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dc.description.abstractMegakaryocytes are precursor cells that undergo differentiation and maturation to produce platelets. Mature megakaryocytes are derived from pluripotent hematopoietic stem cells. Megakaryopoiesis is the complex process that includes cell lineage commitment, increased nucleus numbers through the endomitosis(up to 128N), cytoskeletal development and cytoplasmic maturation leading to the production of proplatelets and release of platelets. I investigated the effect of (R)-N-stearoyl-O-phosphocholine-D-serine methyl ester((R)-NALPCE), a novel synthetic compound derived from Deer Antler on the signal pathways in megakaryopoiesis of human leukemic cell lines K562, HEL cells and primary human stem cell CD34^(+) cells. To enhance the megakaryocytic differentiation and maturation by (R)-NALPCE, we used co-culture system using OP9 stromal cells. Treatment of (R)-NALPCE in K562, HEL and hSC CD34^(+) cells induced morphological and biochemical changes, which are characteristic for megakaryocytes. PMA is broadly used in hematopoietic differentiation system. So I compared the signal pathway in megakaryopoiesis activated by PMA and (R)-NALPCE. PMA-treated K562, HEL and CD34^(+) cells expressed CD41 and CD61 the specific markers of megakaryocyte, neutrophil, and monocyte in concentration- and time-dependent manners. Wheareas (R)-NALPCE treated cells expressed only megakaryocyte markers, CD41 and CD61. The cell size and number of nucleus were increased in (R)-NALPCE-treated cells. However these phenomena were not observed in PMA- treated cells. To elucidate mechanism underlying megakaryocytic differentiation, I carried out several inhibitory experiments. In (R)-NALPCE-treated K562 cells were inhibited by PKC inhibitor, GF109203X and PI3-kinase inhibitor, LY294002 and mTOR inhibitor, Rapamycin. In (R)-NALPCE-treated HEL cells, polyploidization and cytoplasmic maturation were inhibited by PKC inhibitor, GF109203X. Unlike (R)-NALPCE, PMA inhibited GF109203X-induced polyploidization and cytoplasmic maturation both K562 cells and HEL cells. These result suggest that K562 and HEL cells unlike different signal pathways in megakaryopoiesis that is induced by (R)-NALPCE, and that there are considerable differences between PMA- and (R)-NALPCE-induced pathways during megakaryocytic development. To understand the genetic events during megakaryocytic development, I performed a gene chip analysis in (R)-NALPCE-induced K562 cells differentiated. As a result, 67 genes were identified. Among these genes, KLF, INHBE, LOC, GDF15, and DLK1 were chosen as genes whore expression change more than 10 fold. Indeed, it was confirmed that the expression of these genes were markedly changed during (R)-NALPCE-induced megakaryocytic development in the differentiated K562 cells. Furthermore, megakaryocytic development, such as polyploidization and expressions of megakaryocytic specific markers, were suppressed when these genes were knock-downed by shRNA.;거핵구는 분화와 성숙을 거친 후에 혈소판을 만드는 전구세포이다. Megakaryopoiesis는 만능의 조혈 줄기세포로부터 복잡한 과정을 거쳐 거핵구가 만들어지는 과정이다. 이 과정은, 분화세포의 방향 결정, 핵내 유사분열을 통한 핵의 수 증가, 세포 골격의 발달, 전-혈소판의 형성을 위한 세포질의 성숙과 혈소판 생성으로 이루어진다. 녹용으로부터 추출된 물질의 유도체인 새로운 합성 화합물 (R)-NALPCE로 인간 백혈구 세포주인 K562, HEL 세포와 인간 줄기세포인 CD34^(+) 세포에서의 거핵구 발달 작용을 밝히고자 하였다. (R)-NALPCE에 의해 분화된 K562, HEL, CD34^(+) 세포는 거핵구의 특징적인 형상학적 변화와 생화학적 변화를 보였다. 그리고 거핵구로의 분화와 성숙을 증진시키기 위해 기질 세포인 OP9 세포를 사용하여 함께 배양하였다. 그리고 조혈 분화 시 널리 사용되는 PMA와 비교 분석하였다. PMA로 처리된 K562, HEL 그리고 CD34^(+) 세포는 거핵구, 호중구, 단핵구와 같은 다양한 분화 방향 표지 인자를 발현한다. 그러나 (R)-NALPCE를 처리한 세포들은 농도 및 시간에 따라서 거핵구 특이적 인자인 CD41, CD61를 발현한다. 그리고 (R)-NALPCE에 의해 분화된 K562, HEL, CD34^(+) 세포들은 세포질의 부피 증가와, 핵 수의 증가를 보였으나 PMA 처리군 에서는 보이지 않았다. 거핵구 분화 과정에서의 신호 전달과정을 밝히기 위해 세포신호 전달 저해 실험을 수행하였다. (R)-NALPCE로 처리된 K562 세포에서는 핵수의 증가와 세포질의 성숙이 PKC 억제제인 GF109203X와 PI3-kinase 억제제인 LY294002, 그리고 mTOR 억제제인 Rapamycin에 의해 명확하게 저해되었고, HEL 세포의 경우에는 PKC 억제제인 GF109203X에 의해서만 저해되었다. (R)-NALPCE 효과와는 다르게 PMA로 처리된 K562 세포는 GF109203X에 의해 저해되었고, HEL 세포는 비슷한 양상을 보여주었지만 저해 정도가 낮았다. 따라서 우리는 K562 세포와 HEL 세포는 다른 신호전달기작을 보일 것이라 추측한다. 그리고 PMA와 (R)-NALPCE에 의한 두 세포의 거핵구 분화 과정도 확실하게 다른 과정으로 진행될 것으로 추정한다. 거핵구 발달 과정에서의 유전자 변화를 조사하기 위해 (R)-NALPCE로 처리된 분화 K562 세포의 유전자 분석을 수행하였고, 67개의 유전자를 분석하였다. 그 중에서 우리는 (R)-NALPCE로 처리한 후 10배 이상 증가한 KLF, INHBE, LOC, GDF15, DLK1 유전자를 선택하였다. 우리는 (R)-NALPCE에 의해 분화된 K562 세포에서 그들의 발현을 확인하였다. 그리고 shRNA를 이용하여 유전자들의 발현을 억제함으로써, 거핵구 발달 과정에서 거핵구 생성과 표지 인자 발현이 감소되는 것을 확인하였다.-
dc.description.tableofcontentsAbstract = xi 1. Introduction = 1 2. Materials and Methods = 7 2.1. Reagents = 7 2.2. In vitro megakaryopoiesis and thrombosis = 8 2.3. MTT and growth assay = 9 2.4. Flowcytometry and cell sorting = 10 2.5. RNA isolation and oligonucleotide array = 10 2.6. RT-PCR = 11 2.7. Transfection of shRNA(small hairpin RNA) = 12 3. Results = 13 3.1. MTT assay of (R)-NALPCE treated HEL cells = 13 3.2. Growth of (R)-NALPCE treated leukemic cell lines = 13 3.3. Expression of hematopoietic cell surface marker in PMA- and (R)-NALPCE-treated leukemic cell lines = 13 3.4. Concentration- and time-dependent CD41 and CD61 expression of (R)-NALPCE treated HEL cells = 16 3.5. Morphological changes of TPO-, PMA- and (R)-NALPCE-treated leukemic cell lines = 16 3.6. Polyploidization of PMA- and (R)-NALPCE-treated leukemic cell lines = 19 3.7. Inhibition of differentiation by various inhibitors = 23 3.8. Megakaryocytic differentiation of CD34^(+) cells = 23 3.9. Expression of gene in megakaryocytic development = 28 3.10. Inhibition of gene in megakaryocytic development by shRNA = 35 4. Discussion = 40 References = 43 논문개요 = 48-
dc.format.extent1746266 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleMegakaryocytic differentiation and maturation by novel synthetic compound, (R)-N-stearoyl-O-phosphocholine-D-serine methyl ester-
dc.typeMaster's Thesis-
dc.format.pagexiii, 49 p-
dc.identifier.major대학원 나노과학부- 2-
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