Abstract
Chronic myeloid leukemia (CML) is a malignant disease of the human pluripotent hematopoietic stem cell, and is characterized by the Philadelphia chromosome switch which dues to a translocation between the breakpoint-cluster region (bcr) gene on chromosome 22 and the Abelson leukemia (abl) gene on chromosome 9. The bcr-abl gene can translate into an abnormal 210 kDa fusion protein with constitutively-phosphorylated tyrosine kinase activity. The activated kinase activity is suggested to be an important event leading to CML. STI571 is a tyrosine kinase inhibitor, that has a specific growth-inhibitory ability to CML and BCR-ABL positive cells, and can increase the growth-inhibition activity of other drugs to CML. STI571 was on clinical trials in 1998, and better than IFN-a. However, STI571 was reported to have short response duration, and some CML cells could become STI571-resistant. In our experiments, IFN-a could be used to promote the inhibitory effect of STI571 on the growth of K562 cell, in a dose-dependent manner. The combination of STI571 and IFN-a also could increase the apoptosis percentage induced by STI571. We found that STI571 could decrease the phosphorylation level of MAPK, while IFN-a could increase the expression level of PML as well as the phosphorylation level of p53. IFN-a could enhance the homogeneous PML nuclear bodies expression in the presence or absence of STI571. We suggested that IFN-a could increase the cell growth-inhibitory activity of STI571 through enhancement of PML expression in nuclear bodies and at least p53 phosphorylation acts to counter the positive-regulatory effect of BCR-ABL on MAPK and other signalings.Abstract誌謝目錄圖目次前言 一 慢性骨髓性白血病(chronic myeloid leukemia, CML) 1 1-1 病因 1 1-2表徵、病程與治療 2 1-3 Bcr-Abl蛋白質的結構與功能 3 1-4 BCR-ABL與酪胺酸磷酸化脢抑制劑 4二 酪胺酸激脢抑制劑: STI571 4三 前骨髓細胞白血病基因 (Promyelocytic leukemia, PML) 5四 Bcr-Abl傳遞自主的Ras訊號傳遞 6五 實驗目的 7材料與方法 81. 細菌培養 82. DNA 製備 83. 細菌轉形 104. 細胞培養 115. 墨跡轉印 116. 蛋白質偵測 137. 流式細胞儀分析 148. MTT assay 159. 螢光染色 16結果 181. STI571與Interferon-a對白血病細胞株K562的生長抑制。 182. STI571與IFN-a引發K562細胞的細胞凋亡 (apoptosis)。 193 STI571與Interferon-a對K562細胞內細胞凋亡相關蛋白表現的影響。 204. STI571與Interferon-a對K562細胞PML核體的影響。 22結論與討論 24參考文獻 27圖表 33附錄 44圖目次圖一 酪胺酸機脢抑制劑STI571可以抑制白血病細胞株K562的生長 35圖二 Interferon-a可以抑制白血病細胞株K562的生長 36圖三 STI571與Interferon-a合併對白血病細胞株K562的生長抑制 37圖四 STI571與IFN-a對K562細胞的細胞凋亡之影響 38圖五 STI571與IFN-a對K562細胞細胞週期的影響 39圖六 STI571與IFN-a對磷酸化的MAPK和PML蛋白表現量的影響 40圖七 STI571與IFN-a對磷酸化的p53與Rb蛋白質表現量的影響 41圖八 IFN-a單位濃度的增加對PML核體表現的影響 42圖九 IFN-a單位濃度的增加對PML核體數目的影響 43圖十 單獨加入STI571或合併STI571與IFN-a對PML核體數目的影響 44圖十一 STI571與合併STI571和IFN-a對PML核體數目的影響 45附錄一 Bcr-Abl與c-Abl在結構上之差別 46附錄二 STI571對Bcr-Abl影響之示意圖 47附錄三 Bcr-Abl可能影響的訊號傳遞途徑 48附錄四 PML之結構與其可能影響的重要因子 49附錄五 STI571的結構 50