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研究生:林柏每
研究生(外文):LIN, PAI MEI
論文名稱:骨髓性白血病之分子基礎-抑癌基因(FHIT,TSG101,PTEN/MMAC1),細胞週期相關基因,核甘酸重複序列不穩定性之研究
論文名稱(外文):The molecular basis of myeloid leukemia-tumor suppressor genes( FHIT,TSG101,PTEN/MMAC1), cell cyclse related genes, and microsatellite instability
指導教授:林勝豐, 游仲逸
指導教授(外文):SHENG-FUNG LIN, CHUNG-YEE YUO
學位類別:博士
校院名稱:高雄醫學院
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:133
中文關鍵詞:骨髓性白血病抑癌基因細胞週期相關基因核甘酸重複序列之不穩定性
外文關鍵詞:myeloid leukemiatumor suppressor genescycle cycles related genesmicrosatellite instability
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白血病和癌症一樣乃累積抑癌基因和致癌基因的突變所造成. FHIT,
TSG101, PTEN/MMAC1是最近新發現的三個基因, 其分別位於染色體3
p14.2, 11p15.1-15.2, 10q23.3, 極可能為抑癌基因. FHIT轉譯一147個
胺基酸的蛋白質, Ap3A水解媒. Ap3A具有某些細胞內功能, 包含DNA複製
的調控, 細胞受壓力時反應的訊號傳訊. TSG101轉譯含380個胺基酸的蛋
白質,其具有富含脯胺酸之功能基團, transcription factor特性之DNA結
合序列, coiled-coil功能基可和stathmin結合. Stathmin為一細胞內之
磷酸蛋白質, 在許多癌症均有增高的現象, 可調節細胞增生或分化之訊
號. PTEN/MMAC1蛋白質含有蛋白質酪胺酸磷酸媒功能基,其和tensin具有
高度相似性. 為了了解FHIT, TSG101, PTEN/MMAC1基因在骨髓性白血病所
扮演的角色, 利用反轉錄聚合媒連鎖反應及直接定序等方法分析骨髓性白
血病病人及5株白血病細胞株(HL60, U937, Raji, KG-1, K562)此些基因
的表現. 另外經對顯微衛星標記多形性的分析來監測有無基因的掉失. 在
本實驗中, 17位(27%)AML病人, 1位(7%)慢性期CML病人, 3位(16%)急性期
CML病人有不正常的FHIT transcripts. 24位(35%)AML病人, 2位(13%)慢
性期CML病人, 5位(28%)急性期CML病人表現不正常的TSG101
transcripts. 15位(25%)AML病人, 1位(2%)急性期的CML病人表現不正常
的PTEN/MMAC1 transcripts. 而所有的細胞株除K562僅表現正常的PTEN/
MMAC1 transcript外, 所有細胞株均表現正常及不正常的FHIT, TSG101,
PTEN/MMAC1 transcripts. 所有的檢體均無FHIT, PTEN/MMAC1基因的的掉
失. 而不正常的transcripts的產生可能是因為變異的mRNA splicing所造
成. 這些不正常的transcripts可能造成蛋白質量的減少或功能的喪失,
而影響其對細胞週期的控制. 我們建立一掉失變形的內在標準來測定細胞
週期相關基因(cyclin D1, cyclin E, CDK2, CDK4)的表現. 我們於相同
的反應瓶中加入檢體及內在標準再利用特異性的引子來進行競爭性聚合媒
連鎖反應. 我們可以發現CML病人由慢性期進入急性期時, 其細胞週期基
因有過度表現的現象, 另外在AML病人其細胞週期基因亦有過度表現之情
形, 顯示在骨髓性白血病惡化時, 其細胞週期基因之調控有失序之現象.
我們同時研究9位CML病人, 18位AML病人, 3位ALL病人在疾病惡化時有無
核甘酸重複序列不穩定性之存在. 核甘酸重複序列之不穩定性為基因圖譜
中一些小而重複之核甘酸序列之變異, 乃是因配對錯誤修補基因(MSH2,
MLH1, PMS1, PMS2)之突變所造成. 一位急性期之CML病人在D2S123, D5
S299, D17S179 等microsatellite loci發現不穩定性, 一位復發期的AML
病人及一位復發期的ALL病人在D2S123, D5S299, D17S179, D18S34等
microsatellite loci發現不穩定性. 這些結果顯示配對錯誤修補基因的
功能失常, 和骨髓性白血病之惡化有關. 在大腸直腸癌由於配對錯誤修補
基因的缺失造成基因的不穩定性, 而加速APC, RAS, DCC, p53等基因的之
突變, 而和其由正常大腸上皮組織經由腺瘤到癌症之形成有關. 在骨髓性
白血病之致病機轉, 經由我們的研究, 這樣的一個多重基因突變累積形成
的致病理論也可能在白血病的形成過程中扮演著相當重要的角色. 首先我
們在AML, ALL復發病例及CML急性期之病人見到基因之不穩定性之發生,
同時我們也發現多數骨髓性白血病病人存在有大小不等之FHIT, TSG101,
PTEN/MMAC1 變異transcripts, 而可能對正常細胞之週期及生長之調控有
不等之影響,造成了骨髓性白血病之發生.
Leukemia and cancers are caused by accumulation of multiple
genetic alterations, including the gain function of proto-
oncogenes and the inactivation of tumor suppressor genes
controlling the cell cycle. Three novel genes were identified
recently at chromosome 3p14.2, 11p15.1-15.2, 10q23.3, named
FHIT, TSG101, PTEN/MMAC1, and they were designated as potential
human tumor suppressor genes. The FHIT gene encodes a 147 amino-
acid protein, dinucleoside 5'',5''''''-p1,p3-triphosphate (Ap3A)
hydrolase which is homologous to a group of proteins designated
HIT proteins. Ap3A has been proposed to have various
intracellular functions, including regulation of DNA replication
and signaling stress responses. The TSG101 is predicted to
encode a 380 amino-acid protein which contains a proline-rich
domain and DNA-binding motifs characteristic for a transcription
factor and a coiled-coil domain shown to interact with stathmin.
Stathmin is a cytoplasmic phosphoprotein which was elevated in a
variety of malignancies and proposed to coordinate and relay
diverse signals regulating cell proliferation and
differentiation. The PTEN/MMAC1 protein contains protein
tyrosine phosphatase domain and extensive homology to tensin, a
protein that interacts with actin filaments at focal adensions.
To determine the roles of the FHIT, TSG101, PTEN/MMAC1 genes in
leukemia, bone marrow or peripheral blood from myeloid leukemia
patinets and five hematopoietic cell lines (HL60, U937, Raji,
KG-1, K562) were analyzed by reverse transcription of the mRNA
followed by PCR amplification and sequencing of the products. To
detect the deletion of the gnens, 17 cases were evaluated using
microsatellite polymorphism analysis. In this study, 17/62(27%)
AML patients, 1/15(7%) CML patients in the chronic phase, and
3/18(16%) CML patients in the blastic phase expressed aberrant
FHIT transcripts which lack two or more exons of the FHIT
transcripts. 24/68 (35%) AML patietns, 2/15 (13%) CML patients
in the chronic phase, and 5/18 (28%) CML patients in the blastic
phase expressed aberrnat TSG101 transcripts. 15/60(25%) AML
patients, and 1/18(6%) CML patients in the blastic phase
expressed PTEN/MMAC1 aberrant transcripts. All the cell lines
expressed aberrant FHIT, TSG101, and PTEN/MMAC1 transcripts,
excepts K562 displayed only the normal PTEN/MMAC1 transcript. No
case exhibited a loss of the FHIT and PTEN/MMAC1 alleles. The
aberrant transcripts may result from alternative RNA splicing as
evidenced by normal and aberrant transcripts are found in all
specimens examined. The aberrant transcripts may cause the
reduction of the protein or affect the function of the protein,
and alter the control of the cell cycle. We construct a deleted
mutant form of internal standard to detect the expression of the
cell cycle related genes (cyclin D1, cyclin E, CDK2 and CDK4).
We add the specimens, internal standard into the same reaction
tube to perform the competitive polymerase chain reaction with
the specific primers. In our study, we can find the increasd
expression of cyclin D1, cyclin E, CDK2, and CDK4 in CML
patients from the chronic phase to the blastic crisis. We also
find the overexpression of the cell cycle related genes in our
AML patients. These results indicated that the cell cycle
related genes are overexpressed in myeloid leukemia, and may be
related to the progression of leukemia. We also studied
microsatellite instability in 9 CML patients with chronic and
blastic crisis phase, 18 AML patients, and 3 ALL patients with
diagnotic and relapse stage. Microsatellite are highly
polymorphic, short tandem repeat sequences throughout the
genome. Microsatellite instability are mutations of these repeat
sequences, which were resulted from mutation of DNA mismatch
repair genes (MSH2, MLH1, PMS1, PMS2). Three microsatellite
instability loci (D2S123, D5S299, D17S179) were found in one CML
patient with blastic phase, and four microsatllite instability
loci (D2S123, D5S299, D17S179, D18S34) were found in one AML and
one ALL patient with relapse stage. These results indicate that
the dysfunction of mismatch repair genes may be related to the
progression of myeloid leukemia. In colorectal cancer, the
dysfunction of the mistmath repair genes cause the genetic
instability, and accelerate the transformation from the normal
colonic epithelial tissue through adenoma to carcinoma which
appears to be accompanied by mutations in the APC, RAS, DCC, and
p53 genes. Through our study, the accumulation of multiple
genetic alterations may play an important role in myeloid
leukemia. We found the genetic instability in AML, ALL with
relapse stage, and CML with blastic phase. We also found
aberrant FHIT, TSG101, PTEN/MMAC1 transcripts, and it may affect
the control of the cell cycle and cell growth, which lead to the
formation of leukemia.
封面
目錄 (Contents)
中文摘要
英文摘要
第一章 緒論
參考資料
第二章 FHIT、TSG101、PTEN/MMAC1 基因在骨髓性白血病之表現
前言
實驗材料與方法
結果
討論
參考資料
第三章 細胞週期基因在骨髓性白血病之表現
前言
實驗材料與方法
結果
討論
參考資料
第四章 核?酸重複序列不穩定性在骨髓性白血病之表現
前言
實驗材料與方法
結果
討論
參考資料
第五章 總結
參考資料
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