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研究生:游淑琄
研究生(外文):Shu- Chuan Yu
論文名稱:RNA的干擾作用減輕脂多糖所引起的快月太合成
論文名稱(外文):RNA interference attenuates lipopolysaccharide-induced tachykinin synthesis
指導教授:賴義隆賴義隆引用關係
指導教授(外文):Yih-Loong Lai
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生理學研究所
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:2001
畢業學年度:89
語文別:中文
中文關鍵詞:脂多糖RNA干擾做作用快月太P物質即時定量反轉錄聚合酵素鏈反應
外文關鍵詞:LipopolysaccharideRNA interferenceTachykininSubstance Preal-time quantitative RT-PCR
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內毒素 (LPS, lipopolysaccharide) 是革蘭氏陰性細菌細胞壁上的一種脂多糖類成分,此物質和許多肺部及呼吸道疾病的促成有關。先前學者之研究指出,當內毒素的含量在體內增多時,則會促進肺水腫及肺損傷的病理形成及氣喘的惡化。LPS的作用可能經由快 月太,包括由感覺C類神經纖維釋放出來的P物質 (substance P) 及神經激月太A (neurokinin A)。快月太在呼吸道中的生物性效能包括引起血漿蛋白外滲、呼吸道收縮、發炎細胞的匯集、及黏液分泌的增加…等等。而快月太已被證實在內毒素所引起的呼吸道發炎反應中扮演重要之角色。因此,阻止快月太的合成可能是防止由LPS引起肺病變的好方法。
先前學者的研究指出RNAi (RNA interference, double-strand RNA)會干擾基因的post-transcription,或許可以阻止快月太的合成。因此,本實驗的研究目的在於探討內毒素引起的急性肺損傷是否會被RNAi給抑制下來,進而評估RNAi的作用機制。
我們的實驗分成體內實驗 (in vivo) 及體外實驗 (in vitro)。In vivo實驗:我們將Brown Norway品系的大鼠分成兩大部分,第一部分分成四組:Control-1 (不切除結狀神經節,腹腔注射生理食鹽水)、Control-2 (切除結狀神經節,腹腔注射生理食鹽水)、Lipopolysaccharide (LPS-1) (不切除結狀神經節,腹腔注射LPS 4 mg/kg)、LPS-2 (切除結狀神經節,腹腔注射LPS 4 mg/kg)。第二部分分成兩大組 (12小時及24小時),各組再分三小組:Control (腹腔注射生理食鹽水,結狀神經節給予生理食鹽水)、LPS (腹腔注射LPS 4 mg/kg,結狀神經節給予生理食鹽水)、LPS+dsRNA (腹腔給LPS 4 mg/kg、結狀神經結給予2μl dsRNA)。分別從頸部兩側的結狀神經節及肺組織中萃取全部RNA,同時取兩側肺組織測P物質。In vitro實驗:初代培養結狀神經節,分成三組:Control (medium only)、LPS (10μg/ml)、LPS (10μg/ml)+dsRNA,取培養液測P物質,另刮取神經細胞抽全部RNA。RNA利用即時定量反轉錄聚合酵素鏈反應 (real-time quantitative RT-PCR) 的方法來分析preprotachykinin (PPT) mRNA的相對量 (表示快月太 的合成程度)。此法提供具高產量、複製性、及敏感性的技術來偵測極微量的全RNA中PPT mRNA所佔的量。之後我們採用CT比較法 (△△CT),其中CT的定義是PCR過程中的臨界cycle數,而△△CT的計算是從(待測樣本的△CT -校正的△CT)。而P物質是利用EIA的方法去偵測。
我們的結果顯現:在In vivo實驗,LPS處理後,肺組織及結狀神經節中的PPT mRNA的表現增加,P物質的量也上升;而在In vitro實驗中,LPS給予後,結狀神經節細胞中的PPT mRNA與對照組相比表現增加,細胞培養液中P物質也顯示上升。不論在In vitro或In vivo同時給予dsRNA處理後則明顯地減弱了LPS誘使PPT-Ⅰ基因的轉錄及轉譯。
從我們的結果推測:(1)不論是在in vitro或in vivo,結狀神經節的確是表現PPT mRNA的地方;(2)內毒素會使造成肺致病的媒介物,P物質,合成增加,這作用是經由調控PPT-Ⅰ表現所致;(3)我們猜測dsRNA傳送的效力非常好,因此有沒有liposome沒有太大的幫助; (4) dsRNA除了抑制PPT mRNA量的增加 (減少mRNA的合成或增加mRNA的代謝)外,也會抑制P物質蛋白質合成。由我們的實驗結果應可提供臨床上利用dsRNA改善內毒素所引起的敗血症、肺水腫、及成人呼吸窘迫症之可能性。

Endotoxin, a lipopolysaccharide (LPS) component of the cell wall of gram-negative bacteria is implicated in many pulmonary and airway inflammatory diseases. Previous studies have suggested that endotoxin plays a role in the aggravation of asthma and in the pathogenesis of pulmonary edema and lung injury in vivo when a large dose of LPS is administered. Effects of LPS may act via tachykinins, including substance P and neurokinin A, which are released from sensory C-fibers. The biological effects of tachykinins in the airways include plasma protein extravasation, airway constriction, recruitment of inflammatory cells and increased mucous secretion. Furthermore, LPS has previously been demonstrated to play a pivotal role in LPS-induced airway inflammation. For this reason, preventing tachykinins synthesis may be a good method to avoid LPS-induced lung pathology.
Previous studies have suggested that RNAi (RNA interference, double-strand RNA) interferes genetic post-transcription. However,it is not clear whether RNAi suppresses the synthesis of tachykinins. Therefore, the purpose of this study was to determine if RNAi inhibits LPS-induced an increase in tachykinin synthesis.
We used both the in vivo and the in vitro studies. The in vivo study was divided into two portions; the first portion was divided into 4 groups: control-1, control-2, LPS-1, LPS-2. The second portion was divided into 2 groups (LPS 12 hr and LPS 24hr), and each group was further divided into 3 subgroups (control, LPS, and LPS+dsRNA). Total RNA was separately extracted from cervical bilateral nodose ganglia and lung tissues, and substance P was extracted from bilateral lung tissues. For the in vitro study: cultured primary nodose ganglia were divided into three groups: control (medium only), LPS (10μg/ml), and LPS (10μg/ml)+dsRNA. Substance P was extracted from cultured medium, and total RNA was extracted from nodose ganglia cells. The real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was used to assay relative amounts of PPT mRNA (indicating the degree of tachykinin synthesis). This method provides the first high-throughput, reproducible, and sensitive technique capable of determining PPT mRNA levels in nanogram amounts. Subsequently, we evaluated the RT-PCR data using comparative CT (△△CT), where CT is defined as the threshold cycle of PCR amplification period, and △△CT is calculated from the formula of (△CT of unknown-△CT of calibrator). Substance P was measured by a Substance P Enzyme Immunoassay Kit.
Our in vivo results demonstrated that LPS (4 mg/kg) raised PPT mRNA and substance P levels in both nodose ganglia and lung tissue. Also, in comparison with control, LPS (10μg/ml in vitro) elevated PPT mRNA level in nodose ganglia, and increased substance P in culture medium. The LPS-induced increase in PPT-Ⅰgene transcription and translation were significantly decreased by dsRNA treatment.
From these results, we suggest that: (1) PPT mRNA could be expressed in nodose ganglia in vivo and in vitro; (2) endotoxin affects the synthesis of SP, a mediator of pulmonary pathogenesis via preprotachykinin-Ⅰgene expression; (3) dsRNA transfection is effective, even it is mediated with or without liposomes; (4) dsRNA inhibits both mRNA expression and substance P synthesis augmented by LPS. Our findings support the clinical usage of dsRNA to ameliorate the sepsis and adult respiratory distress syndrome, which are commonly induced by endotoxin.

目 錄

致謝………………………………………………….………………….Ⅰ
表次…………………………………………...……………….………..Ⅳ
圖次……………………………………………...…………….………..Ⅴ
中文摘要……………………………………….…………….……..….Ⅶ
英文摘要……………………………………...…………..….…………Ⅸ
緒論及文獻回顧
一、內毒素與肺損傷之間的關係………………….………………1
二、感覺C類神經纖維與快月太在肺臟中的角色….………………3
三、快月太與其接受器及生理效應…………………….…………….4
四、快月太與發炎反應間的交互作用…………………..……………7
五、快月太的前驅物質…………………………….………….………8
六、雙股RNA引發的RNA干擾作用 (RNAi)……………………….9
實驗動機與目的…………………………………….………………….13
材料與方法
一、動物的分組與前處理………………………………………14
二、採取組織樣本……………………………… ………………..14
三、Nodose ganglion cells培養…………… ………………..16
四、RNA 的萃取、定量、品質確認………………………………17
五、dsRNA的製備…………………………………………………19
六、即時定量反轉錄聚合酵素鏈反應…………………………22
七、RNA相對量的計算………………………………….………27
八、測量P物質 ( substance P )………………………………27
九、統計方法………………………………………………..…..29
十、藥品製備及來源………………………………..…………..30
結果
一、全RNA的萃取結果……………………………………31
二、dsRNA的確認結果……………………….……………31
三、即時定量反轉錄聚合酵素鏈的結果分析….…….….31
四、LPS對PPT mRNA的表現量之影響……….…………32
五、右側結狀神經節切除對PPT mRNA的表現量之影響.33
六、右側結狀神經節切除對P物質的表現量之影響…..33
七、dsRNA對PPT mRNA的表現量之影響…………………33
八、 dsRNA對P物質的表現量之影響……………………34
九、不同濃度的LPS對初代培養的結狀神經節細胞之
影響………………….………………………………….35
十、LPS對初代培養的結狀神經節細胞中PPT mRNA之
影響………………………………………………………35
十一、dsRNA對初代培養的結狀神經節細胞中PPT
mRNA之影響………….…………………………………35
十二、dsRNA對初代培養的結狀神經節細胞中P物質之
影響………………………………………………………36
討論
一、內毒素對PPT mRNA的影響……………………………………69
二、迷走神經之結狀神經節對肺組織之神經主控情形…………71
三、利用即時定量反轉錄聚合酵素鏈分析PPT mRNA之探討……74
四、P物質的合成、傳送與分佈……………………………………75
五、雙股RNA所引發的RNAi效應…………………………………76
六、RNAi促使mRNA裂解的可能機制……...………………………80
七、未來研究方向…………………………………………………86
八、總結……………………………………………………………86
參考文獻………………………………………………………………87

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