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研究生:徐泱彤
論文名稱:探討巨噬細胞促發炎與非發炎beta-葡聚醣處理後之對巨噬細胞典型與另類活化的影響
論文名稱(外文):Investigation on the combinatory effects of non-inflammatory and pro-inflammatory beta-glucans in macrophage classical and alternative activations
指導教授:翁博群
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:微生物免疫與生物藥學系研究所
學門:生命科學學門
學類:其他生命科學學類
論文種類:學術論文
畢業學年度:103
語文別:中文
論文頁數:168
中文關鍵詞:巨噬細胞分化葡聚糖發炎反應
外文關鍵詞:Macrophge polarizationbeta-glucansinflammation
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前言:Beta-glucans,以beta醣苷鍵鍵結的葡萄糖聚合體,廣泛分布於自然界,並發現存在於細菌、真菌、蕈類、藻類或高等植物等細胞壁中。現今,已得知beta-glucans具有許多免疫刺激的功能,以beta-(1,3)-glucan及beta-(1,3)(1,6)-glucan的研究最為廣泛,其具有提高抵抗細菌、真菌的能力或抗癌的效果等;beta-(1,3)(1,4)-glucan則有降低心血管疾病發生,直至近期才有免疫相關的研究。在實驗室先前的實驗中發現植物來源為主的beta-(1,3)(1,4)-glucan不具刺激巨噬細胞發炎反應,並提高scavenger receptors表現,刺激巨噬細胞Lys M與Arg-1基因表達,此現象與M2另類巨噬細胞活化表現相類似,後歸類為「非發炎型beta-glucans」。相反地,微生物來源為主的beta-(1,3)-glucan及beta-(1,3)(1,6)-glucan活化巨噬細胞NO與TNF-的分泌,並降低Arg-1與Lys M基因表達,此現象與M1典型巨噬細胞活化表現相似,後歸類稱為「促發炎型beta-glucans」。本篇目的為延續性試驗,分類市售六種且不同來源的高純度beta-glucans為非發炎型beta-glucans或促發炎型beta-glcuans,並以共同活化巨噬細胞方式,以探討兩類beta-glucans間對於不同巨噬細胞之典型或另類途徑活化作用之影響,以進一步瞭解不同來源beta-glucan在巨噬細胞功能與結構上的關係。
材料與方法:六種市售不同來源、高純度的非發炎型beta-glucans來自於昆布 (Laminaria digitata, laminarin, LA)大麥 (barley, GB)與地衣 (lichenan, LI)和促發炎型beta-glucans來自於真菌 (Saccharomyces cerevisiae, zymosan, ZY)、眼蟲 (Euglena gracilis, paramylon, EG)及細菌 (Alcaligenes faecalis, curdlan, CD)與三種不同的巨噬細胞 (RAW264.7、J77A.1與小鼠腹腔巨噬細胞)進行非發炎型與發炎型beta-glucans共同培養,測量NO產生、oxidative burst、lysozyme activity,甚至是iNOS、Lys M、arg-1 mRNA的表達,另外亦測定了TNF-與IL-10細胞激素的表現量。
結果與討論:
受ZY與CD刺激活化的RAW264.7巨噬細胞細胞株與小鼠腹腔巨噬細胞與非發炎型beta-glucans共同培養產生大量促發炎因子及IL-10,此因可能是巨噬細胞具有強烈發炎反應,而使IL-10分泌。受EG刺激活化的RAW264.7巨噬細胞細胞株與小鼠腹腔巨噬細胞與非發炎型beta-glucans共同培養產生比其他兩種促發炎型beta-glucans活化之巨噬細胞產生的發炎反應較輕微,但同時產生IL-10調控發炎反應。另外,促發炎型beta-glucans無法將J774A.1巨噬細胞細胞株極化成M1典型巨噬細胞。
Beta-glucan is a glucose homopolysaccharide naturally occurred in a variety of botanical or microbial sources. Beta-(1,3)-glucan and beta-(1,3)(1,6)-glucan from microbial origins may modulate immunity in a number of beneficial applications such as providing protection against infections agents and anti-tumorignesis. Beta-(1,3)(1,4)-glucan enriched in oat and barley that favorably modify cholesterol and preventions in cardiac vascular disease and diabetes. Previous in our lab, we showed that beta-(1,3)(1,4)-glucan stimulated scavenger receptor expression and upregulated Arg-1 and Lys M gene expressions in macrophages. The outcomes were similar to alternative activated M2-like macrophges, and botanical beta-glucans including beta-(1,3)(1,4)-glucans were categorized into ‘non-inflammatory beta-glucans’. Conversely, beta-(1,3)-glucan and beta-(1,3-(1,6)-glucan of mainly microbial origins could induce NO and TNF-productions of macrophages. The outcomes were classical activated M1-like macrophages and beta-(1,3) and beta-(1,3)(1,6)-glucans of primarily microbial origins were categorized into ‘pro-inflammatory beta-glucans’. The present work utilized macrophage cell lines of RAW264.7, J774A.1 as well as primary peritoneal macrophage to examined the combinatory effects of non-inflammatory beta-glucans (laminarin for LA, barley beta-glucan for GB and lichenan for LI) and inflammatory beta-glucans (zymosan for ZY, paramylon for EG and curldan for CD) on the M1 or M2 macrophages activation. And the structure of beta-glucans and their functional relationship was further discussed. Results showed that ZY and CD induced strongly pro-inflammatory mediators productions whereas IL-10 was produced in RAW264.7 macrophage and mouse peritoneal macrophages treated with non-inflammatory beta-glucans. EG poorly induced pro-inflammatory mediators and IL-10 than ZY and CD. In addition, J774A.1 of a M2-like macrophage cell line was not actisvated into M1-like macrophage by pro-inflammatory beta-glucans.
致謝…………………………………………………………………….....I
中文摘要………………………………………………………………...II
英文摘要………………………………………………………………..IV
目錄……………………………………………………………………..VI
圖次……….……………………………………………………………..X
附錄目錄….……………………………………………………..….XVIII
縮寫表……………………………………………………………..….XIX
壹、 文獻回顧…………………………………………………………..1
一、 Beta-glucan (beta-葡聚醣)……………………………….......1
I. Beta-glucans的分類與生理特性………..……………..2
1. 結構性…………………..……………………………..3
2. 分子量大小……………………………………………4
II. Beta-glucans的受體與傳遞路徑…………………..…..5
III. 市售beta-glucans…..…………………………………...7
(一) Laminarin (LA)…………………………………………7
(二) Barley-derived beta-glucan (GB)……………………….8
(三) Lichenan (LI)……………………………………………8
(四) Zymosan (ZY)…………………………………………..9
(五) Euglena gracilis derive beta-glucan (Paramylon;EG)..10
(六) Curdlan (CD)…………………………………………..10
二、 巨噬細胞的極化……………………………………………11
I. 巨噬細胞的功能及亞群………………………………11
1. 典型巨噬細胞…………………………..……………13
2. 另類巨噬細胞………………………………………..13
3. 小鼠巨噬細胞細胞株:RAW264.7與J774A.1………14
II. L-arginine代謝路徑:NOS與arginase………………..16
III. Lysozyme……………………………………………...18
IV. 前人的相關研究.......................……………………….20
V. Beta-glucans刺激活化的免疫反應..............................21
VI. 參考文獻……………………………………………....24
貳、 前言………………………………………………………………36
參、 材料與方法………………………………………………………42
一、 實驗材料……………………………………………………42
(1) Beta-glucans的來源……………………………………....42
(2) 動物飼養與管理……………………………………….....43
(3) 細胞株來源……………………………………………….43
(4) 試劑藥品與耗材………………………………………….43
(5) 儀器……………………………………………………….46
二、 實驗方法…………………………………………………....47
(1) Mouse macrophage cell lines (細胞組織培養)……...……47
(2) Isolation and purification of BALB/c mice peritoneal macrophage (初代巨噬細胞分離與純化)….…………….47
(3) Preparation of beta-glucans (Beta-glucans製備)…..48
(4) Nitric oxide measurement in cell culture supernatants by Griess reaction assay (細胞上清液中NO濃度的測定)….49
(5) Measurement of intracellular ROS level (細胞內氧化壓力的測試)……………………………………………………51
(6) Gene expression analysis for iNOS and arginase-1 by PCR (iNOS與arginase-1的基因分析)………………………...53
a. Isolation of total RNA (RNA純化)…………………..54
b. Quantification of total RNA (RNA定量)…………….54
c. RNA reverse transcripted to cDNA(RNA反轉錄cDNA)………54
d. Polymerase chain reaction (PCR)…………………….55
e. Gel electrophoresis analysis and semi-quantification of PCR production (瓊脂電泳分析與半定量)………….56
(7) Lysozyme activity assay (Lysozyme活性分析)………….56
(8) TNF-a and IL-10 measurement in cell culture supernatants by ELISA assay (細胞上清液TNF-與IL-10的測定)…58
A. TNF-
B. IL-10……………………………………………………59
(9) Statistical analysis (統計分析)……………………………60
肆、 結果與討論………………………………………………………61
一、 NO production of macrophages stimulatied by beta-glucans (Beta-glucans誘導巨噬細胞產生NO的能力之影響).….....61
二、 Oxidative burst of macrophages stimulated by beta-glucans (Beta-glucans誘導巨噬細胞產生oxidative burst的能力之影響)…………………………………….……………………...68
三、 Lysozyme activity of macrophages stimulated by beta-glucans (Beta-glucans誘導巨噬細胞對lysozyme活性的影響)……72
四、 Gene expression of macrophage stimulated by beta-glucans (Beta-glucans影響巨細胞基因表達的差異)………………75
五、 The secretion of TNF-a and IL-10 from macrophages stimulated by beta-glucans (Beta-glucans對巨噬細胞細胞激素分泌的影響)………………………………………………79
六、 結論………………………………………………………….84
伍、 參考文獻…………………………………………………………87
陸、 結果圖……………………………………………………………92
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