숙을 증진시키기 위해 기질 세포인 OP9 세포를 사용하여 함께 배양하였다. Phosphocholine 유도체에 의해 분화된 K562, HEL 세포는 거핵구의 특징적인 형태적 변화와 생화학적 변화를 보였다. Phosphocholine 유도체가 처리된 세포는 거핵구 표지인자인 CD41, CD61, CD42a, CD42b를 발현하였고, 세포 크기와 핵 수도 증가하였다. 거핵구 분화 과정에서의 신호 전달과정을 밝히기 위해 두 종류의 저해제를 사용하여 세포신호 전달 저해 실험을 수행하였다. Phosphocholine 유도체를 처리한 K562 세포에서는 PI3K 저해제인 LY294002와 mTOR 저해제인 rapamycin에 의해 명확하게 저해되었다. Phosphocholine 유도체를 처리한 HEL 세포에서는 PI3K 저해제인 LY294002에 의해 명확하게 저해되었고, mTOR 저해제인 rapamycin에 의해서는 저해 정도가 약하게 나타났다. 거핵구 발달 과정에서의 유전자 변화를 조사하기 위해 phosphocholine 유도체를 처리하여 분화한 K562 세포의 유전자 분석을 수행한 결과를 바탕으로 10배 이상 증가한 유전자 분석을 수행하였다. 10배 이상 증가한 유전자D, 유전자E, 유전자G, 유전자I, 유전자K, 유전자L, 유전자P2, 유전자P3, 유전자T, 유전자U를 선택하여 실시간 PCR을 수행하였다. 우리는 실시간 PCR을 통해서도 이들 유전자가 유사한 발현양상을 보인다는 것을 확인하였다. 그리고 shRNA를 이용하여 이 중 4가지 유전자인 유전자E, 유전자L, 유전자P3, 유전자U의 발현을 억제함으로써, 거핵구 발달 과정에서 거핵구의 생성과 표지인자 발현이 감소되는 것을 확인하였다. 특히 유전자L, 유전자P3는 거핵구 발달과정에 지배적인 영향을 준다는 것을 확인할 수 있었다.-
dc.description.tableofcontents1. Introduction 1 2. Materials and Methods 6 2.1. Reagents 6 2.2. In vivo megakaryopoiesis 7 2.3. Growth assay 8 2.4. Flowcytometry 8 2.5. RNA isolation 8 2.6. Quantitative Real-time PCR 9 2.7. Synthesis of shRNA(small hairpin RNA) 10 2.8. Transfection of shRNA(small hairpin RNA) 12 2.9. Synthesis of overexpression DNA 13 2.10. Transfection of overexpression DNA 14 3. Results 15 3.1. Growth of phosphocholine derivative-treated K562 and HEL cell lines 15 3.2. Morphological changes of phosphocholine derivative-treated leukemic cell lines 15 3.3. Time-dependent CD41, CD61, CD42a and CD42b expression of phosphocholine derivative-treated K562 and HEL cells 15 3.4. Polyploidization of phosphocholine derivative-treated K562 and HEL cell lines 18 3.5. Inhibition of differentiation by two inhibitors 22 3.6. Expression of genes in megakaryocytic development 27 3.7. Inhibition of gene in megakaryocytic development by shRNA 28 3.8. Overexpression of genes in megakaryocytic development by overexpression DNA 32 4. Discussion 38 5. References 41 논문개요 45-
dc.format.extent3509020 bytes-
dc.publisher이화여자대학교 대학원-
dc.titlePhosphocholine derivative stimulates megakaryopoiesis-
dc.typeMaster's Thesis-
dc.format.pagex, 46 p.-
dc.identifier.major대학원 화학·나노과학과- 2-
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