建立以分子特異性探針辨識臍帶血造血幹原及其先驅細胞分化特性之基礎研究

Establishing a basal study for identifying differential potency of hematopoietic stem and progenitor cells by molecular probes

張麗晴(Lee-Chin Chang)

指導教授 ： 施子弼

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(1999年)

摘要

造血幹原細胞經由複雜的基因調控作用，生成各類型態可辨識之成熟血球細胞。過去的研究，多著重於探討基因在不同染色體位置上的影響，或是在細胞生命力的表現等。而一些特異性基因在分化機能上的調控，尚未完全明瞭，因此更值得進一步研究。由於分化機能的缺損或異常均會導致先天性疾病或是後天性腫瘤癌症等病因。而深入了解這些基因的分化特性，有助於釐清血液細胞多重的分化趨向，尤其是針對初期造血細胞，如造血幹原及先驅細胞，更可藉由分子層次上的研究，進而協助此類病患尋得治療契機。 為了鑑定幹原細胞是否具有分化機能（differential potential），一般實驗室多利用colony formation assay，細胞培養10-14天後觀察是否有colony forming unit (CFU)的形成。 此法除了培養時間冗長外，同時囿於肉眼觀察細胞團簇(colony)的標準不一，在辨別與計數上存在極大的偏差。本研究嘗試利用分子特異性探針技術來完成以下目的：(一) 應用基因表達分析法快速鑑定幹原細胞之分化機能及狀態。(二) 辨識幹原細胞及先驅細胞間之分化趨向(differential lineage)。 本研究選擇GM-CSFR，EPOR二種細胞激素受體之部分序列作為引子(primers)，應用RT-PCR的技術，以細胞數低於103 檢定培養細胞之分化狀態及功能。另一方面，我們利用具路徑特異性(lineage-specific)的基因，例如Bcl-x、CD19、CD34、等, 進一步加以辨識幹原細胞及其先驅細胞間的分化趨向。實驗結果顯示，不同分化程度的細胞，其路徑特異性基因的表現，出現互有消長的現象，並影響其分 化功能。上述結果說明，應用分子生物技術的確優於以型態判斷細胞的分化程度，可更精確地瞭解幹原細胞的分化時機(timing)，對於未來在分化機能的研究或是接受幹原細胞移植的應用上具重要意義。除此之外，我們也做一些關於臍帶血收集、冷凍保存的評估。由於臍帶血銀行(cord blood bank)逐漸興起，為了更有效地長期保存臍帶血，勢必須持續尋求出最佳收集保存模式，期盼能對先天基因缺陷及癌症病患有所助益。

關鍵字

臍帶血 ； 造血系統 ； 幹原細胞 ； 先驅細胞 ； 細胞團簇形成單位

並列摘要

Dys-regulation of genes expression in stem cells and their differentiated progeny has shown involved in several clinical disorders. To maintain the normal hematopoietic stem cell pool is though to be controlled by a wide variety of systemic and local gene regulations. To date, however, the mechanism has only partially been understood. Colony forming assay (CFA) has provided an in vitro mean for the characterization of hematopoietic stem and progenitor cells functions using the morphological examination and evaluation. Objectives of this study have been set to utilize the RT-PCR to detect genes that expressed in the progenitor cells, and to evaluate the functions of colony forming cells in highly sensitive and precise manners. We have identified several genes that expressed in the course of colony formation. i.e. 1) Both Bcl-X and GM-CSFR are not expressed in the day 6 early colonies while GM-CSFR gene start to be detected on the day 10, and no Bcl-X can be detected until the monopoiesis. 2) CD19 is detected in earlier day 6 colonies and day 8 CFU-GEMM but not in BFU-E (days10-12). 3) EPOR is not detected in early days 6-8 colonies and day 9 CFU-G but detected in the same day 10 BFU-E and the later CFU-E (day 14) colonies. We also confirmed that the above tested genes are indeed expressed in a lineage-specific manner. In adapting the single cell RT-PCR protocol, we are able to identify the gene expression by using as little as 10 cells that will enhance our ability to detect progenitors in the course of hematopoiesis with a highly sensitive and precise manner. We conclude that the RT-PCR method can be useful in establishing a phenotype analysis of blood cells and for studying gene expression in the rare population of the blood cells.

並列關鍵字

Umbilical cord blood ； Hematopoiesis ； Stem cell ； Progenitor cell ； Colony forming unit

參考文獻

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