探討活化的Notch受體在K562細胞紅血球生成中的角色

Roles of the Activated Notch Receptors in K562 Erythropoiesis

楊之諄(Chih-Chun Yang)

指導教授 ： 葉添順

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2002年)

摘要

造血作用是一個血球細胞發育的過程，當造血前趨細胞受到許多分子緊密的調控，進行一連串的分化，發育成許多種類成熟的血球細胞。近年來許多研究發現，Notch signaling在造血作用中扮演一個很重要的角色，且與造血前趨細胞的分化和增生相關。Notch受體為細胞表面穿越細胞膜一次的受體蛋白，在演化上被高度的保存。當細胞表面所表現的Notch受體，鄰近細胞所表現的DSL (Delta/Serrate/LAG2 ) ligand相互作用後，使Notch受體活化，進而Notch intracellular domain (NIC)分離，傳遞Notch signaling，進入細胞核內，調節特定基因的表現。關於Notch在lymphopoiesis 和myelopoiesis中的功能，近年來也有初步的研究，但目前對於紅血球生成方面的功能則不是很清楚。此外，在造血的過程中，細胞端粒DNA逐漸的減短，歸因於缺乏端粒酵素的表現，此暗示了DNA複製能力的衰退，所以端粒酵素可能與造血細胞增生的能力相關。 本實驗利用分離自人類胚胎肝臟CD34+CD38-Lin-造血前趨細胞與人類K562 erythroleukemia細胞為材料，探討活化的Notch受體蛋白在紅血球生成時所扮演的角色。當誘導CD34+CD38-Lin-細胞分化，同時以Notch1 antisense oligonucleotides抑制Notch1 signaling，發現對於細胞的分化並沒有明顯的影響。此外建立表現活化的Notch1受體(N1IC)的K562單一細胞株，N1IC的表現調控K562細胞的增生，且降低了細胞進行紅血球分化的比率。另一方面以TRAP (Telomeric Repeat Amplification Protocol) assay分析細胞端粒酵素活性，探討在紅血球分化時， N1IC的表現對端粒酵素活性的影響。結果顯示N1IC的表現並不影響未分化與進行紅血球系分化的K562細胞中端粒酵素活性。根據以上的研究結果顯示，Notch1調節紅血球前趨細胞的增生，抑制細胞進行紅血球的分化，但不影響端粒酵素活性。因此，Notch signaling對於紅血球生成的調控扮演了舉足輕重的角色。

關鍵字

Notch受體 ； 紅血球生成 ； K562細胞

並列摘要

Hematopoiesis is the process by which mature blood cell types are generated from a small population of pluripotent hematopoietic stem cells. How these cells undergo fate selection, however, is not fully understood. The Notch signaling is known to play important roles in precursor survival and cell fate specification during hematopoiesis. Notch is a large, single-pass transmembrane receptor that has been conserved throughout evolution. Notch receptor activation through DSL ligand binding, then Notch intracellular domain translocation to the nucleus, forms a complex with the transcription factor CSL, and acts as a transcriptional modulator of Notch target genes. Recently, roles for Notch signaling in lymphopoiesis and myelopoiesis have been evaluated, but in erythropoiesis are not clear. Furthermore, telomeric DNA shortens in the course of hematopoiesis, which to a major extent is caused by the lack of expression of telomerase, this implicate the replicative senescence, and relates both to the finite capacity of cell proliferation. Hematopoietic progenitor CD34+CD38-Lin- cells isolated from human fetal liver and erythroleukemia K562 cells were used to examine the role of Notch signaling in erythropoiesis. CD34+CD38-Lin- cells were induced toward erythroid-lineage differentiation and then treated with antisense oligonucleotides of Notch1 to block Notch signaling. However, the effect of antisense oligonucleotides was not pronounced. The stable clones of K562 constitutively expressing the active intracellular domain of Notch1 (N1IC) were established. Overexpression of N1IC, the cell proliferation was modulated, and erythroid maturation of K562 cells was suppressed. To determine whether there is a linkage between telomerase activity and Notch signaling in erythropoiesis, the telomerase activity of K562/N1IC stable clones was evaluated by TRAP (Telomeric Repeat Amplification Protocol) assay. The expression of N1IC doesn’t affect the telomerase activity of K562 cells during erythroid maturation. Taken together, these results suggest that Notch1 modulates proliferation, suppresses differentiation, and doesn’t affect the telomerase activity of erythroid cells.

並列關鍵字

Notch receptor ； Erythropoiesis ； K562

參考文獻

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