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研究生:莊皓宇
研究生(外文):Hao-Yu Chuang
論文名稱:Micro-RNAs在惡性神經膠質母細胞瘤與正常腦細胞中之不同表現
論文名稱(外文):Micro-RNAs Express Differentially in Glioblastoma Multiforme and Normal Brain Cells
指導教授:林欣榮林欣榮引用關係
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:臨床醫學研究所碩士班
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:2009
畢業學年度:97
語文別:中文
論文頁數:32
中文關鍵詞:神經腫瘤幹細胞惡性神經膠質瘤免疫細胞治療
外文關鍵詞:AstrocytomaGene expression assayGBMGlioblastoma multiformeMicro-RNAMicro-arraymicro-RNA chip
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試驗主題
本研究主要是研究惡性神經膠質瘤中,microRNA於神經腫瘤細胞及神經腫瘤幹細胞中扮演之角色。我們以組織庫挑選收集病人檢體(已獲得病人同意後),包含惡性神經膠質瘤和正常腦組織檢體細胞,篩檢出有意義的micro-RNA,利用GBM tissue microRNA Chip Data Analysis 瞭解惡性神經膠質瘤和正常腦組織細胞中microRNA之表現及差異程度。

研究背景及目的
即使最先進之治療改變包括先進手術導航手術方法,化學治療,放射線治療, 對 於惡性神經膠質瘤之預後,進展仍十分有限。只能多延長3至6個月生命,腫瘤幹細胞雖然只佔1-2%之惡性腫瘤,它卻有堅強生長分化能力,且對臨床之治療有頑強之抗性。它具有強大自我更新及DNA修護能力,我們認為microRNA在神經腫瘤細胞及神經腫瘤幹細胞中之角色扮演極為重要,有急需研究瞭解之必要。目前已知在不同狀態下的神經腫瘤細胞及神經腫瘤幹細胞(CD133+, CD133-)之中,會有不同的microRNA表現,如能知道microRNA影響神經腫瘤細胞生長分化因子,就可以擬定治療改進之對策,以增進惡性神經膠質瘤預後。

實驗設計
本實驗以組織庫挑選收集病人檢體((檢體6件,實驗檢體取自本院組織庫中已獲得病人同意之檢體,包含惡性神經膠質瘤和正常腦組織等四大類的檢體。)
,包含惡性神經膠質瘤和正常腦組織檢體,利用GBM tissue microRNA Chip Data Analysis 瞭解惡性神經膠質瘤和正常腦組織細胞中microRNA之表現及差異程度。
我們統計並比較microRNA 在不同檢體中的表現差異(miRNA intensity<1, adjust to 1, Ratio=log2 (case#1/case#2) ), 此外我們也利用分析出來的資料, 利用 Gene expression assay datas of Normal Brain Tissue ( GDS596 ) 做基因庫的分析( from Affymetrix U133A and GBM primary culture cell lines (Affymetrix U133 Plus 2.0)). 我們發現有些人類細胞或病毒株之microRNA 具有相當意義之正調控或負調控的表現, 而這些具統計意義之表現存在於惡性神經膠質瘤和正常腦組織細胞之中.

結論
我們可以初步證實microRNA在惡性腦瘤幹細胞中扮演的角色 , 並期待microRNA正調控或負調控的表現, 可以影響惡性神經膠質瘤細胞之表現,並期待可以發展可行之治療策略,以增進治療惡性神經膠質瘤之臨床治療預後。
Object
We hypothesize that Micro-RNAs might play an important roles in glioblastoma glastoma (GBM) cells, and differentially express between GBM and normal brain cells. We compare clinical datas using MicroRNA Chip Data Analysis and statistical analyses by use of the algorithm in miRBase.
Methods
We compare clinical datas using MicroRNA Chip Data Analysis and statistical analyses by use of the algorithm in miRBase. After surgical resection, we make definite GBM pathological specimen examination and to culture GBM cells. The normal brain tissue was donated from a patient expired with intracranial lesions, and we excise the normal brain tissue within 30 minutes just after his death.
We want to find some differently expressed miRs (If miRNA intensity<1, adjust to 1, Ratio=log2 (case#1/case#2) ) in these 4 types of cells, and adopt to molecular- biological research for malignant brain tumors. Furthermore, we also adopt our preliminary datas to compare with Gene expression assay datas of Normal Brain Tissue ( GDS596 ) from Affymetrix U133A and GBM primary culture cell lines (Affymetrix U133 Plus 2.0)
Results
We have obtained a promising preliminary miRNA profiling which resulted from comparing with GBM and non-tumor brain tissues. By use of the algorithm in miRBase Targets, some miRNAs have been computationally predicted to show the significantly change of expression ratio in our preliminary miRNA data.
Conclusion
Some Micro-RNAs significantly expressed differentially in GBM cells and normal brain cells. Some express as up-regulation, and others express as down-regulation. Further study including Micro-RNAs RT- PCR and gene expression assay are ongoing. Micro-RNAs might play an important
目次
書名頁-------------------------------------------------
口試委員審定書-----------------------------------------
中文摘要----------------------------------------------- I
英文摘要----------------------------------------------- IV
致謝--------------------------------------------------- VI
目錄------------------------------------------------- VIII

第一章 Background (研究背景)---------------------------- 1
第二章 Methods and Materials (研究材料與研究設計) ------ 3
第三章 Results (研究結果)-------------------------------- 5
第四章 Discussion (結果討論)----------------------------- 7
第五章 Conclusion (結論)--------------------------------- 11

Figures and Tables (圖表)---------------------------------------------------- 12
Figure 1.RNA QA / QC information------------------------------------ 12
Figure 2. ---------------------------------------------------------------------- 13
2-1. All miRNAs in miRNAs Expression Profiles ----------------- 13
2-2. Represented as miRNAs flaged (Ratio> 0) --------------------14
2-3. Significantly represented as miRNAs flaged ( Ratio>0 , P-value p=0.05) ---------------------------------------------------- 15
Figure 3.---------------------------------------------------------------------- 16
3-1. 2 folds changed expression on miRNA chip in all condition ------------------------------------------------------------------------- 16
3-2. 2-folds-change Referenced by Normal Brain ( Ratio> = 2folds in condition 1-3 ) ------------------------------------------ 17
3-3. Brain-specific miRNA Expression Profile in GBM miRNA Chips------------------------------------------------------------------18
3-4. 94 miRNAs are noticed as 2-folds change reference by normal brain tissue-------------------------------------------------19
Figure 4. ---------------------------------------------------------------------- 20
4-1. Filter out intensity<=1: 448 miRNAs) ------------------------- 20
4-2. Intensity>=1 and ratio>=1 in any GBM referenced by normal: 390 miRNAs ------------------------------------------- 21
4-3. Intensity>=1 and Flag=’P, Present’ or Flag¹’A, Absent’in all conditions and ratio>=1 in any GBM referenced by norma: 295 miRNAs -------------------------------------------------------- 22
Figure 5.---------------------------------------------------------------------- 23
5-1. Virus GBM miRNA Expression Profiles (76 virus mi-RNAs)-------------------------------------------------------------23
5-2. Differentially Expressed miRNA (Up-regulation: green, down-regulation: red.)---------------------------------------------24
Table 1.------------------------------------------------------------------------ 25
1-1. In comparison of GDS596 Normal brain tissue gene expression assay datas (All genes: 11,934 [ log2 ( GBM /Normal ) ])------------------------------------------------------ 25
1-2. In comparison of GDS596 Normal brain tissue gene expression assay datas (Filters: intensity>=1 and Flag=’P’)--------------------------------------------------------- 26
Table 2. hsa-miR-425, hsa-miR-425*, hsa-miR-451,
hsa-miR-486-5p and hsa-miR-486-3p are expressed as down-regulated ------------------------------------------------- 27
Table 3. hsa-miR-21 are expressed as up-regulated ----------------- 28
Table 4. MicroRNA-124 and microRNA-137 express as up-regulated ------------------------------------------------------ 29
第六章References (參考文獻)--------------------------------------------- 30
1.Aurora Esquela-Kerscher. Frank J. Slack. Oncomirs — microRNAs with a role in cancer. Nat. Rev. Cancer, 2006, v6:259.
2.Benjamin Kefas, Jakub Godlewski, Laurey Comeau . microRNA-7 Inhibits the Epidermal Growth Factor Receptor and the Akt Pathway and Is Down-regulated in Glioblastoma Cancer Res. 68 (2008) 3566–3572.
3.Calin GA, Croce CM. MicroRNA signatures in human cancers[J].Nat Rev Cancer, 2006, 6(11): 857-866
4.Chan JA, Krichevsky AM, Kosik KS. MicroRNA 21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res,2005,65:6029 6033
5.Charles D. Stiles, David H. Rowitch. Glioma Stem Cells: A Midterm Exam Neuron, 2008, v58:832
6.Esquela-Kerscher A, Slack FJ. Oncomirs -- microRNAs with a role in cancer. Nat Rev Cancer 2006;6:259-269
7.Gabriely G, Wurdinger T, Kesari S. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol. 2008 Sep;28(17):5369-80.
8.George A. Calin & Carlo M. Croce. MicroRNA signatures in human cancers. Nat. Rev. Cancer, 2006, v6:857
9.Hilah Gal, Arik Makovitzki b, Ninette Amariglio.. A rapid assay for drug sensitivity of glioblastoma stem cells. Biochemical and Biophysical Research Communications 358 (2007) 908–913.
10.Hilah Gal, Gopal Pandi, Andrew A. Kanner. MIR-451 and Imatinib mesylate inhibit tumor growth of Glioblastoma stem cells. Biochemical and Biophysical Research Communications 376 (2008) 86–90.
11.Lewis BP, Burge CB, Bartel DP., Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005 Jan 14;120(1):15-20
12.Liang Y, Ridzon D, Wong L.Characterization of microRNA expression profiles in normal human tissues.BMC Genomics 2007 Jun 12;8:166
13.Rosenfeld N, Aharonov R, Meiri E, et al: MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol 26:462-469, 2008
14.Selbach M, Schwanhäusser B, Thierfelder N . Widespread changes in protein synthesis induced by microRNAs.Nature. 2008 Sep 4;455(7209):58-63. Epub 2008 Jul 30.
15.Shingo Mitomo, Chihaya Maesawa, Satoshi Ogasawara . Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines. Cancer Sci, 2008, v99:280-286
16.Silber J, Lim DA, Petritsch C. miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med. 2008 Jun 24;6:14.
17.Volinia S, Calin GA, Liu CG. A microRNA expression signature of human solid tumors defines cancer gene targets[J]. Proc Natl Acad Sci USA,2006,103(7):2257-2261.
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