臺灣博碩士論文加值系統

一氧化氮是一種小型氣體分子，具有特殊的反應性，目前已知與血管舒張、神經訊息傳遞、毒殺細菌及抑制腫瘤細胞生長等作用有關。過去實驗結果顯示，生物體內若產生過量的一氧化氮，可能會危害到正常細胞而造成不良病理狀態。在此過程中，一氧化氮對於細胞之毒性主要來自於許多重要酵素系統受到影響。白血病是一種極重要的血液疾病，患者血液中含有許多未成熟的細胞，功能不健全且未成熟細胞的過度增生是此疾病的特徵。過去研究發現，利用藥物誘發病態細胞的分化是治療此疾病的有效療法，由於在骨髓微環境中有多種細胞可釋放一氧化氮，科學家曾推測一氧化氮可能對於血液細胞的分化過程具有調節作用。本論文利用多種血癌細胞株來探討一氧化氮對於血液細胞生長、分化及活化等功能的調節，並希望能從中瞭解一氧化氮之作用機制。
由於過去曾有實驗結果顯示一氧化氮可刺激髓性系列細胞的生長，本論文第一部分利用一氧化氮釋放劑SNP來研究一氧化氮對於人類血癌細胞株生長的影響，結果發現1 mM的SNP會抑制細胞生長，但是0.1 mM的SNP卻可顯著刺激細胞生長。細胞生長增加的同時，細胞多處於S/G2+M期；相對的，處於G0/G1期的細胞較少。SNP刺激細胞生長的效應是因其釋放出的一氧化氮，而不是其他如ferricyanide之類的副產物。此外，一氧化氮刺激細胞生長的訊息傳遞，是經由活化細胞內的環氧化(cyclooxygenase)，進而產生PGE2所造成的，此訊息傳遞過程並不涉及其他kinase。除了利用indomethacin和dexamethasone 等環氧化脢抑制劑來証明環氧化參與SNP誘發之細胞生長外，若直接以環氧化產物PGE2處理細胞則可發現：10-7 M的PGE2可刺激細胞生長，但10-5 M則會抑制U937細胞的生長，似乎與SNP在高劑量抑制細胞生長之趨勢有平行關係。SNP (0.1 mM) 可刺激4種髓性系列血癌細胞株(U937, HL-60, NB4, THP-1)的生長，1 mM 的SNP會抑制生長；然而，SNP (0.1 mM) 對紅血球(K562)或淋巴球(Jurkat, RAMOS)系列的細胞生長則沒有類似效應。不同氧化還原態的一氧化氮釋放劑會對細胞生長造成不同的影響。總結以上結果，外源性一氧化氮可經由活化環氧化產生的PGE2刺激細胞生長。一氧化氮刺激生長的效應與其濃度及氧化還原態有直接的關聯。
第一部份論文顯示1 mM 以上的SNP可顯著的抑制U937及HL-60等血癌細胞株的生長。由於細胞生長遲滯是分化過程的一項表徵，而過去已有文獻報告一氧化氮可誘導血癌細胞株HL-60的分化，但誘導之機制並不清楚。因此本論文第二部份利用一氧化氮釋放劑SNP來探討一氧化氮造成人類血癌細胞株HL-60分化的可能機制。結果發現：SNP (1-5 mM) 處理24至72小時均可使細胞顯現出如下之分化表徵：(1)核/質比顯著縮小；(2)數目顯著減少；(3)表面抗原CD14及CD11b顯著增加；(4)吞噬能力顯著增加;(5)NBT還原活性增加；(6)c-myc mRNA表現顯著降低。由以上分化表徵可知HL-60細胞會因SNP的刺激朝向單核球/巨噬細胞系列而分化。由於過去文獻曾暗示MAP kinase可能參與分化過程的調節，所以本論文利用PD98059 (ERK 抑制劑，10 M)和SB203580 (p38抑制劑，10-20 M)與SNP共同處理細胞，發現PD98059在不具細胞毒性的劑量下具有抑制SNP誘發細胞分化之趨勢，而SB203580則可顯著地抑制由SNP所誘發之細胞分化。進一步分析由SNP所引起的p38 MAP kinase活性變化可知，隨著SNP劑量的增加，p38 MAP kinase活性逐漸增加，而SNP所誘發p38 MAP kinase活性的增加可被SB203580前處理所抑制。c-myc基因表現的調降(down-regulation)被認為是分化過程中的重要指標，若以c-myc之反序列核酸與SNP來模擬c-myc基因表現過度調降時對分化的影響，則發現c-myc反序列核酸與SNP共同處理組之分化表徵沒有顯著改變。另外，本論文發現SNP造成c-myc基因表現調降是因為：(1) c-myc mRNA半生期縮短；(2) c-myc基因表現抑制者(如本論文所觀察之PRDI-BF1)的表現增加。以上結果顯示：SNP產生的一氧化氮可誘發HL-60血癌細胞株分化成為單核球/巨噬細胞。一氧化氮誘發單核球/巨噬細胞分化的機制是經由直接活化p38 MAP kinase。
巨噬細胞在生物體內消滅外來病源前的活化(activation)過程，已被認為是其功能的評估指標，而以適量內毒素(endotoxin; lipopolysaccharide)刺激巨噬細胞則是最常見的活化實驗模式。過去曾有許多研究探討巨噬細胞經內毒素活化後之訊息傳遞路徑，結果顯示一氧化氮合成及環氧化均可被內毒素激活，而MAPK可能在此激活過程中扮演重要的調節角色，但其結論並不一致。由於人類細胞在離體的環境下不易進行活化的研究，故在本論文第三部份，改利用小鼠巨噬細胞株J774來研究一氧化氮及MAPK在內毒素所引起細胞活化過程中所扮演之調節作用。結果發現，內毒素(0.1-1g/ml)處理後可使J774細胞產生：(1)細胞表面突起與細胞質中大量的液泡；(2) 一氧化氮及PGE2濃度上升等現象，這些現象顯示J774細胞正處於活化階段。將PD98059 (ERK抑制劑)前處理之細胞再以內毒素刺激，則發現其環氧化活性及PGE2含量顯著降低。本論文也發現內毒素(1g/ml)引發 ERK 活性變動的趨勢會在20分鐘時達到高峰爾後回到基礎值，此種ERK 活性變動會因預先處理一氧化氮合成抑制劑而消失。此外，內毒素引發 ERK 活性的變動可受到 protein phosphatase 2A、protein tyrosine phosphatase、MAP kinase phosphatase 1等的調節。以上結果顯示：由內毒素所引起之巨噬細胞活化過程中，環氧化活性受 ERK 之調節；而內毒素所引發之ERK活性又會受到一氧化氮及多種protein phosphatase 之調節。
總結以上所有實驗結果：一氧化氮在調節血癌細胞生長及分化的過程中，都扮演著極重要的角色，其調節作用係藉由影響不同的酵素系統而完成。一氧化氮與生物體內酵素系統交互作用之研究可用以解釋一氧化氮在生物體內所具有之獨特生物效應，並可提供未來臨床治療的依據。

Nitric oxide (NO), a gaseous reactive molecule, is involved in various biological functions including vasodilation, neurotransmission, and antimicrobial activities. Inadequate production of NO, however, may be detrimental to normal cells and responsible for certain pathological conditions. It is believed that the cell-damaging effect of NO is mediated by the inhibition of crucial cellular enzyme systems. Because various cell types in the bone marrow release NO, it is postulated that NO may play an important role in hematopoiesis. The aim of this study is to explore the regulatory mechanisms of NO in cell growth, differentiation, and activation using various leukemic cell lines.
It has been shown that under certain conditions NO stimulated the growth of myeloid hematopoietic cells. To examine the mechanism(s) involved in the growth-promoting effect of NO, sodium nitroprusside (SNP, a NO donor) was used in the culture of the U937 human promonocytic leukemic cells. Treatment of U937 cells with 0.1 mM SNP for 72 h caused a 45(2% increase in U937 cell growth with significantly increased S/G2+M-phase and decreased G0/G1-phase of the cell cycle. The growth-enhancing effect of SNP was blocked by indomethacin, a cyclooxygenase (COX) inhibitor. SNP treatment resulted in a dose-dependent increase in prostaglandin E2 (PGE2) production. Furthermore, the addition of exogenous PGE2 not only enhanced U937 cell growth but also restored the indomethacin-inhibited mitogenic effect of SNP. Stimulatory effect of SNP on cell growth was limited in myeloid leukimia lines, but not in erythroid or lymphoid lines. NO donors releasing different redox status of NO appeared to have differential effects on cell growth. These data indicate that NO can enhance cell growth via PGE2 by activating the COX pathway.
A recent study showed that SNP-derived NO suppressed the growth and induced the monocytic differentiation of a human leukemia cell line, HL-60. To explore the mechanism of NO-induced differentiation, HL-60 cells were cultured in the presence of SNP under various conditions. Treatment with SNP (1-5 mM) for 24-72 hrs caused changes that are characteristics of differentiation, including: decreased nucleus/cytoplasm ratio, decreased viable cells, increased expression of CD14 and CD11b, increased phagocytosis, increased NBT reduction activity, and decreased c-myc expression. MAP kinases have been implicated in the regulation of monocytic cell differentiation; however, the role of p38 MAP kinase in monocytic differentiation is unclear. Using SB203580 (p38 inhibitor) in combination with SNP, it was found that SB203580 significantly inhibited SNP-induced differentiation. SNP activated p38 MAP kinase in a time- and dose-dependent manner. Since down-regulation of c-myc has been considered as an important index of cell differentiation, the mechanism of SNP-induced c-myc down-regulation was further studied. Using a kinetic analysis, it was found that SNP-induced c-myc down-regulation was associated with shortened mRNA halflife of c-myc. Furthermore, the expression of PRDI-BF1, an inhibitory factor of c-myc expression, was increased. In summary, SNP-induced monocytic differentiation appears to involve the activation of p38 MAP kinase.
Stimulation with lipopolysaccharide (LPS) is a widely accepted experimental model for macrophage activation. It has been shown that NO synthase and COX were activated during LPS-induced cell activation. However, the role of MAP kinase in macrophage activation remains controversial. To determine the role of NO in LPS-induced activation, J774 murine macrophages were cultured in the presence of various modulators. Treatment with LPS (0.1-1 g/ml) for 24 hrs increased the surface processes, cytoplasmic vacuoles, and production of NO and PGE2 in J774 macrophages, indicating the activation of cells in response to LPS. Pretreatment of PD98059 (ERK inhibitor) decreased LPS-stimulated levels of PGE2. LPS-induced ERK activity peaked at 30 minutes and then declined to the basal values. Preatment of L-NAME (NOS inhibitor) abolished the LPS-induced ERK activity. In summary, in LPS-activated macrophages, LPS-stimulated ERK activity appears to regulate the COX activity and may involve mediation by NO.
In conclusion, NO plays an important role in the regulation of myeloid cell growth, differentiation, and activation. NO acts by activating multiple enzyme systems. Interactions between NO and enzyme systems may account for the diverse biological effects of NO.

封面
目錄
中文摘要
英文摘要
縮寫對照表
第一章、緒論
I、一氧化氮之簡介
II、單核吞噬細胞系統 (mononuclear phagocyte system)(Furth et al., 1972)
III、研究動機
第二章 一氧化氮刺激人類血癌細胞株 U937 增生之機制探討
I、引言
II、實驗方法
III、實驗結果
IV、討論
第三章、一氧化氮誘導人類血癌細胞株 HL-60 分化之機制探討
I、引言
II、實驗方法
III、結果
IV、討論
第四章、一氧化氮在巨噬渦胞活化過程中扮演之角色
I、引言
II、實驗方法
III、 結果
IV、討論
第五章、總結論
附錄
參考文獻

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