作家專欄

黃麴毒素B1對鴨周邊血液淋巴細胞
之致害作用及β-胡蘿蔔素對其保護效果

鄭永祥(1)    龐    飛(2)

收件日期:84年10月26日;接受日期:85年1月29日

摘   要

        本試驗之目的為建立以MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)方法進行鴨周邊血液淋巴細胞增殖作用之測定,並利用此法探討黃麴毒素B1 (AFB1)對鴨周邊血液淋巴細胞之致害作用和β-胡蘿蔔素(BC)對其保護效果。

        試驗一:在以MTT法進行裂殖原的比較中顯示,三種裂殖原中OD值以phytohaemagglutinin,(PHA;0.603)的增反應最佳。pokeweed mitogen,(PWM;0.421)次之,而concanavalin A,(Con A;0.395)最差。迴歸方程式經一次微分計算增殖反應所須最佳裂殖原濃度分別為PHA 23.68μg/ml、Con A 34.48 μg/ml和PWM 23.57 μg/ml。試驗二:淋巴細胞數對MTT之反應呈二次曲線,當細胞濃度自1×104提高至1×107 cell/ml時,隨著細胞數目的增加其OD值則呈降低的趨勢。試驗三:結果顯示AFB1處理組自高量(1×104 ng/ml)至低量時(1×101 ng/ml),其OD值顯著(P<0.05)較單獨PHA處理組為低,但AFB1在極低量(1×10-2 ng/ml)時對淋巴細胞增殖無抑制作用。當高濃度AFB1 (1×104 ng/ml)微粒體代謝後,較未經微粒體代謝AFB1顯著(P<0.05)抑制淋巴細胞增殖作用,但低濃度(1×101、1×10-2 ng/ml)時則抑制作用不顯著。試驗四:結果顯示單獨高濃度BC (0.1 μM)添加時,OD值顯著(P<0.05)較培養基對照組為高。且中低濃度BC (0.05、0.025μM)添加於AFB1經微粒體代謝之培養系統中時對細胞增殖率則無改善之效果。然而高濃度BC (0.1 μM)添加於AFB1經微粒體代謝之培養系統中時,顯著的(P<0.05)較中低濃度BC者之細胞增殖率為高,且改善至與單獨PHA刺激的對照組相同之細胞增殖率。

        綜觀本試驗結果顯示高濃度AFB1 (104 ng/ml)經代謝後對鴨淋巴細胞增殖作用的抑制性有增強的效果,而高濃度B-胡蘿蔔素(0.1 μM)可保護鴨淋巴細胞免除此一毒害作用。(關鍵語:鴨、黃麴毒素B1、淋巴細胞、β-胡蘿蔔素)

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(1)國立宜蘭農工專科學校畜產科。

(2)國立台灣大學獸醫學系。

緒   言

        黃麴毒素B1 (AFB1)具有免疫抑制性(Pier et al., 1977),且抑制phytohaemagglutinin (PHA)所誘發之鴨淋巴細胞轉型作用(鄭與龐,1994),其致害可能為AFB1經肝臟微粒體作用(鄭與龐,1994-5),其致害可能為AFB1經肝臟微粒體作用代謝為毒性較高之氧衍生自由基(oxygen-derived free radical)所致(Neldon-Ortiz and Qureshi, 1991),過去β-胡蘿蔔素 (β-carotene)一直局限於維生素原A (Provitamin A)的角色,但最近的研究發現,β-carotene不須先轉變為維生素A即可有效清除新生態氧和過氧化自由基(Chew, 1993aKadian et al., 1988),在家畜日糧中具有免疫調節的功能,先前的研究顯示β-carotene可促進鼠毒殺性T-細胞的增殖、增加裂殖原所誘發之鼠(Bendich and Shapiro, 1986)、豬淋巴細胞轉型反應及巨噬細胞之tumor necrosis factor (TNF)活性(Bendich, 1991)及與維生素E協同增強雞對疾病抵抗力和降低因Escherchia Coli感染之死亡率(Tengerdy et al., 1990)。

        生物學上常需測定細胞存活率及增殖作用,其方法包括有trypan blue染色法、放射法核W酸併入法(3H-thymidine incorperation)。另外;快速呈色法,其主要依賴粒線體中琥珀酸去氫A之作用將MTT 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)之tetrazolium轉為藍色之產物MTT formazan (Mosmann, 1983;Denizot and Lang, 1986)。MTT轉變僅在存活細胞中進行,且formazan形成量與數目呈正比,由於擁有快速、經濟和無放射性元素污染之問題,故常被使用為細胞存活率及巨噬細胞吞噬能力之測定,且經比較MTT呈色法所測定禽類細胞存活率與放射性核W酸併入法之結果相關性可達0.8 (Bounous et al., 1992)。

        本試驗之目的即在以MTT方法探討試管內鴨淋巴細胞最佳裂殖原及濃度、細胞數及黃麴毒素B1對鴨周邊血液淋巴細胞之致害作用和β-carotene對其保護效果。

材料與方法

一、實驗動物及試驗液製備

        以成熟之產蛋菜鴨做為血液供應鴨,採血期間鴨隻採籠飼,飼料及飲水均自由採食。

        鴨淋巴細胞之分離與純化係採用鄭與龐(1)之方法。先自鴨翼下靜脈採血10ml,以EDTA (0.2%)為抗凝劑。該血液,經4℃,1300×g下離心30分鐘,收集白血球層,與合0.2%EDTA之等量 0.2M phosphate buffer solution (PBS, pH 7.2, 41℃)混合。再將之置於等量Ficoll-Paque (density:1.077;Sigma, St Louis, Mo., USA)上於4℃,200×g下離心25分鐘,將細胞層吸出,先以0.2 M PBS清洗一次,於4℃,200×g下離心10分鐘後再以含EDTA (0.2%)之RPMI-1640清洗兩次,取得純度95%以上淋巴細胞。將細胞懸浮液以含20%胎牛血清、青黴素(100U/ml)、鏈黴素(100 μg/ml)及 1% L-glutamine之RPMI-1640調整最終細胞濃度為1×107淋巴細胞/ml。

        MTT溶液製備:將MTT (Sigma, M2128)以0.2 M PBS調整為 5 mg/ml之原液,經0.2μm millipore過濾,以鋁箔紙密封遮光置於4℃保存備用。

        裂殖原之製備:phytohaemagglutinin (PHA)、pokeweed mitogen (PWM)及concanavalin A (Con A)等三種裂殖原。先以含20%非動化胎牛血清之培養用RPMI-1640調整為5mg/ml之原液備用。

        黃麴毒素B1製備:將純品AFB1結晶5 mg (Sigma, A6636),先以0.5ml二甲亞楓(dimethyl sulfoxide, DMSO)溶解,再以0.2 M PBS調整為1mg/ml之原液,以鋁箔紙密封遮光置於-20℃保存備用。

        β-carotene製備:將β-carotene 10 mg之純品(Sigma, C-0126)先以絕對酒精溶解,再以0.2 M PBS 10 ml調整為1 mg/ml之原液,以鋁箔紙密封遮光置於-20℃保存備用。

        肝臟微粒體之製備:取鴨肝臟40g置於200ml冰冷之0.2 M PBS中,將肝臟剪碎。使用均質機(Ace homogenizer, Nissei AM-5)予以均質,再以組織研磨機(Wheaton overhead stirrer)研磨來回三次。均質液以10000×g離心15分鐘(HIMAC, CR21)使細胞核及粒線體沉澱,上清液以100000×g離心60分鐘,倒去上清液後將沉澱之肝細胞微粒體,以0.2 M PBS稀釋為5mg/ml微粒體蛋白質之原,微粒體蛋白質含量係依Peterson (1977)修定Lowry et al. (1951)所發表的方法測定之,以牛血清白蛋白做為蛋白質標準。

        NADPH之製備NADPH (Sigma, St. Louis, MO., USA),以0.2 M PBS調整為0.25 mM溶液,再以鋁箔紙密封遮光置於-80℃保存備用。

        淋巴細胞存活率分析:將50ul,1×107 cells/ml之鴨淋巴細胞和20μl濃度為125μg/ml之PHA,並以RPMI-1640調整使各孔之總體積為250μl置入96孔U型之微量培養皿中,於41℃,含5% CO2之濕壓恆溫箱中培養72小時,培養結束前4小時,經800×g離心5分鐘,並吸除上清液,將濃度5mg/ml的MTT 20 μl及RPMI-1640  80μl加入微量培養皿中,時間到後經800×g離心5分鐘,並吸除上清液且於每孔中加100μl之acid isopropanol (0.04 N HCl/isopropanol)。經由振盪以溶解所形成之藍色formazan結晶,於波長560nm下以ELISA reader讀取OD值,每一試驗之處理使用6個孔。

二、試驗處理

        試驗一:不同裂殖原PHA、Con A及PWM於不同濃度(0、5、10、20和40 μg/ml)加入培養系統中,最終細胞濃度為1×107淋巴細胞/ml,以探討最佳MTT反應之裂殖原與濃度。

        試驗二:不同鴨淋巴細胞濃度(1×104、1×105、1×106、1×107和1×108 cell/ml)加入培養系統中,以探討最佳MTT反應之細胞濃度。

        試驗三:培養系統加入不同最終濃度之黃麴毒素B1 (1×104、1×101、1×10-2 ng/ml)及有無添加肝臟微粒體,以探討黃麴毒素B1代謝與否對鴨淋巴細胞毒害作用,最終細胞濃度為1×107淋巴細胞/ml。試驗處理詳如表1。

 

表一 以MTT法測定黃麴毒素B1對鴨淋巴細胞毒害作用之試驗處理

Table 1. The experimental design of cytotoxic effect of aflatoxin B1 on duck lymphocytes by MTT assays

  CTRL *- CTRL+ MIC AFB1 104 ng/ml MIC AFB1 104 ng/ml AFB1 101 ng/ml MIC AFB1 101 ng/ml AFB1 10-2 ng/ml MIC AFB1 10-2 ng/ml
RPMI-1640

Lymphocytes

(1×107/ml)

AFB1

PHA

(200μg/ml)

MIC

(12.5 mg/ml)

NADPH

(0.25 mM)

Total

200

50

 

 

 

 

250

175

50

 

25

 

 

 

250

170

50

 

 

20

 

10

 

250

100

50

 

45

25

 

20

 

10

 

250

130

50

 

45

25

 

 

 

250

100

50

 

45

25

 

20

 

10

 

250

130

50

 

45

25

 

 

 

20

100

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45

25

 

20

 

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250

130

50

 

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25

 

 

 

250

*CTRL-=medium control;CTRL+=PHA alone;MIC=microsome alone;Volume unit=μl.
試驗四:培養系統加入不同最終濃度之β-胡蘿蔔素(0.025、0.05、0.1μM)和添加肝臟微粒體與黃麴毒素B1 (1×104 ng/ml),最終細胞濃度為1×107淋巴細胞/ml,以探討黃麴毒素B1對鴨周邊血液淋巴細胞之致害作用及β-胡蘿蔔素對其是否具保護作用。試驗處理詳如表2。

表二 以 MTT 法 測 定 黃 麴 毒 素 B1 對 鴨 周 邊 血 液 淋 巴 細 胞 之 致 害 作 用 及 β- 胡 蘿 蔔 素 對 其 保 護 效 果 之 驗 處 理

Table 2.  The experimental design of cytotoxic effect of aflatoxin B1 on duck lymphocytes and protective activity of β-carotene by MTT assays

  CTRL- CTRL+ MIC BCH AFB1 104 ng/ml AFB1 104 ng/ml MIC AFB1 104 ng/ml MIC+ BCL AFB1 104 ng/ml MIC+ BCM AFB1 104 ng/ml MIC+ BCH
RPMI-1640

Lymphocytes

(1×107/ml)

AFB1

PHA

(200μg/ml)

betacarotene

MIC

(12.5 mg/ml)

NADPH

(0.25 mM)

Total

200

50

 

 

 

 

250

175

50

 

25

 

 

 

250

170

50

 

 

20

 

10

 

250

170

50

 

 

30

 

 

250

130

50

 

45

25

 

 

 

250

100

50

 

45

25

 

20

 

10

 

250

70

50

 

45

25

 

30

20

 

10

 

250

70

50

 

45

25

 

30

20

 

10

 

250

70

50

 

45

25

 

30

20

 

10

 

250

*CTRL-=medium control;CTRL+=PHA alone;MIC=microsome alone;BCH=betacarotene alone (0.1μM);Volume unit=μl.

三、統計分析

        實驗所得之資料先經一般線性模式(general linear model;GLM)進行變方分析,再以鄧肯氏新多項變域法(Duncan's new multiple range test)比較各組間差異顯著性。

結果與討論

        不同裂殖原PHA、Con A及PWM於不同濃度(0、5、10、20和40μg/ml)加入培養系統中,以探討最佳MTT反應之裂殖原與濃度,結果示於圖1。資料經多項式(polynominal)迴歸所繪之迴歸曲線圖,由曲線圖可知裂殖原以PHA刺激增殖反應最佳,PWM次之,而ConA最差。迴歸方程式經一次微分求得增殖反應所須最大裂殖原濃度分別為PHA 23.86μg/ml、Con A 23.57 μg/ml和PWM 34.48 μg/ml。鴨周邊血液淋巴細胞不同濃度對MTT存活率之反應,示於圖2淋巴細胞數對MTT之反應呈二次曲線,當細胞濃度自1×104提高至1×107 cell/ml時,淋巴細胞數目持續增加其OD值亦升高,當細胞濃度再增加到1×108 cell/ml時OD值呈降低的趨勢,推測此一細胞濃度在經72小時培養後,營養分可能為其增殖之限制因子,亦即胎牛血清已用盡,由此一結果顯示以MTT方法測定鴨淋巴細胞之存活率以1×107 cell/ml最為適當,此一結果與Lessard and Dupuis (1994)試驗使用之細胞數一致。不同家畜禽之淋巴細胞表面之裂殖原受體數目與特性不一,對同一裂殖原反應時之最佳細胞數互異(Talebi et al., 1995)。

             wpeA6.jpg (42977 bytes)

                    wpe90.jpg (30185 bytes)

以不同濃度之黃麴毒素B1經有無微粒體代謝時,對鴨周邊血液淋巴細胞存活率之影響,結果示於圖3。單獨淋巴細胞或微粒體處理淋巴細胞增生數目相似,顯示微粒體對其並無刺激效果,但當PHA (20μg/ml)加入時,淋巴細胞受其刺激而大量增殖。黃麴毒素B1自高量(1×104 ng/ml)至低量時(1×101 ng/ml)OD值顯著(P<0.05)較單獨PHA處理者為低,此兩濃度顯然已造成淋巴細胞存活率之降低,但黃麴毒素B1 (1×10-2 ng/ml)時對淋巴細胞增殖無抑制作用,可能為低量黃麴毒素且經淋巴細胞代謝後產生之毒性代謝物低至不影響其增殖作用。當高濃度黃麴毒素B1 (1×104 ng/ml)經微粒體代謝後,顯著(P<0.05)抑制淋巴細胞增殖作用,由本試驗結果顯示黃麴毒素B1 (104 ng/ml)經代謝後可產生淋巴細胞致害作用,但濃度降低(1×101、1×10-2 ng/ml)時致害作用不顯著。

       wpe97.jpg (48973 bytes)

  肝臟微粒體為細胞經均質化裂解而來,內質網斷裂後,再組合而成大小約100nm之小泡(Forrester et al., 1990),其富含多種酵素,其中以cytochrome p450在黃麴毒素B1代謝轉化為8,9-epoxide毒性增強(Ramsdell and Eation, 1990)。Neldon-Ortiz and Qureshi (1991)以經微粒體代謝之黃麴毒素B1作用雞腹腔巨噬細胞1小時,則發現微粒體使黃麴毒素B1毒性增強,甚至0.5μg/ml之濃度亦造成細胞形態改變,黏附力及吞噬能力亦明顯降低。相較本試驗使用之濃度104 ng/ml等於10 μg/ml,意即以淋巴細胞存活率來評估黃麴毒素B1經代謝之毒性上比以巨噬細胞之形態學、黏附力及吞噬力為評估指標較不敏感。Pang and Pan (1994)以豬淋巴細胞為對象,結果顯示黃麴毒素B1濃度於5×104 ng/ml處理12小時,豬淋巴細胞死亡率即達50%。

  經微粒體代謝之AFB1 (104 ng/ml)同時加入β-胡蘿蔔素(BC)對鴨周邊血液淋巴細胞存活率之影響,結果示於圖4。單獨微粒體處理與培養基對照組間無顯著差異,而當單獨高濃度BC (0.1 μM)添加時,OD值顯著(P<0.05)較培養基對照組為高,顯示BC為一潛在性裂殖原。單獨AFB1 (104 ng/ml)顯著(P<0.05)較AFB1經微粒體代謝者之細胞存活率為高,且中低濃度BC (0.05、0.025 μM)添加於AFB1經微粒體代謝之培養系統中時對細胞存活率並無改善之效果。然而高濃度BC (0.1 μM)添加於AFB1經微粒體代謝之培養系統中時極顯著的(P<0.05)較中低濃度BC者之細胞存活率為高,且改善至單獨PHA對照組相同之細胞存活率。

  Chew et al., (1993b)實驗指出仔牛一次口服膠囊化BC 200 mg時,可提高淋巴細胞對BC之攝取量,但白血球及紅血球對BC攝取量無影響。進一步分析淋巴細胞之胞器。發現粒線體、胞核及微粒體中BC含量著增加,但仔牛血漿中BC及視覺醇含量無影響。本試驗之BC (0.1 μM)添加可有效的提高淋巴細胞存活率,可能與提高淋巴細胞對BC之攝取有關;雖然對於BC在調節免疫力上所扮演的角色尚不瞭解,但BC可能以自由基清除者抵抗自由基導致之細胞受損。事實上,生體內和生體外研究指出,BC可促進淋巴細胞增殖作用(Hoskinson et al., 1989)及增加助手T細胞數目(Prabhala et al., 1989)。而本試驗之淋巴細胞含高濃度BC可能保護或對抗AFB1經代謝後產生之自由基,此外BC也可能直接調節淋巴細胞之增殖。Chew et al., (1993b)實驗發現視覺醇並未隨之升高,顯示BC有別於一般人認為僅是維生素A原之刻板印象,而是有其獨特的作用。

  雖然AFB1造成淋巴細胞死亡的確實機制未知,但AFB1經微粒體酵素代謝之二次產物造成細胞膜不穩定可能扮演一重要地位(Pokrovsky et al., 1972)。曾有報告指出,氧自由基造成細胞膜受損,而致脂質過氧化、蛋白質及DNA之破壞(Perera et al., 1987)。近來研究顯示AFB1及其代謝物可經酵素非酵素作用形成自由基(Kodama et al., 1990),是否自由基為造成淋巴細胞存活率降低之主因,須加以進一步證實。Lessard and Dupuis (1994)生體試驗也陳述日糧中BC含量增加,雞淋巴細胞增殖作用及自然殺手細胞(natural killer)活性亦提高。但亦有試驗指出BC或BC與維生素E協同時無法提高以E. coli免疫雞隻的凝集抗體力價,維生素E或A卻可以提高,此可能與BC對疾病保護機制異於維生素E或A有關。

        生體外試驗,當添加BC濃度為10-8M對鼠脾臟淋巴細胞增殖無影響,添加BC10-5M抑制牛週邊血液淋巴細胞之增殖作用(Tjoelker et al., 1988),此一結果與本試驗結果相去甚遠,其原因可能為不宣品種動物對BC攝取程度差異及free radical存在與否時造成之影響。此一推論基礎可由Lawlor and O'brien (1995)應用paraquat 0.25 mM處理雞胚纖維母細胞(chicken embryo fibroblast, CEF)18小時,造成其氧化性緊迫,當0.1μM BC添加時,superoxide dimutase及glutathione peroxidase (GSH-px)活性回復至與對照組相同水準,且降低catalase活性。當10μM BC添加時superoxide dimutase及catalase顯著升高且GSH-px活性降低至與對照組相當。顯示BC有效保護CEF對抗paraquat誘發之氧化性緊迫。

        綜觀本試驗結果顯示,高濃度AFB1 (104 ng/ml)經代謝後對鴨淋巴細胞毒性增強,高濃度胡蘿蔔素(0.1 μM)可保護鴨淋巴細胞免除此一毒害作用,是否為抑制氧自由基之產生,須進一步探討。


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參考文獻

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The Cytotoxic Effect of Aflatoxin B1 on Duck Lymphocytes and the Protective Effect of β-Carotene

Yeong-Hsiang Cheng(1) and Victor-Fei Pang(2)

Received Oct. 26, 1995; Accepted Jan. 29, 1996

ABSTRACT

        The purpose of this experiment was to determine duck peripheral blood lymphocyte proliferation by a MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This assay was also used to evaluate the cytotoxic effect of aflatoxin B1 (AFB1) on duck lymphocyte and the protective effect of β-carotene (BC).

        Trial 1: Three mitogens were examined and the results showed that phytohaemagglutinin (PHA) had the best proliferation response, pokeweed mitogen (PWM) intermediate, and Concanavalin A (Con A) the least. Using best-fit regression, the best mitogen concentrations for proliferation response were PHA, 23.68 μg/ml;Con A, 34.48μg/ml and PWM, 23.57 μg/ml, respectively.

        Trial 2: The proliferation response of lymphocyte number by a MTT assay showed a quadratic fashion. When cell numbers were raised from 1×104 to 1×107 cell/ml, the OD values were also increased. However, the OD values tended to decrease when cell numbers were increased to 1×108 cell/ml.

        Trial 3:The results showed that AFB1 treatment from high dose (1×104 ng/ml) to low dose (1×101 ng/ml) had OD values significantly lower (P<0.05) than PHA treatment alone. But, there was no inhibitory effect when AFB1 Aat an extremly low dose (1×10-2 ng/ml) was introduced. High dose of AFB1 which was metabolized by microsome inhibited lymphocyte proliferation to a greater extent than AFB1 which was without microsome metabolization. There was no significant inhibition at low dose (1×101,1×10-2 ng/ml) treatments.

        Trial 4: The results showed that when high BC level (0.1μM) was added, OD values of lymphocyte proliferation were higher (P<0.05) than medium control. Treatment of AFB1 alone had higher (P<0.05) proliferation than AFB1 metabolized by microsome. There was no improvement on lymphocyte proliferation when medium and low levels of BC (0.05, 0.025 μM) were added to culture system of AFB1 metabolized by microsome. However, when high level of BC was added to the same culture system, the proliferation was higher (P<0.05) than medium and low levels of BC and the improvement was the same as PHA control group.

        This experiment revealed that high dose of AFB1 metabolized by microsome had an intensified effect on inhibition of duck lymphocyte proliferation. However, high levels of BC could alleviate this detrimental effect on duck lymphocyte. (Key Words: Duck, Aflatoxin B1, Lymphocyte, β-Carotene)

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(1)Department of Animal Science, National 1-Lan Institute of Agriculture and Technology, I-Lan, Taiwan, R.O.C.

(2)Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, R.O.C.




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