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研究生:張建裕
研究生(外文):Jan-Yi Chang
論文名稱:板藍根對於細胞激素相關基因的影響
論文名稱(外文):THE EFFECT OF BANLANGEN (ISATIS INDIGOTICA FORT) ON THE EXPRESSION OF CYTOKINE-RELATED GENES
指導教授:陳建先陳建先引用關係
指導教授(外文):Chien-Hsien Chen
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:67
中文關鍵詞:板藍根細胞激素相關基因晶片即時定量反轉錄PCR西方墨點法
外文關鍵詞:real-time RT-PCRarray chipcytokine-related genesBan-Lan-GenWestern blot.
相關次數:
  • 被引用被引用:1
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中文摘要
在2003年SARS病毒流行期間的大陸地區,有一部分的人利用板藍根來治療或預防SARS的感染,其療效及真正的作用機轉仍有待探討。我們篩選cytokine有關的基因248個,利用RT-PCR的方法,製造出120-bp左右的DNA片段,然後利用microarray的機器將它們點在nylon membrane上。此membrane即用於探討板藍根對於A549肺癌細胞株所分泌的cytokine相關基因的作用。其結果再進一步利用即時定量RT-PCR証實。結果顯示,板藍根可抑制EphA3,EphA1及DLK1的作用,但可促進Tyro3,IL30及CXCL12的作用,我們進一步利用Western blot確定Tyro3之表現。由以上的結果看來,板藍根可能是經由這些cytokine相關基因而發揮作用。我們的結果將利用動物實驗進一步証實。
Abstract
The severe acute respiratory (SARS) was originated from China and became a pandemic disease in early 2003 and has claimed thousands of deaths worldwide.
During the pandemic period, people in China believed that Banlangen (Isatis indigotica Fort) could cure or prevent the infection of SARS corona virus (SARS-CoV). However, the detail of the mechanism on how it functions on the treatment of SARS still remained unknown. Onepossibility is that Banlangen may influence cytokine signal pathway by regulating cytokine-related genes. In this study, we utilized RT-PCR to synthesize approximately 120-bp DNA fragment each for 248cytokine-related genes. The lung cancer cell line, A549, was treated with Banlangen, and the total mRNAs were then extracted. The expression difference of these gens in the presence and absence of Banlangen was determined by the array chip and was further by real-time RT-PCR. Our results revealed that Banlangen inhibited the expression of EphA1, EphA3, and DLK, whereas it enhanced the expression of Tyro3, IL30, and CXCL12 The expression of Tyro3 was also confirmed by Western bolt. These results will be further confirmed by animal study.
目錄
中文摘要………………………………………………………………………….i
英文摘要………………………………………………………………………….ii
目錄..................................................................................................iii
圖目錄.............................................................................................. vi
表目錄...............................................................................................vii
第一章 緒論……………………………………………………………………..1
1.1 動機.......................................................................................1
1.2 文獻回顧................................................................................1
1.2.1 板藍根的功用.....................................................................1
1.2.2 關於SARS病毒....................................................................5
1.2.2.1 病毒的發現......................................................................5
1.2.2.2 SARS的臨床症狀..............................................................5
1.2.3 關於248個細胞激素相關基因的分類....................................6
1.2.3.1 細胞激素.........................................................................7
1.2.3.2 細胞激素受體..................................................................8
1.2.4 基因晶片的基本原理及應用..................................................9
1.2.4.1 基本原理.........................................................................9
1.2.4.2 晶片的應用....................................................................10
1.2.5 即時定量PCR的基本原理………………………………………………...13
1. 2. 6 西方墨點法的基本原理……………………………………………………15
1.3 本文目的..............................................................................17
第二章 材料與方法..........................................................................18
2.1 藥物的萃取...........................................................................18
2.2 細胞株的培養........................................................................18
2.3 RNA的萃取及cDNA的製備…………………………………………18
2.4 細胞激素相關基因晶片的製作實驗及分析..............................19
2.4.1 chip的製作........................................................................20
2.4.2 螢光探針(probe)的製作.....................................................20
2.4.3 雜交(Hybridization)...........................................................21
2.4.3.1 方法.............................................................................21
2.4.3.2 試劑製備......................................................................22
2.4.4 分析.................................................................................23
2.5 Real-time quantitative RT-PCR(qRT-PCR)的分析......................23
2.6 Western blot analysis………………………………………………….24
第三章 結果與討論..........................................................................26
3.1 結果.....................................................................................26
3.1.1 Cytokine chip 分析的結果……………………………………….26
3.1.2 Real-time quantitative RT-PCR的結果.................................31
3.1.3 Western blot analysis的結果...............................................35
3.2 討論......................................................................................37
第四章 結論.....................................................................................39
參考文獻..........................................................................................40
附錄.................................................................................................52

TABLES OF CONTENTS

ENGLISH ABSTRACT……….……………….………………………..…..i
CHINESE ABSTRACT…….……………...............……………………......ii
TABLE OF CONTENTS….……………......…….…………..…………….iii
LIST OF FIGURES………………….......………………..………………..vi
LIST OF TABLES……………….......…………………….……………….vii
CHAPTER
I INTRODUCTION……………………..................…………….1
1.1 Motivation..……………………….................…………….1
1.2 Literature Review…………….................………………....2
1.2.1 The effects of Banlangen…………….................…….2
1.2.2 About SARS virus……………................…………….5
1.2.2.1 SARS virus discovery…………….................……5
1.2.2.2 SARS Clinical features…………….............……..5
1.2.3 Classification of the 248 cytokine-related genes……...6
1.2.3.1 Cytokine …………….................………………....6
1.2.3.2 Cytokine receptors ……………........…………….9
1.2.4 Application and principle of chip arrays....…….……10
1.2.4.1 Principle…………….................………………..10
1.2.4.2 Principal application..……….……..……………11
1.2.5 Principle of Real-Time PCR…................……………15
1.2.6 Principle of Western Blotting…….......………………17
1.3 Purpose.......…………………………………………..…20
II MATERIALS AND METHOES………….…….……………21
2.1 Purification of Ban-Lan-Gen…….………….…………..21
2.2 Cell culture and drug treatment………………..………..21
2.3 RNA extraction and generation cDNA….…..…………..22
2.4 Microarray analysis…………….................…………….22
2.4.1 Microarrays prepare ……...........................................22
2.4.2 Probe-labeled samples prepare………………………23
2.4.3 Hybridization…………………………………………24
2.4.4 Analysis …………….................…………………….24
2.5 Real-time quantitative reverse transcriptase polymerase chain reaction (real-time qRT-PCR) …………………...25
2.6 Western blot analysis………................…………………26
III RESULTS AND DISCUSSION..……………………….....….27
3.1 Results…………………………………………....………27
3.1.1 Cytokine chip analysis……….................……………27
3.1.2 Real-time quantitative RT-PCR analysis…………….32
3.1.3 Western blot analysis…………...........………………36
3.2 Discussion….……………….…….……….……………...37
IV CONCLUSION S….........................................................………40
REFERENCES…........…………………….…………………...…………..41
APPENDIXES..............................................................................................53
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