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研究生:蘇真真
研究生(外文):Su, Jen-Jen
論文名稱:大鼠腦星狀細胞與人類單核性白血球間交互作用產生單核球趨化激素-1和第二介白質及其受體之研究
論文名稱(外文):Interactions between rat brain astrocytes and human mononuclear cells to produce MCP-1 ( monocyte chemoattractant protein-1 ) ; interleukin-2 and interleukin-2 receptor
指導教授:余家利余家利引用關係
指導教授(外文):Yu, Chia-Li
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子醫學研究所
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:單核球趨化蛋白質-1趨化激素第二介白質
外文關鍵詞:MCP-1( Monocyte chemoattractant protein-1)chemokineIL-2 ( Interleukin 2 )
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單核球趨化蛋白質-1 ( Monocyte chemoattractant protein-1, MCP-1 )是屬於趨化激素( chemokine )β家族的一員。星狀細胞( astrocyte ) 於中樞神經膠質系統的一部份,它的足突 ( process ), 除擔任分子的運送外, 也組成部份的腦血管障壁( blood-brain-barrier )。趨化激素一般被認為和組織的發炎﹑感染﹑甚至腫瘤的發生有關。在中樞神經系統的發炎的方面, 一般認為趨化激素具有吸引白血球到組織而造成發炎的功能。MCP-1 更在一些動物實驗中證實可導致神經系統的發炎反應, 與產生類似人類的多發性硬化症的病變有關.在細胞激素中, 第二介白質( Interleukin 2, IL-2) 為一種由T-淋巴球所產生的T-/B-淋巴球的活化因子。第二介白質在發炎反應不僅扮演重要的角色, 同時在一些的研究中顯示在第二介白質與多發性硬化症的致病機轉也有相當的關連性。
但是,有關何種訊息活化星狀細胞產生MCP-1機轉的研究仍然相當有限.在本實驗,我以MTT的方法測定各種中樞神經疾病的腦脊髓亦對老鼠腦星狀細胞存活的影響, 以酵素免疫吸附法( ELISA )定量腦老鼠腦星狀細胞-單核性細胞共同的培養上清液中MCP-1與IL-2的產生, 以反轉錄-聚合酶連鎖反應( RT-PCR )的方法測定UPAR (urokinase-type plasminogen activator receptor )訊息核苷酸的表現, 以流式細胞儀測定細胞表面趨化激素受體的表現.經由以上實驗來探討單核球及星狀細胞間的交互作用。
在我們的實驗中,我們發現老鼠腦星狀細胞和單核球共同培養後; MCP-1的產量有明顯的增加。即使兩種細胞分開培養, MCP-1產量也有增加的現象.但是以多形性白血球或是紅血球和老鼠腦星狀細胞共同培養則對MCP-1產量沒有顯著的影響.同時在加入各種細胞激素後, 發現TNF-α, IL-6, IFN-γ對MCP-1產量的影響較為顯著.但濃度過高的IFN-γ反而抑制MCP-1的產生.在IL-2方面,我們也得到相同的結果: 在老鼠腦星狀細胞和單核球共同培養時較星狀細胞單獨培養時產生較多的IL-2,同時我們測定上清液中的sIL-2R也得到相同結果. 即是兩種細胞分開或共同培養的實驗也有近似的結果。在UPAR實驗中,在星狀細胞ma單獨培養或星狀細胞與老鼠腦星狀細胞共同培養時,都有 UPAR訊息核苷酸的表現.而老鼠腦星狀細胞單獨培養時則無。
另外, 我們嘗試將各種神經科疾病, 包括發炎﹑血管病變﹑免疫性疾病病人的腦脊髓液中與腦星狀細胞共同培養, 再測定MCP-1的產生. 初步的結果顯示並無明顯疾病的相關性; 因此MCP-1在神經疾病扮演的角色, 仍有待進一步的研究.
綜合以上的實驗結果我們發現,老鼠腦星狀細胞和單核性細胞球的共同培養, 無論有無細胞-細胞之間的接觸, 皆會使MCP-1及IL-2/sIL-2的產量較單獨培養時增加.而我們知道MCP-1只有一種受體CCR2, 平時只表現在單核球細胞, 其他實驗曾報告在IL-2的刺激下, 淋巴球除了表現CCR2也會表現CCR1.從以上的的實驗知道單核球能使腦星狀細胞MCP-1的產量增加,IL-2/sIL-2R的產量也增加. 推測腦星狀細胞可能藉由MCP-1活化單核性細胞產生IL-2, 再藉由單核性細胞產生的TNF-α, IL-6,IFN-γ形成放大迴路進一步刺激腦星狀細胞產生更多的MCP-1.至於其真正的機轉則有待進一步探討。

Monocyte chemoattractant protein —1 ( MCP-1 ) is belonged to chemokine βfamily. Astrocytes are part of glial system of the central nervous system( CNS ). The foot process of astrocyte is responsible for molecular transport and composes part of blood-brain-barrier ( BBB ). Chemokines are thought to be related with tissue inflammation, infection and tumor. As about the CNS infection, chemokines are thought to attract leukocytes to tissue then induce inflammation. In some animal model; MCP-1 is identified to induce CNS inflammation. Interleukin-2 ( IL-2 ), produced by T lymphocyte, is a kind of T-/B- lymphocyte growth factor. Except being the important role in inflammation, IL-2 also shows significant correlation with multiple sclerosis ( MS ) in a series of studies.
Anyway, what kind of signals to activate astrocyte or produce MCP-1 is still unknown. In our study, we used cell culture and MTT test to test the survival of rat neroblastoma cell ( RBA-1 ); ELISA to quantity the production of MCP-1 and IL-2 from super ant; RT-PCR to estimate UPAR expression and flow cytometry to estimate chemokine receptor. We wish to clarify the interaction between monocyte and RBA-1 and the effect to produce IL-2 and MCP-1. Then we estimate MCP-1 form co-culture of RBA-1 with CSF from lot patients with neurological diseases for the possible pathogenesis.
In our experiment; we found there is significant increase of MCP-1 after RBA-1 co-culture with MNC. Also it is same when we used transwell to separate both kinds of cells. And we found TNF-α,IFN-βand IL-6 influence RBA-1 to produce more MCP-1. We also had more IL-2 and sIL-2R produced when co-culture with RBA-1 and MNC. As to UPAR experiment, we found no UPARmRNA expression when RBA-1 alone.
According to the above experiments, we conclude that MCN can make RBA-1 produce increased MCP-1 even without direct contact; then MCP-1 activate MNC to produce IL-2, and it will make an amplification recruitment to make further inflammation.

中文摘要
英文摘要
緒論
方法
結果
討論
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參考文獻

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