酵母葡聚糖衍生物對(duì)凡納濱對(duì)蝦血細(xì)胞免疫反應(yīng)的影響

白 楠 谷 珉 張文兵 麥康森

(中國(guó)海洋大學(xué)水產(chǎn)動(dòng)物營(yíng)養(yǎng)與飼料農(nóng)業(yè)部重點(diǎn)實(shí)驗(yàn)室, 海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室, 青島 266003)

酵母葡聚糖衍生物對(duì)凡納濱對(duì)蝦血細(xì)胞免疫反應(yīng)的影響

白 楠 谷 珉 張文兵 麥康森

(中國(guó)海洋大學(xué)水產(chǎn)動(dòng)物營(yíng)養(yǎng)與飼料農(nóng)業(yè)部重點(diǎn)實(shí)驗(yàn)室, 海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室, 青島 266003)

酵母葡聚糖是水產(chǎn)養(yǎng)殖中使用最廣泛的免疫增強(qiáng)劑之一, 但其不溶解性不利于其免疫增強(qiáng)作用的發(fā)揮。為了增加酵母葡聚糖的溶解性, 研究共制備了8種酵母葡聚糖衍生物, 即4種不同取代度的羧甲基葡聚糖和磺乙基葡聚糖。將葡聚糖和其8種衍生物分別按照5、25和100μg/mL的濃度分別添加到原代培養(yǎng)凡納濱對(duì)蝦血細(xì)胞的培養(yǎng)液中。以空白血細(xì)胞作為對(duì)照。孵育6h、12h和24h后分別取樣, 測(cè)定血細(xì)胞的酚氧化酶和呼吸暴發(fā)活力。結(jié)果表明, 在 6h時(shí), 所有葡聚糖衍生物處理組的酚氧化酶活力均顯著高于相同濃度下的未衍生葡聚糖處理組(P＜0.05)。而25μg/mL酵母葡聚糖衍生物處理組的呼吸暴發(fā)活力顯著高于同濃度未經(jīng)衍生葡聚糖處理組(P＜0.05)。在12h時(shí), 所有酵母葡聚糖衍生物處理組的酚氧化酶和呼吸暴發(fā)活力均顯著高于未衍生葡聚糖處理組(P＜0.05)。在6h和12h時(shí), 同濃度各葡聚糖衍生物處理組的血細(xì)胞酚氧化酶和呼吸暴發(fā)活力并無(wú)顯著差異(P＞0.05)。研究結(jié)果表明, 羧甲基葡聚糖和磺乙基葡聚糖均比未衍生葡聚糖具有更強(qiáng)的免疫促進(jìn)作用, 而且這種免疫促進(jìn)作用在兩種衍生物之間沒(méi)有顯著差異; 另一方面, 酵母葡聚糖衍生物的免疫促進(jìn)作用與其使用濃度有關(guān), 而與其取代度沒(méi)有明顯的關(guān)系。

葡聚糖衍生物; 羧甲基葡聚糖; 磺乙基葡聚糖; 凡納濱對(duì)蝦; 細(xì)胞培養(yǎng)

對(duì)蝦養(yǎng)殖在我國(guó)水產(chǎn)養(yǎng)殖中占有舉足輕重的地位。但在過(guò)去的數(shù)十年中, 對(duì)蝦疾病的發(fā)生嚴(yán)重制約了該產(chǎn)業(yè)的持續(xù)健康發(fā)展[1]。酵母葡聚糖作為水產(chǎn)養(yǎng)殖中廣泛使用的免疫增強(qiáng)劑, 能夠激活對(duì)蝦的免疫系統(tǒng), 提高其免疫力[2], 從而減少對(duì)蝦疾病的發(fā)生[3, 4]。然而葡聚糖富含羥基, 羥基之間易形成具有疏水性的氫鍵, 從而造成葡聚糖在水中的不溶解性[5]。因此, 對(duì)蝦飼料中添加的酵母葡聚糖很難從飼料中溶出, 穿過(guò)腸道并作用于血細(xì)胞。另外, 在哺乳動(dòng)物中的研究已經(jīng)證實(shí), 葡聚糖的不溶解性會(huì)降低其免疫活性[6]。因此, 增加葡聚糖的溶解性有利于提高其免疫活性。

通過(guò)化學(xué)改性將多糖中的羥基被某些化學(xué)基團(tuán)取代, 制備多糖衍生物是提高多糖溶解性的有效途徑[7]。近年來(lái)研究表明, 多糖衍生物相比多糖具有更高的免疫活性, 且這種活性與引入化學(xué)基團(tuán)的種類和取代度有關(guān)[8]。目前, 尚沒(méi)有關(guān)于葡聚糖衍生物對(duì)于甲殼動(dòng)物生長(zhǎng)、存活、免疫力和抗病力的報(bào)道。

本研究擬采用羧甲基化和磺乙基化2種化學(xué)改性法分別制備2類酵母葡聚糖衍生物——羧甲基葡聚糖和磺乙基葡聚糖, 通過(guò)調(diào)節(jié)反應(yīng)條件, 分別制備2類衍生物4種不同的取代度, 考察其對(duì)凡納濱對(duì)蝦血細(xì)胞免疫反應(yīng)的影響, 篩選出更有效的酵母葡聚糖類免疫增強(qiáng)劑。

1 材料與方法

1.1 酵母葡聚糖的提取和不同取代度酵母葡聚糖衍生物的制備

葡聚糖提取于干酵母(湖北安琪酵母股份有限公司), 提取方法參考Suphantharika, et al.[9]的方法。以此酵母葡聚糖為原料, 制備了 4種不同取代度的羧甲基葡聚糖(分別命名為CMGA、CMGB、CMGC和 CMGD)和 4種不同取代度的磺乙基酵母葡聚糖(分別命名為SEGA、SEGB、SEGC和SEGD)。制備方法參考Machová, et al.[10]和?andula, et al.[5]的方法。

1.2 酵母葡聚糖衍生物取代度的測(cè)定

羧甲基葡聚糖取代度的測(cè)定參考 Stojanovi?, et al.[11]的方法。具體步驟如下: 首先將1 g羧甲基葡聚糖溶解于15 mL丙酮中, 充分混勻。在上述體系中加入3 mL 6 mol/L鹽酸溶液, 反應(yīng)30min, 在此期間不斷攪拌使之充分反應(yīng)。將反應(yīng)液過(guò)濾, 以 80%的甲醇洗滌濾渣, 使濾渣呈中性。將濾渣在 50℃下烘烤2h, 使其充分干燥。取0.5 g濾渣, 溶于20 mL 0.2 mol/L氫氧化鈉溶液中, 并用蒸餾水定容到100 mL。取25 mL定容液, 加入1滴酚酞試劑, 以0.05 mol/L鹽酸滴定至無(wú)色, 記下所用體積 V1。另取 20 mL 0.2 mol/L氫氧化鈉溶液, 并用蒸餾水定容到100 mL。取25 mL定容液, 加入1滴酚酞試劑, 以0.05 mol/L鹽酸溶液滴定至無(wú)色, 記下所用體積 V2。取代度的計(jì)算公式如下:

磺乙基葡聚糖的取代度的測(cè)定參考Zhang, et al.[12]的方法。用元素分析儀(CHNS/O Analyzer, Vario EI III, Perkin Elmer)測(cè)定樣品中的碳元素和硫元素的百分含量。取代度的計(jì)算公式如下:

1.3 葡聚糖及其衍生物溶解率的測(cè)定

溶解率的測(cè)定方法參考Byun, et al.[13]的方法。稱取大約2 g(精確到0.0001 g, 記為M)酵母葡聚糖或其衍生物, 加入到 10 mL去離子水中, 振蕩20min。在振蕩結(jié)束后, 將上述混合液于 3500×g速度下離心20min, 取上清液, 于105℃下烘干。記下干燥出的粉末重量M1(精確到0.0001 g)。溶解率的計(jì)算公式如下:

1.4 凡納濱對(duì)蝦血細(xì)胞的原代培養(yǎng)

凡納濱對(duì)蝦血細(xì)胞原代培養(yǎng)的方法參考 Zhou, et al.[14]的方法。實(shí)驗(yàn)用蝦購(gòu)買于青島寶榮水產(chǎn)科技發(fā)展有限公司, 平均體重8 g左右。將購(gòu)買的蝦放于室內(nèi)海水循環(huán)養(yǎng)殖系統(tǒng)中進(jìn)行暫養(yǎng)。在暫養(yǎng)過(guò)程中,每天2次(08: 00和18: 00)投喂商業(yè)對(duì)蝦飼料至表觀飽食。每天清理殘餌和糞便。水溫保持在20 , pH ℃ 7.5—8.0, 溶解氧為6—7 mg/L。

在兩周暫養(yǎng)結(jié)束后, 取健康對(duì)蝦 20只, 先在4℃無(wú)菌海水中浸泡 5—10min, 再用 75%的酒精噴灑體表, 用無(wú)菌紗布拭干體表后, 再用1 mL注射器吸入抗凝劑(10 mmol/L EDTA Na2, 450 mmol/L NaCl, 10 mmol/L KCl, 10 mmol/L HEPES, pH調(diào)至7.3, 滲透壓調(diào)至850 mOsm/kg, 以0.22 μm濾膜濾過(guò)除菌,分裝, 于4℃保存), 等比自腹血竇抽取血淋巴。將抽取的血淋巴離心10min (4 ℃ , 400×g), 棄上清, 加入無(wú)血清培養(yǎng)基(2×Leibovitz’s L-15, 并補(bǔ)充 100 U/mL青霉素和100 μg/mL鏈霉素。調(diào)節(jié)pH至7.2, 經(jīng)0.22 μm濾膜濾過(guò)除菌, 分裝, 于 4℃保存, 用前添加 20%胎牛血清), 打散細(xì)胞, 離心 10min(400×g, 4 ℃) , 棄上清。用添加有 20%胎牛血清的培養(yǎng)基重懸血細(xì)胞, 充分輕柔吹散細(xì)胞, 吸取少量細(xì)胞懸液用血球計(jì)數(shù)板計(jì)數(shù), 調(diào)整細(xì)胞密度至1×106個(gè)/mL。然后, 加200 μL細(xì)胞懸液于96孔細(xì)胞培養(yǎng)板, 28℃恒溫培養(yǎng)24h, 使血細(xì)胞充分貼壁。

1.5 實(shí)驗(yàn)設(shè)計(jì)和指標(biāo)測(cè)定

在血細(xì)胞培養(yǎng) 24h后, 將培養(yǎng)板離心(800×g, 10min), 棄上清。然后加入含有特定免疫增強(qiáng)劑的培養(yǎng)液。免疫增強(qiáng)劑(酵母葡聚糖或其衍生物)的濃度如下: 0(對(duì)照組)、5、25和100 μg/mL。分別培養(yǎng)6h、12h和24h后, 取樣, 測(cè)定血細(xì)胞酚氧化酶和呼吸暴發(fā)活力。加入免疫增強(qiáng)劑 24h后, 測(cè)定血細(xì)胞活力。免疫增強(qiáng)劑濃度和取樣時(shí)間的選擇點(diǎn)參考Gu, et al.[15]的方法。

酚氧化酶活力的測(cè)定 酚氧化酶活力的測(cè)定方法參考Zhou, et al.的方法[14]。取樣時(shí), 首先將離心培養(yǎng)板離心10min (4000×g, 28 ℃)后棄上清, 加入等體積的預(yù)冷PBS緩沖液, 超聲破碎細(xì)胞(40 amplitude, 2s×4 次, 0 ℃)。然后再次將培養(yǎng)板離心10min (800×g, 0 ℃ ), 取上清液用于酚氧化酶活力檢測(cè)。

取50 μL上述血細(xì)胞破碎液與50 μL胰蛋白酶溶液(0.1 mg/mL 溶于CAC 緩沖液: 10 mmol/L二甲胂酸鈉, 10 mmol/L氯化鈣, 將pH調(diào)節(jié)至7.0)加入到96孔細(xì)胞培養(yǎng)板中, 室溫下溫育 10min。然后加入50 μL L-DOPA 溶液(3 mg/mL溶于CAC 緩沖液),室溫下溫育10min后, 立刻放入酶標(biāo)儀(Model Multiskan spectrum, Thermo MA, Waltham, USA)中, 在492 nm波長(zhǎng)下測(cè)定酶活力。在此反應(yīng)條件下, 每分鐘每毫升血細(xì)胞上清液OD值增加0.001 為一個(gè)酶活力單位。

呼吸暴發(fā)活力的測(cè)定 呼吸暴發(fā)活力的測(cè)定參考Song和Herish[16]的方法并略有改動(dòng)。首先于4000×g條件下離心細(xì)胞培養(yǎng)板10min, 棄上清, 加入100 μL 0.3% NBT, 37℃溫育30min。溫育結(jié)束后,以800×g, 4℃離心10min, 去除上清, 加入200 μL純甲醇終止反應(yīng)。10min后, 以800×g, 4℃離心10min,去除上清, 并以70%甲醇洗滌三次, 離心去除上清后, 室溫晾干。干燥后, 加入 120 μL 2 mol/L KOH和 140 μL DMSO, 充分溶解, 測(cè)定溶液在波長(zhǎng)630 nm下的吸光值。呼吸暴發(fā)活力表示為OD 630 nm。

細(xì)胞活力的測(cè)定 細(xì)胞活力以比色法測(cè)定[17]。待免疫增強(qiáng)劑處理血細(xì)胞24h后, 將50 μL MTT (購(gòu)買于Sigma-Aldrich, 產(chǎn)品編號(hào)M5655)溶液(5 mg/mL溶于PBS溶液)加入每個(gè)細(xì)胞培養(yǎng)孔中, 在黑暗中反應(yīng)5h。在反應(yīng)結(jié)束后, 離心細(xì)胞培養(yǎng)板, 棄上清, 加入200 μL DMSO溶解MTT顆粒。然后在酶標(biāo)儀(Model Multiskan spectrum, Thermo MA, Waltham, USA)中讀取570 nm波長(zhǎng)下的吸光值。細(xì)胞活力的計(jì)算公式如下:

細(xì)胞活力%=(各實(shí)驗(yàn)處理組吸光值/對(duì)照組吸光值)×100

1.6 數(shù)據(jù)處理

實(shí)驗(yàn)結(jié)果以平均數(shù)±標(biāo)準(zhǔn)差來(lái)表示。采用 SPSS 17.0軟件包進(jìn)行數(shù)據(jù)分析。采用雙因素方差分析酵母葡聚糖衍生物的取代度和濃度對(duì)凡納濱對(duì)蝦血細(xì)胞免疫反應(yīng)的影響, 當(dāng)差異顯著時(shí)(P＜0.05), 采用Tukey檢驗(yàn)進(jìn)行多重比較。在每個(gè)取樣時(shí)間點(diǎn), 分別從羧甲基酵母葡聚糖處理組和磺乙基酵母葡聚糖處理組中選取免疫指標(biāo)最高的處理組進(jìn)行獨(dú)立樣本 T檢驗(yàn), 從而比較羧甲基葡聚糖和磺乙基葡聚糖免疫活力的高低。當(dāng)P＜0.05時(shí)視為差異顯著。

2 結(jié)果

2.1 酵母葡聚糖衍生物的取代度和溶解率

經(jīng)過(guò)測(cè)定和計(jì)算(表1), 酵母葡聚糖溶解率僅為4.07%。隨著取代度的增加, 溶解率顯著增加。羧甲基葡聚糖的溶解率從 33.76%增加到 86.56%; 而磺乙基葡聚糖的溶解率從27.56%增加到81.69%。

2.2 酵母葡聚糖及其衍生物對(duì)凡納濱對(duì)蝦血細(xì)胞活力的影響

從圖 1可以看出, 經(jīng)酵母葡聚糖和酵母葡聚糖衍生物作用24h后, 對(duì)蝦血細(xì)胞活力仍然高于90%,且各處理組之間沒(méi)有顯著差異(P＞0.05)。這表明,酵母葡聚糖和葡聚糖衍生物對(duì)細(xì)胞活力沒(méi)有顯著影響。

圖 1 與葡聚糖(GLU)、羧甲基葡聚糖(CMG, 圖 A)或磺乙基葡聚糖(SEG, 圖B)孵育24h后凡納濱對(duì)蝦血細(xì)胞的細(xì)胞活力Fig. 1 Viability of haemocytes of L. vannamei, which were incubated 24h with β-glucan (GLU), carboxymethylglucan (CMG, Fig. A) or sulfoethylglcuan (SEG, Fig. B) at three concentrations

2.3 酵母葡聚糖及其衍生物對(duì)凡納濱對(duì)蝦血細(xì)胞酚氧化酶活力的影響

從圖2中可以看出, 在6h時(shí), 免疫增強(qiáng)劑處理組的酚氧化酶活力顯著高于對(duì)照組(P＜0.05)。在相同的濃度下, 葡聚糖衍生物處理組的酚氧化酶活力顯著高于未衍生葡聚糖處理組(P＜0.05, 圖2)。葡聚糖衍生物的濃度顯著影響了血細(xì)胞的酚氧化酶活力(P＜0.05, 表 2), 酚氧化酶活力隨著葡聚糖衍生物的濃度的增加而顯著增加(P＜0.05, 圖2)。在此時(shí)間點(diǎn),羧甲基葡聚糖和磺乙基葡聚糖的取代度對(duì)血細(xì)胞酚氧化酶活力無(wú)顯著影響(P＞0.05, 表 2), 二者相比也無(wú)顯著差異(P＞0.05, 表3)。

表1 葡聚糖及其衍生物的取代度和溶解率Tab. 1 The degree of substitution and solubility of glucan and its derivatives

圖 2 與葡聚糖(GLU)、羧甲基葡聚糖(CMG, 圖 A)或磺乙基葡聚糖(SEG, 圖B)孵育6h后凡納濱對(duì)蝦血細(xì)胞的酚氧化酶活力Fig. 2 Phenoloxidase (PO) activity of haemocytes of L. vannamei, which were incubated 6h with β-glucan (GLU), carboxymethylglucan (CMG, Fig. A) or sulfoethylglcuan (SEG, Fig. B) at three concentrations

在12h時(shí), 5、25 μg/mL酵母葡聚糖處理組的血細(xì)胞的酚氧化酶活力仍然顯著高于對(duì)照組(P＞0.05,圖3)。酵母葡聚糖衍生物處理組的血細(xì)胞的酚氧化酶活力顯著高于同濃度未衍生葡聚糖處理組(P＜0.05, 圖3)。羧甲基酵母葡聚糖的取代度和濃度都顯著影響了對(duì)蝦血細(xì)胞的酚氧化酶活力(P＜0.05,表 2)。但是, 磺乙基酵母葡聚糖的取代度和濃度并不能顯著影響對(duì)蝦血細(xì)胞的酚氧化酶活力(P＞0.05,表 2)。在此時(shí)間點(diǎn), 羧甲基酵母葡聚糖和磺乙基酵母葡聚糖對(duì)于血細(xì)胞酚氧化酶活力的影響無(wú)顯著差異(P＞0.05, 表3)。

在 24h時(shí), 所有處理組之間的酚氧化酶活力無(wú)顯著差異(P＞0.05, 圖4)。

圖 3 與葡聚糖(GLU)、羧甲基葡聚糖(CMG, 圖 A)或磺乙基葡聚糖(SEG, 圖B)孵育12h后凡納濱對(duì)蝦血細(xì)胞的酚氧化酶活力Fig. 3 Phenoloxidase (PO) activity of haemocytes of L. vannamei, which were incubated 12h with β-glucan (GLU), carboxymethylglucan (CMG, Fig. A) or sulfoethylglcuan (SEG, Fig. B) at three concentrations

表2 葡聚糖衍生物的取代度、濃度及其交互作用對(duì)于凡納濱對(duì)蝦血細(xì)胞酚氧化酶活力和呼吸暴發(fā)活力的影響Tab. 2 Effects of degree of substitution, concentration and their interaction on the phenoloxidase activity and respiratory burst of haemocytes of white shrimp Litopenaeus vannamei

表3 羧甲基葡聚糖和磺乙基葡聚糖的比較Tab. 3 The comparison of carboxymethylglucan and suloethylglucan

2.4 酵母葡聚糖及其衍生物對(duì)凡納濱對(duì)蝦血細(xì)胞呼吸暴發(fā)活力的影響

從圖5中可以看出, 在6h時(shí), 所有免疫增強(qiáng)劑處理組的血細(xì)胞呼吸暴發(fā)活力顯著高于對(duì)照組(P＜0.05)。其中25 μg/mL羧甲基酵母葡聚糖處理組和磺乙基葡聚糖處理組的血細(xì)胞呼吸暴發(fā)活力顯著高于同濃度未衍生葡聚糖處理組(P＜0.05)。在此時(shí)間點(diǎn), 酵母葡聚糖衍生物的濃度顯著影響了呼吸暴發(fā)活力(P＜0.05, 表 2), 但取代度沒(méi)有顯著影響(P＞0.05,表2)。羧甲基酵母葡聚糖和磺乙基酵母葡聚糖對(duì)于血細(xì)胞呼吸暴發(fā)活力的影響無(wú)顯著差異(P＞0.05, 表3)。

圖 4 與葡聚糖(GLU)、羧甲基葡聚糖(CMG, 圖 A)或磺乙基葡聚糖(SEG, 圖B)孵育24h后凡納濱對(duì)蝦血細(xì)胞的酚氧化酶活力Fig. 4 Phenoloxidase (PO) activity of haemocytes of L. vannamei, which were incubated 24h with β-glucan (GLU), carboxymethylglucan (CMG, Fig. A) or sulfoethylglcuan (SEG, Fig. B) at three concentrations

在12h 時(shí), 所有酵母葡聚糖處理組的血細(xì)胞呼吸暴發(fā)活力與對(duì)照組相比無(wú)顯著差異(P＞0.05, 圖6)。但是, 酵母葡聚糖衍生物處理組的血細(xì)胞呼吸暴發(fā)活力仍然顯著高于對(duì)照組(P＜0.05, 圖6)。酵母葡聚糖衍生物處理組的血細(xì)胞呼吸暴發(fā)活力顯著高于未衍生葡聚糖處理組(P＜0.05, 圖 6)。在此時(shí)間點(diǎn),酵母葡聚糖衍生物的取代度和濃度均不能顯著影響血細(xì)胞呼吸暴發(fā)活力(P＞0.05, 表2)。羧甲基酵母葡聚糖和磺乙基酵母葡聚糖對(duì)于血細(xì)胞呼吸暴發(fā)活力的影響無(wú)顯著差異(P＞0.05, 表3)。

圖5 與葡聚糖(GLU)、羧甲基葡聚糖(CMG, 圖A)或磺乙基葡聚糖(SEG, 圖B)孵育6h后凡納濱對(duì)蝦血細(xì)胞的呼吸暴發(fā)活力Fig. 5 Respiratory burst (RB) of haemocytes of L. vannamei, which were incubated 6h with β-glucan (GLU), carboxymethylglucan (CMG, Fig. A) or sulfoethylglcuan (SEG, Fig. B) at three concentrations

圖6 與葡聚糖(GLU)、羧甲基葡聚糖(CMG, 圖A)或磺乙基葡聚糖(SEG, 圖B)孵育12h后凡納濱對(duì)蝦血細(xì)胞的呼吸暴發(fā)活力Fig. 6 Respiratory burst (RB) of haemocytes of L. vannamei, which were incubated 12h with β-glucan (GLU), carboxymethylglucan (CMG, Fig. A) or sulfoethylglcuan (SEG, Fig. B) at three concentrations

在 24h時(shí), 所有處理組之間的呼吸暴發(fā)活力無(wú)顯著差異(P＞0.05, 圖7)。

圖 7 與葡聚糖(GLU)、羧甲基葡聚糖(CMG, 圖 A)或磺乙基葡聚糖(SEG, 圖B)孵育24h后凡納濱對(duì)蝦血細(xì)胞的呼吸暴發(fā)活力Fig. 7 Respiratory burst (RB) of haemocytes of L. vannamei, which were incubated 24h with β-glucan (GLU), carboxymethylglucan (CMG, Fig. A) or sulfoethylglcuan (SEG, Fig. B) at three concentrations

3 討論

3.1 化學(xué)改性葡聚糖對(duì)凡納濱對(duì)蝦血細(xì)胞免疫反應(yīng)的影響

本研究結(jié)果表明, 羧甲基葡聚糖和磺乙基葡聚糖在適宜的孵育時(shí)間內(nèi)均能顯著提高葡聚糖的免疫活性。研究表明, 化學(xué)改性法可以提高葡聚糖的生物活性, 如羧甲基化可以顯著提高靈芝葡聚糖[18]和茯苓葡聚糖[19]的抗氧化力; 羧甲基化可以提高桑黃菌多糖[20]、虎奶菇葡聚糖[21,22]和靈芝多糖[23]的抗腫瘤能力; 硫酸化可以提高虎奶菇葡聚糖抗腫瘤能力[24]。Wang, et al.[19]認(rèn)為, 衍生物生物活性的提高主要是因?yàn)榛瘜W(xué)改性方法提高了多糖的溶解性, 并改變了多糖的結(jié)構(gòu)。Zhang, et al.[21—23]也發(fā)現(xiàn)具有較高生物活性的羧甲基葡聚糖比未衍生的葡聚糖具有更伸展的構(gòu)象。

在本研究中, 酵母葡聚糖表現(xiàn)出了免疫增強(qiáng)作用缺失的現(xiàn)象, 即酵母葡聚糖組的血細(xì)胞酚氧化酶和呼吸暴發(fā)活力與對(duì)照組相比無(wú)顯著差異。這與在仿刺參體腔細(xì)胞實(shí)驗(yàn)中的結(jié)果一致[14]。但是在 12h時(shí), 酵母葡聚糖衍生物處理組并未表現(xiàn)出免疫增強(qiáng)作用缺失的現(xiàn)象, 在各添加濃度中其酚氧化酶和呼吸暴發(fā)活力仍然顯著高于對(duì)照組。這說(shuō)明化學(xué)改性不僅能夠提高酵母葡聚糖的免疫增強(qiáng)作用, 而且能夠延長(zhǎng)其免疫作用的時(shí)間。這可能與溶解性的提高和結(jié)構(gòu)的改變有關(guān), 但具體的原因仍然需要進(jìn)一步的分析。

3.2 葡聚糖衍生物的取代度對(duì)凡納濱對(duì)蝦血細(xì)胞免疫反應(yīng)的影響

化學(xué)基團(tuán)的引入會(huì)改變多糖分子內(nèi)和分子間的氫鍵, 影響其電子排斥力, 從而使多糖分子的結(jié)構(gòu)發(fā)生變化[23,25]。化學(xué)取代會(huì)造成多糖結(jié)構(gòu)的改變,而改變的程度與取代度有關(guān)[22,23]。多糖的結(jié)構(gòu)決定著多糖功能和生物活性[26]。取代度是決定多糖衍生物生物活性的最重要的因素之一[8]。但是, 關(guān)于葡聚糖衍生物的最適取代度尚無(wú)統(tǒng)一結(jié)論。?andula, et al.[6]制備了不同取代度(0.56、0.75、0.89、1.08和1.15)的羧甲基葡聚糖, 并發(fā)現(xiàn)取代度為0.75的羧甲基葡聚糖在小鼠胸腺細(xì)胞的有絲分裂實(shí)驗(yàn)中顯示出最高的活力, 而取代度大于 1的羧甲基葡聚糖則無(wú)生物活性。Bao, et al.[27]制備了7種羧甲基葡聚糖, 其取代度從0.17到1.44。與?andula, et al.[6]的報(bào)道不同, Bao, et al.[27]發(fā)現(xiàn)最低取代度(小于0.28)的羧甲基葡聚糖表現(xiàn)出最高的生物活力。與Bao, et al.[27]類似, Zhang, et al.[23]也發(fā)現(xiàn)低取代度(0.38)的羧甲基葡聚糖比較高取代度(0.43和 0.54)的葡聚糖的抗腫瘤活力要高。對(duì)于磺乙基葡聚糖, 目前尚沒(méi)有關(guān)于最適取代度的報(bào)道。但是, Jung, et al.[28]發(fā)現(xiàn), 取代度對(duì)于硫酸化杏鮑菇葡聚糖的抗癌細(xì)胞生長(zhǎng)的性能無(wú)顯著影響。

本研究證明, 不同取代度的羧甲基葡聚糖和磺乙基葡聚糖處理組的血細(xì)胞酚氧化酶和呼吸暴發(fā)活力并無(wú)顯著差別。這與Jung, et al.[28]所報(bào)道的結(jié)果類似, 表明取代度的不同并不能夠顯著影響原代培養(yǎng)凡納濱對(duì)蝦血細(xì)胞的免疫反應(yīng)。

3.3 酵母葡聚糖的羧甲基和磺乙基衍生物對(duì)凡納濱對(duì)蝦血細(xì)胞免疫反應(yīng)的影響的比較

羧甲基葡聚糖和磺乙基葡聚糖生物活性的不同與實(shí)驗(yàn)動(dòng)物或細(xì)胞系、實(shí)驗(yàn)?zāi)康幕虮O(jiān)測(cè)指標(biāo)有關(guān)。注射羧甲基葡聚糖和磺乙基葡聚糖都可以提高斷奶仔豬嗜中性粒細(xì)胞還原酶的活力, 而羧甲基葡聚糖對(duì)還原酶活力的提高作用高于磺乙基葡聚糖[29]。但是, 當(dāng)以小鼠胸腺細(xì)胞的有絲分裂實(shí)驗(yàn)進(jìn)行二者比較時(shí), 磺乙基葡聚糖比羧甲基葡聚糖表現(xiàn)出更高的生物活力[6]。Slameňová, et al.[30]也證明磺乙基葡聚糖保護(hù) DNA不被過(guò)氧化氫氧化損壞的能力要高于羧甲基葡聚糖。但是, Mucksová, et al.[31]的研究表明,羧甲基葡聚糖和磺乙基葡聚糖對(duì)于小鼠腹膜黏附細(xì)胞的過(guò)氧化物酶活力和一氧化氮合成酶活力的提高能力并無(wú)顯著差別。

在本研究中, 羧甲基酵母葡聚糖和磺乙基酵母葡聚糖處理組的血細(xì)胞酚氧化酶和呼吸暴發(fā)活力并無(wú)顯著差異。這與Mucksová, et al.[31]的研究相似,表明酵母葡聚糖衍生物對(duì)于原代培養(yǎng)凡納濱對(duì)蝦血細(xì)胞的免疫促進(jìn)作用不受本實(shí)驗(yàn)中所用衍生方法的影響。

總的來(lái)說(shuō), 研究證明羧甲基酵母葡聚糖和磺乙基酵母葡聚糖在適宜的孵育時(shí)間內(nèi)均比酵母葡聚糖對(duì)凡納濱對(duì)蝦血細(xì)胞有更高的免疫促進(jìn)作用。這種免疫促進(jìn)作用不受本研究中所用衍生方法的影響, 每種衍生物的取代度之間也沒(méi)有表現(xiàn)出顯著的差異。

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THE EFFECTS OF YEAST β-GLUCAN DERIVATIVES ON THE IMMUNE RESPONSES OF THE HAEMOCYTES OF WHITE SHRIMP LITOPENAEUS VANNAMEI

BAI Nan, GU Min, ZHANG Wen-Bing and MAI Kang-Sen
(Key Laboratory of Aquaculture Nutrition and Feeds (Ministry of Agriculture), Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, China)

Yeast β-glucan is a widely-used immunostimulant in aquaculture. However, the insolubility of β-glucan reduces its efficiency as an immunostimulant. In our study, carboxymethylglucan (CMG) and sulfoethylglcuan (SEG) were generated to increase the solubility of β-glucan. Both CMG-type and SEG-type of glucans had four derivatives with different degree of substitution (DS), so in total eight kinds of immunostimulants and β-glucan as the control were tested in our study. Each immunostimulant was added to the primary culture of the haemocytes of white shrimp Litopenaeus vannamei at different concentrations of 5, 25 and 100 μg/mL. With each concentration of every immunostimulant the primary culture was incubated for 6, 12 and 24 hours, before the haemocytes were sampled and their phenoloxidase (PO) activity and respiratory burst (RB) were assayed. After 6 hours, the PO activity of the haemoctyes incubated with all the eight β-glucan derivatives was significantly higher than those incubated with β-glucan control at the same concentration (P＜0.05). The RB of the haemocytes incubated with 25 μg/mL of all the β-glucan derivatives was significantly higher than those incubated with 25 μg/mL of β-glucan (P＜0.05). After 12 hours, PO activity and RB of the haemoctyes incubated with all the β-glucan derivatives were significantly higher than those with β-glucan (P＜0.05). However, the eight β-glucan derivatives showed no significant difference among each other in their effects on the PO activity or RB of the haemoctyes (P＞0.05). Our study revealed that the immune-enhancing effects of β-glucan derivatives were higher than β-glucan. However, there was no significant difference in immune-enhancing effects between CMG and SEG. The concentration of β-glucan derivatives could significantly influence their immune-enhancing effects, but DS did not affect the immune-enhancing effects of β-glucan derivatives.

Glucan derivative; Carboxymethylglucan; Sulfoethylglcuan; Litopenaeus vannamei; Cellculture

S963.73

A

1000-3207(2014)04-0642-09

10.7541/2014.91

2013-03-25;

2013-12-10

國(guó)家公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)經(jīng)費(fèi)(201103034)資助

白楠(1984—), 男, 河南鄭州市人; 博士; 主要從事水生動(dòng)物營(yíng)養(yǎng)生理與免疫方面的研究。E-mail: bainan668@gmail.com

張文兵, 教授, E-mail: wzhang@ouc.edu.cn