斷奶應(yīng)激對幼齡反芻動物免疫系統(tǒng)的影響及其機(jī)理

張　千　李發(fā)弟,2　李　飛*

(1.草地農(nóng)業(yè)生態(tài)系統(tǒng)國家重點實驗室，蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院，蘭州730020；2.甘肅省肉羊繁育生物技術(shù)工程實驗室，民勤733300)

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斷奶應(yīng)激對幼齡反芻動物免疫系統(tǒng)的影響及其機(jī)理

張千1李發(fā)弟1,2李飛1*

(1.草地農(nóng)業(yè)生態(tài)系統(tǒng)國家重點實驗室，蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院，蘭州730020；2.甘肅省肉羊繁育生物技術(shù)工程實驗室，民勤733300)

摘要：斷奶初期，幼齡反芻動物的消化和免疫系統(tǒng)等尚未發(fā)育完全，斷奶應(yīng)激會導(dǎo)致幼畜體內(nèi)激素水平及免疫功能改變，引起免疫系統(tǒng)抑制，誘發(fā)炎癥反應(yīng)，阻礙幼畜生長發(fā)育，增加患病風(fēng)險。本文從糖皮質(zhì)激素、免疫細(xì)胞、急性期蛋白和相關(guān)細(xì)胞因子4個方面闡述了斷奶應(yīng)激對幼齡反芻動物免疫系統(tǒng)的影響及其機(jī)理，旨在為相關(guān)研究提供科學(xué)依據(jù)。

關(guān)鍵詞：幼齡反芻動物；斷奶應(yīng)激；免疫系統(tǒng)；糖皮質(zhì)激素；細(xì)胞因子

幼齡反芻動物斷奶時，親子紐帶關(guān)系的破壞、飼料成分及物理形態(tài)的改變、社群的重組和外環(huán)境的變化[1]，會造成幼畜心理[2]、生理和免疫應(yīng)激[3]。研究表明，斷奶刺激會導(dǎo)致幼畜出現(xiàn)鳴叫、不安、走動頻率增加、采食和反芻時間減少[4]等行為學(xué)改變。由于反芻動物生長早期神經(jīng)體液調(diào)節(jié)功能尚不健全，機(jī)體穩(wěn)態(tài)易遭受破壞且恢復(fù)能力較差，發(fā)生應(yīng)激時會導(dǎo)致幼畜出現(xiàn)心率和血壓異常[5]、直腸溫度上升等一系列生理變化，嚴(yán)重時會抑制動物的正常生長發(fā)育[6]。斷奶應(yīng)激不僅影響固有免疫，也會對動物的體液免疫與細(xì)胞調(diào)節(jié)產(chǎn)生顯著影響，激素水平和免疫功能的改變會導(dǎo)致自身免疫調(diào)節(jié)異常[7]。此外，斷奶應(yīng)激與反芻動物呼吸道疾病的發(fā)病率及嚴(yán)重程度有關(guān)[8-10]。目前，有關(guān)反芻動物斷奶應(yīng)激的研究眾多，但多集中在生產(chǎn)性能、胃腸道發(fā)育和微生物類群方面，免疫功能方面的研究較少，尤其是在分子水平以及作用機(jī)理上的研究鮮有報道。本文就斷奶應(yīng)激對幼齡反芻動物免疫系統(tǒng)的影響及其機(jī)理作一綜述，為明確幼齡反芻動物斷奶過程中免疫功能的調(diào)節(jié)及其作用機(jī)制和途徑提供理論依據(jù)。

1斷奶應(yīng)激對幼齡反芻動物免疫功能的影響

1.1降低獲得性免疫屏障功能

幼齡反芻動物斷奶時，免疫系統(tǒng)發(fā)育尚不成熟，適應(yīng)性免疫尚未完全建立，此時免疫功能主要以固有免疫為主。斷奶后，幼畜無法從母乳中獲得免疫球蛋白和谷胱甘肽過氧化物酶、溶菌酶等酶類[11-12]，導(dǎo)致獲得性免疫屏障功能降低，幼畜抗病能力下降，增加患呼吸道疾病的風(fēng)險。研究發(fā)現(xiàn)，大約44%的犢牛死亡與斷奶時發(fā)生的呼吸道疾病有關(guān)[8]。因此，增強斷奶階段固有免疫功能是降低幼畜患病風(fēng)險的關(guān)鍵。

1.2損害免疫系統(tǒng)功能

幼齡反芻動物斷奶會導(dǎo)致急性的免疫應(yīng)激，并引起一系列的生理變化。急性應(yīng)激還有可能發(fā)展成為長期慢性應(yīng)激，而急性和慢性應(yīng)激都會影響免疫系統(tǒng)，損害動物健康(圖1)。犢牛斷奶后血液中淋巴細(xì)胞數(shù)、中性粒細(xì)胞數(shù)以及紅細(xì)胞數(shù)和血小板數(shù)會發(fā)生顯著變化，但單核細(xì)胞數(shù)的變化罕見報道[13-20]。O’Loughlin等[20]研究發(fā)現(xiàn)犢牛斷奶后2 d血液中淋巴細(xì)胞數(shù)由斷奶前的7.2×103cells/μL下降到6.8×103cells/μL，在斷奶后11 d內(nèi)持續(xù)降低至6.5×103cells/μL，并且在試驗期內(nèi)沒有恢復(fù)到正常水平；中性粒細(xì)胞數(shù)在斷奶后24 h由斷奶前的2.3×103cells/μL升高到3.5×103cells/μL，并在斷奶后7 d內(nèi)保持高水平，在斷奶后11 d恢復(fù)到斷奶前水平；犢牛斷奶后11 d紅細(xì)胞數(shù)由斷奶前的10.6×106cells/μL降低到6.6×106cells/μL；血小板數(shù)由斷奶前的815.9×106cells/μL降低到495.6×106cells/μL。同時，斷奶應(yīng)激可以通過改變糖皮質(zhì)激素水平影響多種細(xì)胞因子的分泌，犢牛斷奶后血液中白細(xì)胞介素1(IL-1)、白細(xì)胞介素8(IL-8)、干擾素-γ(IFN-γ)、腫瘤壞死因子-α(TNF-α)、Toll樣受體4(TLR4)、

糖皮質(zhì)激素受體α(GRα)和細(xì)胞凋亡因子(如Fas)的基因表達(dá)量顯著上調(diào)，引起全身的炎癥反應(yīng)[20]。此外，糖皮質(zhì)激素水平的升高會對免疫系統(tǒng)產(chǎn)生抑制作用，增加動物患病的危險。研究發(fā)現(xiàn)犢牛斷奶后24 h血液中糖皮質(zhì)激素受體(GR)的基因表達(dá)量較斷奶前升高了3倍，并且在整個試驗期內(nèi)高于正常水平2倍以上[16]，表明斷奶應(yīng)激不僅可以誘導(dǎo)糖皮質(zhì)激素分泌增加，還可以促進(jìn)其受體相關(guān)基因的表達(dá)。正常水平的糖皮質(zhì)激素對T淋巴細(xì)胞與CD4+和CD8+T淋巴細(xì)胞的調(diào)節(jié)功能十分有限[21]，但高水平的糖皮質(zhì)激素不僅可以限制白細(xì)胞發(fā)揮免疫學(xué)功能，還可以誘導(dǎo)未成熟的T淋巴細(xì)胞與B淋巴細(xì)胞過早凋亡，導(dǎo)致胸腺萎縮[22-23]，損害免疫系統(tǒng)功能，在小鼠和犢牛上都有相似的研究報道[24-26]。

圖1應(yīng)激與免疫功能和健康的關(guān)系

Fig. 1The relationships among stress, immune function and health

2斷奶應(yīng)激對幼齡反芻動物免疫功能影響的機(jī)理

2.1斷奶應(yīng)激對免疫細(xì)胞的影響

2.1.1對細(xì)胞因子分泌的影響

斷奶應(yīng)激會通過內(nèi)部和外部感受器同時刺激動物下丘腦-垂體-腎上腺軸(HPA)引起交感神經(jīng)興奮[27]，下丘腦釋放促腎上腺皮質(zhì)激素釋放激素(CRH)與血管增壓素(VP)并協(xié)同調(diào)控腎上腺皮質(zhì)分泌糖皮質(zhì)激素[28-30]，隨后糖皮質(zhì)激素通過與GR結(jié)合發(fā)揮其生物學(xué)功能，調(diào)節(jié)免疫系統(tǒng)(圖2)。研究發(fā)現(xiàn)，斷奶應(yīng)激通過糖皮質(zhì)激素抑制犢牛細(xì)胞轉(zhuǎn)錄因子核因子κB(NFκB)的活性，從而抑制細(xì)胞內(nèi)相關(guān)靶基因(IL-1、IL-8、TNF-α和IFN-γ等)的表達(dá)[31-32]，阻礙免疫系統(tǒng)對炎癥的應(yīng)答[33-36]。犢牛在接受糖皮質(zhì)激素處理后，應(yīng)激引起的促炎性細(xì)胞因子IL-1、白細(xì)胞介素6(IL-6)、TNF-α和IFN-γ的基因表達(dá)量升高會延遲30～120 min[37]，IFN-γ信號通路會被抑制數(shù)分鐘至1 h[38]。小鼠受到脂多糖刺激后1 h促炎性細(xì)胞因子表達(dá)量明顯升高，但促炎性細(xì)胞因子的分泌會隨著糖皮質(zhì)激素水平的升高而受到抑制[39]。Goujon等[40]在糖皮質(zhì)激素抑制炎癥應(yīng)答信號通路的研究中獲得了相似研究結(jié)果，表明糖皮質(zhì)激素可以通過減少炎性細(xì)胞因子的分泌減輕炎癥反應(yīng)，但同時對免疫系統(tǒng)產(chǎn)生抑制作用。

圖2中樞神經(jīng)系統(tǒng)和下丘腦-垂體-腎上腺軸對應(yīng)激的響應(yīng)

Fig.2The responses of stress on the sympathetic nervous system and the HPA axis

2.1.2對中性粒細(xì)胞數(shù)和淋巴細(xì)胞數(shù)的影響

斷奶應(yīng)激會導(dǎo)致血液中淋巴細(xì)胞數(shù)顯著下降和中性粒細(xì)胞數(shù)顯著上升[3,13-20]，血液中免疫細(xì)胞數(shù)量的變化(表1)是斷奶應(yīng)激導(dǎo)致炎癥發(fā)生的有力證據(jù)[41]。血液中淋巴細(xì)胞數(shù)減少的原因，一方面是糖皮質(zhì)激素抑制胸腺細(xì)胞成熟和分化，誘導(dǎo)淋巴細(xì)胞過早凋亡[26]；另一方面是淋巴細(xì)胞游離出循環(huán)血液，進(jìn)入發(fā)生炎癥組織及受感染部位發(fā)揮其免疫學(xué)功能[42]。然而，當(dāng)細(xì)胞黏附分子L選擇素(CD62L)的基因表達(dá)量受到糖皮質(zhì)激素的抑制時，會導(dǎo)致中性粒細(xì)胞著邊能力變?nèi)?，不能黏附于血管壁遷移進(jìn)入炎癥部位[42]，從而引起血液中性粒細(xì)胞數(shù)的升高。此外，炎癥會刺激骨髓中干細(xì)胞分化，大量成熟的中性粒細(xì)胞進(jìn)入循環(huán)血液也是血液中性粒細(xì)胞數(shù)上升的原因之一[43]。犢牛斷奶后中性粒細(xì)胞數(shù)會在7～14 d恢復(fù)到斷奶前水平[13,20]，這可能是由于免疫系統(tǒng)具有一定的自我調(diào)節(jié)能力，當(dāng)炎癥發(fā)生后免疫系統(tǒng)會控制炎癥反應(yīng)的程度，避免出現(xiàn)過度的炎癥反應(yīng)對機(jī)體造成新的損傷。綜上所述，動物對斷奶應(yīng)激有一個適應(yīng)過程，在這個過程中應(yīng)激對免疫系統(tǒng)造成的影響是不可避免的。

2.1.3對單核細(xì)胞數(shù)穩(wěn)定控制的調(diào)節(jié)

在斷奶應(yīng)激誘導(dǎo)的炎癥反應(yīng)中，單核細(xì)胞率先啟動免疫應(yīng)答，非特異性的吞噬殺傷病原微生物并分泌多種細(xì)胞因子，對免疫系統(tǒng)產(chǎn)生廣泛的調(diào)節(jié)作用[42,44]。單核細(xì)胞還參與多種抗原加工過程并為T細(xì)胞呈遞抗原，其表面黏附分子還可與T細(xì)胞表面的協(xié)同刺激分子受體結(jié)合，產(chǎn)生協(xié)同刺激信號誘導(dǎo)T細(xì)胞的活化，啟動免疫應(yīng)答。但是由于反芻動物的單核細(xì)胞表面缺少對應(yīng)激激素敏感的受體[45]，且單核細(xì)胞從骨髓進(jìn)入循環(huán)血液后只停留36～48 h，甚至更短時間便游離出血管進(jìn)入周圍組織器官，導(dǎo)致斷奶后單核細(xì)胞數(shù)的變化很難被觀察到，因此在過去的研究中大多忽略了單核細(xì)胞的功能。目前，尚不明確斷奶應(yīng)激誘導(dǎo)的免疫應(yīng)答中是否存在抑制單核細(xì)胞分化增殖的因素，今后應(yīng)對相關(guān)的免疫信號通路進(jìn)行研究，以解答這一問題。

表1　斷奶應(yīng)激對血液中白細(xì)胞數(shù)的影響Table 1　Effects of weaning stress on leukocyte number in the blood

↑表示升高；↓表示降低；?表示無顯著變化。

↑= increase; ↓=decrease; ?=no significant change.

2.2斷奶后紅細(xì)胞和血小板參與的免疫反應(yīng)

紅細(xì)胞和血小板是病理生理反應(yīng)的敏感指標(biāo)，通常可以用來確定反芻動物的亞臨床疾病[45]。紅細(xì)胞與血小板具有“血細(xì)胞免疫黏附作用”，抗原抗體免疫復(fù)合物與表面受體的補體結(jié)合后可以黏附于紅細(xì)胞或血小板表面，后被巨噬細(xì)胞一同吞噬，這可能是斷奶應(yīng)激導(dǎo)致血液中紅細(xì)胞數(shù)和血小板數(shù)減少的主要途徑。此外，紅細(xì)胞具有呈遞抗原和增強T細(xì)胞活性等免疫學(xué)功能，其細(xì)胞表面附著有過氧化物酶，可以直接殺傷病原微生物，本身也具有一定的吞噬能力[46]。血小板還具有調(diào)節(jié)免疫應(yīng)答和炎癥反應(yīng)的功能[47-49]，當(dāng)抗原抗體免疫復(fù)合物黏附于血小板表面時，血小板功能發(fā)生改變，伸出樹突附著于血管壁并發(fā)生黏性形變，最終導(dǎo)致血管壁出現(xiàn)炎性損傷[50]。

2.3斷奶應(yīng)激與血漿葡萄糖和胰島素濃度異常

一些研究發(fā)現(xiàn)犢牛斷奶后血漿中葡萄糖濃度顯著升高，這可能是應(yīng)激引起的兒茶酚胺和糖皮質(zhì)激素分泌增加導(dǎo)致肝糖原分解造成的[51]。但另一項研究發(fā)現(xiàn)，犢牛斷奶后2 d血漿葡萄糖濃度由斷奶前的3.2 mmol/L升高到3.7 mmol/L，并且在35 d的試驗期內(nèi)維持高水平，而血漿皮質(zhì)醇濃度卻沒有顯著變化[16]，這表明此時動物可能出現(xiàn)了胰島素抵抗(IR)[52]。斷奶應(yīng)激誘導(dǎo)的炎癥反應(yīng)使胰島素靶器官對胰島素的敏感性低于正常水平，引起胰腺的代償性增生及胰島素的過量分泌，導(dǎo)致血漿中胰島素濃度異常升高。持續(xù)的炎癥反應(yīng)會使抵抗程度增加，隨后胰島素分泌量逐漸減少，導(dǎo)致血漿中胰島素濃度降低而葡萄糖濃度升高[53]。此外，由于犢牛斷奶后采食量急劇下降，體脂動員增加，導(dǎo)致血漿中甘油三酯濃度降低(35.0 mg/dL vs. 18.3 mg/dL)，β-羥基丁酸濃度升高(0.29 mmol/L vs. 0.39 mmol/L)[16]，非酯化脂肪酸濃度升高，從而增加幼畜酮病的發(fā)生幾率[54]，這種代謝障礙會直接影響幼齡反芻動物的生長發(fā)育和生產(chǎn)性能[55]。斷奶后，觀測動物血漿葡萄糖和胰島素濃度，有助于我們通過營養(yǎng)調(diào)控的手段緩解斷奶應(yīng)激。

2.4斷奶應(yīng)激對急性期蛋白的影響

急性期蛋白(酶、蛋白酶抑制劑、凝固蛋白、纖維蛋白原和轉(zhuǎn)運蛋白等)對免疫系統(tǒng)有調(diào)節(jié)功能，如激活巨噬細(xì)胞和參與組織修復(fù)重建等[56-58]。急性期蛋白通常處在相對穩(wěn)定的狀態(tài)[59]，斷奶應(yīng)激可增加IL-1、TNF-α和IL-6等促炎性細(xì)胞因子的分泌，而促炎性細(xì)胞因子會刺激肝臟使急性期蛋白的分泌量急劇增加，導(dǎo)致體蛋白質(zhì)沉積減少，間接影響幼齡反芻動物的生長發(fā)育。Arthington等[60]和Horadagoda等[61]提出急性期蛋白可作為判斷免疫應(yīng)激的一個指標(biāo)。但是，由于目前國際上缺乏統(tǒng)一的判斷標(biāo)準(zhǔn)，并且不能排除由肝臟發(fā)育和肝臟損傷等因素造成的干擾，所以在相關(guān)研究中尚不能確定是否可以使用急性期蛋白作為一個穩(wěn)定的指標(biāo)。目前在反芻動物上研究較多的是觸珠蛋白和纖維蛋白原[13-14,16-19,60]：犢牛斷奶后2 d血漿纖維蛋白原濃度由斷奶前的408 mg/dL升高到458 mg/dL，斷奶后21 d升高到493 mg/dL；犢牛斷奶后2 d血漿觸珠蛋白濃度由斷奶前的0.33 mg/dL升高到0.43 mg/dL，斷奶后14 d升高到0.72 mg/dL，這2種急性期蛋白的濃度在斷奶后35 d的試驗期內(nèi)都保持著高水平[16]。但目前各類急性期蛋白與應(yīng)激反應(yīng)的聯(lián)動機(jī)制尚不清楚[58]，今后應(yīng)加強急性期蛋白的分類研究，找出斷奶應(yīng)激啟動急性期蛋白表達(dá)的免疫信號通路，從機(jī)理上解釋這一生理變化。

2.5與幼齡反芻動物免疫調(diào)節(jié)有關(guān)的細(xì)胞因子的變化

2.5.1IFN-γ

IFN-γ具有抗病毒和免疫調(diào)節(jié)的作用。犢牛斷奶后24 h血液中IFN-γ的表達(dá)量較斷奶前升高了近3倍，并在整個試驗期內(nèi)維持在高水平，明顯增強了細(xì)胞調(diào)節(jié)的炎癥應(yīng)答[20]。此外，IFN-γ與抗炎因子白細(xì)胞介素4(IL-4)之間存在拮抗作用，二者都可以調(diào)節(jié)T細(xì)胞輔助細(xì)胞的分化，其中一種細(xì)胞因子的表達(dá)上調(diào)會導(dǎo)致另一種細(xì)胞因子的分泌量減少[62]。IFN-γ可通過增強固有細(xì)胞免疫，活化中性粒細(xì)胞和單核細(xì)胞，刺激CD4+T細(xì)胞分化為Th1細(xì)胞從而抑制Th2細(xì)胞分泌IL-4[63]，增強炎癥反應(yīng)，損害動物健康。目前在幼齡反芻動物斷奶應(yīng)激的研究中針對IFN-γ的報道較少，這可能成為今后研究幼齡反芻動物免疫調(diào)節(jié)的新方向。

2.5.2白細(xì)胞介素

白細(xì)胞介素在斷奶應(yīng)激引起的炎癥反應(yīng)和免疫調(diào)節(jié)中起重要作用，如IL-8被認(rèn)為是非常重要的中性粒細(xì)胞分化誘導(dǎo)物。犢牛斷奶后24 h血液中IL-8的表達(dá)量較斷奶前升高了2倍[20]，這也從另一個角度解釋了斷奶后中性粒細(xì)胞數(shù)顯著升高的原因。在炎癥反應(yīng)中，IL-8表達(dá)量升高最早出現(xiàn)于發(fā)生炎癥部位的巨噬細(xì)胞中，意味著炎癥反應(yīng)的出現(xiàn)可能早于斷奶后24 h[64]。因此，在今后的研究中有必要在斷奶后更短的時間內(nèi)對相關(guān)細(xì)胞因子的變化進(jìn)行觀察。

2.5.3腫瘤壞死因子和細(xì)胞凋亡因子

斷奶應(yīng)激會導(dǎo)致TNF-α和Fas的表達(dá)量上調(diào)。O’Loughlin等[20]研究發(fā)現(xiàn)，犢牛斷奶后24 hTNF-α的表達(dá)量較斷奶前顯著升高；斷奶后24 hFas的表達(dá)量比正常水平升高了近4倍，斷奶后這2種細(xì)胞因子表達(dá)量上調(diào)的持續(xù)時間可能長于預(yù)期。TNF-α主要介導(dǎo)急性炎癥應(yīng)答，在斷奶應(yīng)激誘導(dǎo)的炎癥反應(yīng)中TNF-α起到了關(guān)鍵作用[65]。TNF-α可與靶細(xì)胞表面受體結(jié)合形成TNF-R三聚體，誘導(dǎo)胞漿內(nèi)的死亡結(jié)構(gòu)域形成，隨后與死亡域蛋白結(jié)合，并激活半胱氨酸天冬氨酸蛋白水解酶，最終促使細(xì)胞凋亡。Fas對細(xì)胞凋亡的調(diào)控與TNF-α類似，細(xì)胞膜表面的Fas蛋白與其配體結(jié)合導(dǎo)致細(xì)胞過早凋亡，引起炎癥和疾病。IFN-γ與TNF-α表達(dá)量的上調(diào)可增強Fas在多種細(xì)胞中的表達(dá)[66]，而Fas表達(dá)量的升高可促進(jìn)其他促炎細(xì)胞因子的分泌，增強炎癥反應(yīng)[67]。此外，在應(yīng)激誘導(dǎo)的炎癥反應(yīng)中觀察到了細(xì)胞周期負(fù)調(diào)控因子(P21)的基因表達(dá)量的上調(diào)[68-70]，P21通過細(xì)胞周期的阻滯作用和激活T淋巴細(xì)胞誘導(dǎo)的Fas信號通路參與細(xì)胞凋亡過程[71-72]。斷奶應(yīng)激加速細(xì)胞凋亡的現(xiàn)象，可能是免疫系統(tǒng)對動物生理平衡被破壞做出的反應(yīng)，而這種現(xiàn)象對動物健康的潛在危害目前尚不清楚，加速細(xì)胞凋亡是否會抑制幼齡反芻動物生長發(fā)育甚至引起死亡還有待進(jìn)一步研究。

2.5.4Toll樣受體家族

Toll樣受體家族是參與適應(yīng)性免疫的重要蛋白質(zhì)分子，可以在單核細(xì)胞、T細(xì)胞、B細(xì)胞和自然殺傷(NK)細(xì)胞等多種細(xì)胞中表達(dá)，也是連接固有免疫與適應(yīng)性免疫的橋梁[73]。TLR4可識別脂多糖和宿主細(xì)胞壞死釋放的熱休克蛋白[74]，并在抗原呈遞和抗體識別過程中起關(guān)鍵的調(diào)控作用。犢牛斷奶后7 d血液中TLR4的表達(dá)量比斷奶前升高了2倍[20]，長期慢性應(yīng)激亦可激活TLR4基因使之表達(dá)量顯著升高[75-76]。研究證實，TLR4的激活可上調(diào)數(shù)種促炎細(xì)胞因子的表達(dá)[77]，TLR4表達(dá)量的升高可能導(dǎo)致嚴(yán)重的炎癥反應(yīng)和慢性疾病[78]，并在非傳染性炎癥疾病的發(fā)生中起著重要作用[79]。Toll樣受體引發(fā)的炎癥反應(yīng)的持續(xù)時間可能長于預(yù)期，這意味著斷奶后炎癥可能會長期存在于動物體內(nèi)并對動物的健康造成損害。但目前對斷奶應(yīng)激的研究多局限于斷奶后7～14 d[3,15,18-20]。因此，在今后的研究中有必要適當(dāng)延長試驗時間，以便更準(zhǔn)確地評估斷奶對幼齡反芻動物的影響。

2.5.5細(xì)胞黏附分子

CD62L對中性粒細(xì)胞的著邊和向感染部位移動起著關(guān)鍵性作用。CD62L同時參與免疫細(xì)胞識別、誘導(dǎo)未致敏淋巴細(xì)胞歸巢以及多種細(xì)胞間附著與信號傳導(dǎo)[80]。斷奶應(yīng)激引起的糖皮質(zhì)激素分泌量的增加會明顯抑制CD62L基因的表達(dá)[15,43,81]，CD62L分泌量減少會直接影響免疫細(xì)胞的著邊和遷移，使其不能發(fā)揮正常的免疫學(xué)功能，降低動物的抗病能力。因此，通過營養(yǎng)調(diào)控解除CD62L基因表達(dá)的抑制因素，可能是增強斷奶幼畜免疫力和抗病力的有效途徑。

3小結(jié)

幼齡反芻動物斷奶后血漿中糖皮質(zhì)激素濃度升高會影響多種細(xì)胞因子的分泌，而細(xì)胞因子分泌量的改變是斷奶應(yīng)激引起免疫系統(tǒng)調(diào)節(jié)異常的主要原因。目前，幼齡反芻動物斷奶應(yīng)激的研究多集中于器官發(fā)育和生產(chǎn)性能，對免疫系統(tǒng)功能和動物健康的研究較少，且研究多局限于激素與細(xì)胞水平。今后需要進(jìn)一步從激素、免疫細(xì)胞和細(xì)胞因子3個水平對斷奶應(yīng)激影響免疫系統(tǒng)的機(jī)制進(jìn)行研究，為通過營養(yǎng)調(diào)控手段緩解斷奶應(yīng)激和保障動物健康提供科學(xué)依據(jù)。

致謝：

感謝蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院劉婷博士對論文撰寫的指導(dǎo)。

參考文獻(xiàn)：

[2]GREENWOOD P L,CAFE L M.Prenatal and pre-weaning growth and nutrition of cattle:long-term consequences for beef production[J].Animal,2007,1(9):1283-1296.

[3]O’LOUGHLIN A,MCGEE M,DOYLE S,et al.Biomarker responses to weaning stress in beef calves[J].Research in Veterinary Science,2014,97(2):458-463.

[4]LAMBERTZ C,FARKE-R?VER A,GAULY M.Effects of sex and age on behavior and weight gain in beef calves after abrupt weaning[J].Animal Science Journal,2015,86(3):345-350.

[5]OBRIST P A,GAEBELEIN C J,TELLER E S,et al.The relationship among heart rate,carotid dP/dt,and blood pressure in humans as a function of the type of stress[J].Psychophysiology,1978,15(2):102-115.

[6]GREENWOOD P L,CAFE L M,HEARNSHAW H,et al.Consequences of nutrition and growth retardation early in life for growth and composition of cattle and eating quality of beef[J].Recent Advances in Animal Nutrition in Australia,2005,15:183-195.

[7]HULBERT L E,COBB C J,CARROLL J A,et al.The effects of early weaning on innate immune responses of Holstein calves[J].Journal of Dairy Science,2011,94(5):2545-2556.

[8]CALLAN R J,GARRY F B.Biosecurity and bovine respiratory disease[J].Veterinary Clinics of North America:Food Animal Practice,2002,18(1):57-77.

[9]DUFF G C,GALYEAN M L.Board-invited review:recent advances in management of highly stressed,newly received feedlot cattle[J].Journal of Animal Science,2007,85(3):823-840.

[10]SNOWDER G.Genetics,environment and bovine respiratory disease[J].Animal Health Research Reviews,2009,10(2):117-119.

[11]NOCEK J E,BRAUND D G,WARNER R G.Influence of neonatal colostrum administration,immunoglobulin,and continued feeding of colostrum on calf gain,health,and serum protein[J].Journal of Dairy Science,1984,67(2):319-333.

[12]XU R J.Development of the newborn GI tract and its relation to colostrum/milk intake:a review[J].Reproduction Fertility and Development,1996,8(1):35-48.

[13]HICKEY M C,DRENNAN M,EARLEY B.The effect of abrupt weaning of suckler calves on the plasma concentrations of cortisol,catecholamines,leukocytes,acute-phase proteins and in vitro interferon-gamma production[J].Journal of Animal Science,2003,81(11):2847-2855.

[14]KIM M H,YANG J Y,UPADHAYA S D,et al.The stress of weaning influences serum levels of acute-phase proteins,iron-binding proteins,inflammatory cytokines,cortisol,and leukocyte subsets in Holstein calves[J].Journal of Veterinary Science,2011,12(2):151-157.

[15]LYNCH E M,EARLEY B,MCGEE M,et al.Effect of abrupt weaning at housing on leukocyte distribution,functional activity of neutrophils,and acute phase protein response of beef calves[J].BMC Veterinary Research,2010,6(1):39.

[16]LYNCH E M,EARLEY B,MCGEE M,et al.Characterisation of physiological and immunological responses in beef cows to abrupt weaning and subsequent housing[J].BMC Veterinary Research,2010,6(1):37.

[17]LYNCH E M,MCGEE M,DOYLE S,et al.Effect of post-weaning management practices on physiological and immunological responses of weaned beef calves[J].Irish Journal of Agricultural and Food Research,2011,50(2):161-174.

[18]LYNCH E M,MCGEE M,DOYLE S,et al.Effect of pre-weaning concentrate supplementation on peripheral distribution of leukocytes,functional activity of neutrophils,acute phase protein and behavioural responses of abruptly weaned and housed beef calves[J].BMC Veterinary Research,2012,8(1):1.

[19]O’LOUGHLIN A,LYNN D J,MCGEE M,et al.Transcriptomic analysis of the stress response to weaning at housing in bovine leukocytes using RNA-seq technology[J].BMC Genomics,2012,13(1):250.

[20]O’LOUGHLIN A,MCGEE M,WATERS S M,et al.Examination of the bovine leukocyte environment using immunogenetic biomarkers to assess immunocompetence following exposure to weaning stress[J].BMC Veterinary Research,2011,7(1):45.

[21]PAZIRANDEH A,XUE Y,PRESTEGAARD T,et al.Effects of altered glucocorticoid sensitivity in the T cell lineage on thymocyte and T cell homeostasis[J].The FASEB Journal,2002,16(7):727-729.

[22]BOMMHARDT U,BEYER M,HüNIG T,et al.Molecular and cellular mechanisms of T cell development[J].Cellular and Molecular Life Sciences CMLS,2004,61(3):263-280.

[23]FRAKER P J,KING L E.Reprogramming of the immune system during zinc deficiency[J].Annual Review of Nutrition,2004,24(1):277-298.

[24]BIOLATTI B,BOLLO E,CANNIZZO F T,et al.Effects of low-dose dexamethasone on thymus morphology and immunological parameters in veal calves[J].Journal of Veterinary Medicine Series A,2005,52(4):202-208.

[25]BISWAS R,ROY T,CHATTOPADHYAY U.Prolactin induced reversal of glucocorticoid mediated apoptosis of immature cortical thymocytes is abrogated by induction of tumor[J].Journal of Neuroimmunology,2006,171(1/2):120-134.

[26]RODRIGUES-MASCARENHAS S,FERNANDES DOS SANTOS N,RUMJANEK V M.Synergistic effect between ouabain and glucocorticoids for the induction of thymic atrophy[J].Bioscience Reports,2006,26(2):159-169.

[27]GLASER R,KIECOLT-GLASER J K.Stress-induced immune dysfunction:implications for health[J].Nature Reviews Immunology,2005,5(3):243-251.

[28]MINTON J E,PARSONS K M.Adrenocorticotropic hormone and cortisol response to corticotropin-releasing factor and lysine vasopressin in pigs[J].Journal of Animal Science,1993,71(3):724-729.

[29]RIVIER C,VALE W.Interaction of corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin secretioninvivo[J].Endocrinology,1983,113(3):939-942.

[30]WATABE T,TANAKA K,KUMAGAE M,et al.Role of endogenous arginine vasopressin in potentiating corticotropin-releasing hormone-stimulated corticotropin secretion in man[J].The Journal of Clinical Endocrinology & Metabolism,1988,66(6):1132-1137.

[31]AUPHAN N,DIDONATO J A,ROSETTE C,et al.Immunosuppression by glucocorticoids:inhibition of NF-κB activity through induction of IκB synthesis[J].Science,1995,270(5234):286-290.

[32]WRIGHTON C J,HOFER-WARBINEK R,MOLL T,et al.Inhibition of endothelial cell activation by adenovirus-mediated expression of I kappa B alpha,an inhibitor of the transcription factor NF-kappa B[J].Journal of Experimental Medicine,1996,183(3):1013-1022.

[33]CONNOR T J,BREWER C,KELLY J P,et al.Acute stress suppresses pro-inflammatory cytokines TNF-α and IL-1β independent of a catecholamine-driven increase in IL-10 production[J].Journal of Neuroimmunology,2005,159(1/2):119-128.

[34]CURTIN N M,BOYLE N T,MILLS K H G,et al.Psychological stress suppresses innate IFN-γ production via glucocorticoid receptor activation:reversal by the anxiolytic chlordiazepoxide[J].Brain,Behavior,and Immunity,2009,23(4):535-547.

[35]MARX J.Immunology:how the glucocorticoids suppress immunity[J].Science,1995,270(5234):232-233.

[36]MELTZER J C,MACNEIL B J,SANDERS V,et al.Stress-induced suppression ofinvivosplenic cytokine production in the rat by neural and hormonal mechanisms[J].Brain,Behavior,and Immunity,2004,18(3):262-273.

[37]CARROLL J A,ARTHINGTON J D,CHASE C C.Early weaning alters the acute-phase reaction to an endotoxin challenge in beef calves[J].Journal of Animal Science,2009,87(12):4167-4172.

[38]HU X Y,LI W P,MENG C,et al.Inhibition of IFN-γ signaling by glucocorticoids[J].Journal of Immunology,2003,170(9):4833-4839.

[39]JOHNSON J D,O’CONNOR K A,DEAK T,et al.Prior stressor exposure sensitizes LPS-induced cytokine production[J].Brain,Behavior,and Immunity,2002,16(4):461-476.

[40]GOUJON E,PARNET P,LAYE S,et al.Stress downregulates lipopolysaccharide-induced expression of proinflammatory cytokines in the spleen,pituitary,and brain of mice[J].Brain,Behavior,and Immunity,1995,9(4):292-303.

[41]ZAHOREC R.Ratio of neutrophil to lymphocyte counts—rapid and simple parameter of systemic inflammation and stress in critically ill[J].Bratislavské Lekárske Listy,2001,102(1):5-14.

[42]DHABHAR F S.A hassle a day may keep the pathogens away:the fight-or-flight stress response and the augmentation of immune function[J].Integrative and Comparative Biology,2009,49(3):215-236.

[43]TEMPELMAN R J,SAAMA P M,FREEMAN A E,et al.Genetic variation in bovine neutrophil sensitivity to glucocorticoid challenge[J].Acta Agriculturae Scandinavica,Section A-Animal Science,2002,52(4):189-202.

[44]SAUL A N,OBERYSZYN T M,DAUGHERTY C,et al.Chronic stress and susceptibility to skin cancer[J].Journal of the National Cancer Institute,2005,97(23):1760-1767.

[45]JONES M L,ALLISON R W.Evaluation of the ruminant complete blood cell count[J].Veterinary Clinics of North America:Food Animal Practice,2007,23(3):377-402.

[46]EMLEN W,CARL V,BURDICK G.Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes[J].Clinical & Experimental Immunology,1992,89(1):8-17.

[47]LAZARUS A H,ELLIS J,SEMPLE J W,et al.Comparison of platelet immunity in patients with SLE and with ITP[J].Transfusion Science,2000,22(1/2):19-27.

[48]LIPPI G,FRANCHINI M.Platelets and immunity:the interplay of mean platelet volume in health and disease[J].Expert Review of Hematology,2015,8(5):555-557.

[49]WAZNA E.Platelet-mediated regulation of immunity[J].Postepy Higieny Ⅰ Medycyny Doswiadczalnej,2006,60:265-277.

[50]GARRAUD O,COGNASSE F.Platelet immunology and the immune response[J].Transfusion Clinique et Biologique,2009,16(2):106-117.

[51]MCDOWELL G H.Hormonal control of glucose homoeostasis in ruminants[J].Proceedings of the Nutrition Society,1983,42(2):149-167.

[52]SHOELSON S E,LEE J,GOLDFINE A B.Inflammation and insulin resistance[J].Journal of Clinical Investigation,2006,116(7):1793-1801.

[53]AKBARI H,DALIR-NAGHADEH B,ASRI-REZAEI S,et al.Experimental hyperlipidemia induces insulin resistance in sheep[J].Domestic Animal Endocrinology,2015,53:95-102.

[54]VOSOOGHI-POOSTINDOZ V,FOROUGHI A R,DELKHOROSHAN A,et al.Effects of different levels of protein with or without probiotics on growth performance and blood metabolite responses during pre- and post-weaning phases in male Kurdi lambs[J].Small Ruminant Research,2014,117(1):1-9.

[55]BACH A,DOMINGO L,MONTORO C,et al.Short communication:insulin responsiveness is affected by the level of milk replacer offered to young calves[J].Journal of Dairy Science,2013,96(7):4634-4637.

[56]GODSON D L,BACA-ESTRADA M E,VAN KESSEL A G,et al.Regulation of bovine acute phase responses by recombinant interleukin-1 beta[J].Canadian Journal of Veterinary Research,1995,59(4):249-255.

[57]GRUYS E,TOUSSAINT M J,NIEWOLD T A,et al.Acute phase reaction and acute phase proteins[J].Journal ofZhejiangUniversity:Science B,2005,6(11):1045-1056.

[58]PETERSEN H H,NIELSEN J P,HEEGAARD P M H.Application of acute phase protein measurements in veterinary clinical chemistry[J].Veterinary Research,2004,35(2):163-187.

[59]CARROLL J A,FORSBERG N E.Influence of stress and nutrition on cattle immunity[J].Veterinary Clinics of North America:Food Animal Practice,2007,23(1):105-149.

[60]ARTHINGTON J D,EICHERT S D,KUNKLE W E,et al.Effect of transportation and commingling on the acute-phase protein response,growth,and feed intake of newly weaned beef calves[J].Journal of Animal Science,2003,81(5):1120-1125.

[61]HORADAGODA N U,KNOX K M G,GIBBS H A,et al.Acute phase proteins in cattle:discrimination between acute and chronic inflammation[J].Veterinary Record,1999,144(16):437-441.

[62]MORINOBU A,KUMAGAI S.Cytokine measurement at a single-cell level to analyze human Th1 and Th2 cells[J].The Japanese Journal of Clinical Pathology,1998,46(9):908-914.

[63]BOEHM U,KLAMP T,GROOT M,et al.Cellular responses to interferon-γ[J].Annual Review of Immunology,1997,15(1):749-795.

[64]SPORER K R B,BURTON J L,EARLEY B,et al.Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis,tissue remodeling,margination,and anti-bacterial function[J].Veterinary Immunology and Immunopathology,2007,118(1/2):19-29.

[65]BAILEY M T,KINSEY S G,PADGETT D A,et al.Social stress enhances IL-1β and TNF-α production byPorphyromonasgingivalislipopolysaccharide-stimulated CD11b+cells[J].Physiology & Behavior,2009,98(3):351-358.

[66]NAGATA S,GOLSTEIN P.The Fas death factor[J].Science,1995,267(5203):1449-1456.

[67]KIM J M,KIM S S,LEE Y D.Fas-associated factor 1 promotes in neurofibrillary tangle-mediated cell death of basal forebrain cholinergic neurons in P301L transgenic mice[J].Neuroreport,2015,26(13):767-772.

[68]SPORER K R B,XIAO L,TEMPELMAN R J,et al.Transportation stress alters the circulating steroid environment and neutrophil gene expression in beef bulls[J].Veterinary Immunology and Immunopathology,2008,121(3/4):300-320.

[69]YIN D L,TUTHILL D,MUFSON R A,et al.Chronic restraint stress promotes lymphocyte apoptosis by modulating CD95 expression[J].Journal of Experimental Medicine,2000,191(8):1423-1428.

[70]YIN D L,ZHANG Y,STUART C,et al.Chronic restraint stress modulates expression of genes in murine spleen[J].Journal of Neuroimmunology,2006,177(1/2):11-17.

[71]COQUERET O.New roles for p21 and p27 cell-cycle inhibitors:a function for each cell compartment[J].Trends in Cell Biology,2003,13(2):65-70.

[72]HINGORANI R,BI B Y,DAO T,et al.CD95/Fas signaling in T lymphocytes induces the cell cycle control protein p21cip-1/WAF-1,which promotes apoptosis[J].The Journal of Immunology,2000,164(8):4032-4036.

[73]GAN L,LI L W.Regulations and roles of the interleukin-1 receptor associated kinases (IRAKs) in innate and adaptive immunity[J].Immunologic Research,2006,35(3):295-302.

[74]LEHNARDT S,LACHANCE C,PATRIZI S,et al.The toll-like receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS[J].Journal of Neuroscience,2002,22(7):2478-2486.

[75]ZHANG Y,WOODRUFF M,ZHANG Y,et al.Toll-like receptor 4 mediates chronic restraint stress-induced immune suppression[J].Journal of Neuroimmunology,2008,194(1/2):115-122.

[76]ZHANG Y,ZHANG Y,MIAO J Y,et al.Chronic restraint stress promotes immune suppression through Toll-like receptor 4-mediated phosphoinositide 3-kinase signaling[J].Journal of Neuroimmunology,2008,204(1/2):13-19.

[77]SHAHRARA S,PARK C C,TEMKIN V,et al.RANTES modulates TLR4-induced cytokine secretion in human peripheral blood monocytes[J].Journal of Immunology,2006,177(8):5077-5087.

[78]LU Y C,YEH W C,OHASHI P S.LPS/TLR4 signal transduction pathway[J].Cytokine,2008,42(2):145-151.

[79]SEKI E,DE MINICIS S,?STERREICHER C H,et al.TLR4 enhances TGF-β signaling and hepatic fibrosis[J].Nature Medicine,2007,13(11):1324-1332.

[80]KANSAS G S.Selectins and their ligands:current concepts and controversies[J].Blood,1996,88(9):3259-3287.

[81]WEBER P S D,TOELBOELL T,CHANG L C,et al.Mechanisms of glucocorticoid-induced down-regulation of neutrophilL-selectin in cattle:evidence for effects at the gene-expression level and primarily on blood neutrophils[J].Journal of Leukocyte Biology,2004,75(5):815-827.

(責(zé)任編輯菅景穎)

doi：10.3969/j.issn.1006-267x.2016.07.003

收稿日期：2016-01-21

基金項目：國家自然科學(xué)基金項目(31501975)；公益性行業(yè)(農(nóng)業(yè))科研專項經(jīng)費(201503134)；長江學(xué)者和創(chuàng)新團(tuán)隊發(fā)展計劃(IRT13019)

作者簡介：張千(1990—)，男，內(nèi)蒙古呼和浩特人，碩士研究生，研究方向為反芻動物營養(yǎng)學(xué)。E-mail: zhangqian14@lzu.edu.cn *通信作者：李飛，副教授，碩士生導(dǎo)師，E-mail: lfei@lzu.edu.cn

中圖分類號：S816

文獻(xiàn)標(biāo)識碼：A

文章編號：1006-267X(2016)07-1988-10

*Corresponding author, associate professor, E-mail: lfei@lzu.edu.cn

Effects and Mechanism of Weaning Stress on Immune System in Young Ruminants

ZHANG Qian1LI Fadi1,2LI Fei1*

(1. Key State Laboratory of Agro-Ecosystems, College of Pastoral Agriculture Science and Technology,Lanzhou University, Lanzhou 730020, China; 2. Biotechnology Engineering Laboratory of Gansu Meat Sheep Breeding, Minqin 733300, China)

Abstract:The digestive and immune systems have not yet developed completely, when young ruminants were weaned. Weaning stress leaded to adolescent alterations in hormone levels and immune function, which could suppress immune system and cause inflammatory response, consequently restricted their growth and increased the risk of disease in ruminants. In this article, the effects and mechanism of weaning stress on the immune system in young ruminants were discussed, focusing on glucocorticoid, immune cell, acute phase proteins and related cytokine, with the aim of providing a scientific basis for the relevant research.[Chinese Journal of Animal Nutrition, 2016, 28(7)：1988-1997]

Key words:young ruminants; weaning stress; immune system; glucocorticoid; cytokine