奶山羊?qū)嶒?yàn)性乳房炎的病理組織學(xué)觀察及促炎性細(xì)胞因子的檢測(cè)

韓炎森,牟 珊,陳德坤*

(1.西北農(nóng)林科技大學(xué)生命科學(xué)學(xué)院,陜西楊陵 712100;2.西北農(nóng)林科技大學(xué)動(dòng)物醫(yī)學(xué)院,陜西楊陵 712100)

奶山羊?qū)嶒?yàn)性乳房炎的病理組織學(xué)觀察及促炎性細(xì)胞因子的檢測(cè)

韓炎森1,牟 珊2,陳德坤2*

(1.西北農(nóng)林科技大學(xué)生命科學(xué)學(xué)院,陜西楊陵 712100;2.西北農(nóng)林科技大學(xué)動(dòng)物醫(yī)學(xué)院,陜西楊陵 712100)

選健康泌乳期關(guān)中奶山羊8只,分成金黃色葡萄球菌感染組和大腸埃希菌感染組,每只羊分別經(jīng)右乳頭灌注病原性大腸埃希菌(3×103cfu)和金黃色葡萄球菌(3×102cfu),對(duì)照乳區(qū)灌注等量無菌PBS。于灌注細(xì)菌前后不同時(shí)間采集血樣和乳樣,測(cè)定血清和乳樣中的促炎性細(xì)胞因子水平,并于72 h后取山羊乳腺組織制備病理切片觀察組織變化。結(jié)果顯示,金黃色葡萄球菌感染組奶山羊乳樣中IL-1β、IL-6、IL-8、IL-12、IL-17和TNF-α水平不斷升高,血樣中相同細(xì)胞因子水平均表現(xiàn)先上升后下降特點(diǎn);大腸埃希菌感染奶山羊乳樣中細(xì)胞因子表現(xiàn)為先上升后下降趨勢(shì),血樣中變化趨勢(shì)不明顯。組織病理切片觀察顯示乳腺被金黃色葡萄球菌感染后腺泡腔內(nèi)可見脫落的腺泡細(xì)胞、淋巴細(xì)胞、中性粒細(xì)胞及巨噬細(xì)胞;間質(zhì)水腫,結(jié)締組織間隙可見大量淋巴細(xì)胞、中性粒細(xì)胞及巨噬細(xì)胞浸潤。

乳房炎;組織病理學(xué);細(xì)胞因子

*通訊作者

乳房炎是泌乳動(dòng)物主要疾病之一[1-2],在奶牛和奶山羊養(yǎng)殖中最為常見。近年來隨著奶山羊養(yǎng)殖業(yè)的迅速發(fā)展,乳房炎問題也日顯突出。乳房炎的發(fā)生不僅導(dǎo)致動(dòng)物泌乳量降低,乳汁質(zhì)量下降,嚴(yán)重的還會(huì)導(dǎo)致動(dòng)物死亡,不僅給養(yǎng)殖業(yè)造成經(jīng)濟(jì)損失,還使得乳制品品質(zhì)下降,有害微生物含量超標(biāo)等,危害消費(fèi)者身體健康。

乳腺在乳房炎發(fā)生時(shí)具有一系列防御機(jī)制保護(hù),由粒細(xì)胞和細(xì)胞因子所介導(dǎo)的非特異性免疫反應(yīng)在這個(gè)過程的早期起著大部分的保護(hù)作用。雖然綿羊、奶牛、豬和小鼠實(shí)驗(yàn)性乳房炎[3-7]及其免疫保護(hù)因子的變化規(guī)律和機(jī)理[7-9]都有過報(bào)道,但由于免疫機(jī)制的復(fù)雜性,乳房炎一直都難以得到有效預(yù)防。本研究利用人工誘發(fā)山羊?qū)嶒?yàn)性乳腺炎,研究乳房發(fā)病過程中的病理變化及血液和乳汁中細(xì)胞因子的變化規(guī)律。為闡明乳腺的防御體系并通過人工干預(yù)提高乳腺的健康水平提供理論依據(jù)。

1 材料與方法

1.1 材料

1.1.1 試驗(yàn)用動(dòng)物 8只健康泌乳期關(guān)中奶山羊,購自關(guān)中地區(qū)養(yǎng)殖戶,處于第2個(gè)～3個(gè)泌乳期,體重35 kg左右。持續(xù)檢測(cè)1周,乳樣菌檢陰性,動(dòng)物無乳房炎表現(xiàn)。

1.1.2 病原菌 西北農(nóng)林科技大學(xué)動(dòng)物醫(yī)學(xué)院免疫學(xué)實(shí)驗(yàn)室從患乳房炎奶山羊乳汁中分離鑒定所得2種病原菌,大腸埃希菌(E.coli)O117血清型和金黃色葡萄球菌(S.aureus)。

1.2 方法

1.2.1 奶山羊乳房炎模型建立 調(diào)整大腸埃希菌濃度為3×103cfu/mL,葡萄球菌濃度為3×102cfu/mL。奶山羊分為大腸埃希菌感染組和金黃色葡萄球菌感染組,每組4只。采用乳頭管灌注法于奶山羊右乳區(qū)灌注 1 mL菌液,左乳區(qū)灌注等量無菌PBS。

1.2.2 樣品采集及處理 分別于灌注前(0h)和灌注后4、8、24、48、72 h采集靜脈血和左右乳區(qū)乳汁。乳汁3 000r/min離心30 min,棄去上層乳脂和沉淀物。靜脈血1 500 r/min離心10 min,收血清。脫脂乳和血清分裝后-20℃保存待檢。感染葡萄球菌的奶山羊于72 h后處死,取乳腺組織,40 g/L多聚甲醛固定,石蠟包埋,切片,HE染色,進(jìn)行病理組織學(xué)觀察。

1.2.3 細(xì)胞因子檢測(cè) 用購自美國R&D公司的細(xì)胞因子ELISA試劑盒按說明書所述:加樣,加酶,顯色,終止,分別檢測(cè)血清和脫脂乳中細(xì)胞因子IL-1β,IL-6,IL-8,IL-12,IL-17和 TNF-α的濃度。

2 結(jié)果

2.1 乳房灌注病原菌后山羊的臨床變化

金黃色葡萄球菌感染組山羊在灌注細(xì)菌后2 h開始出現(xiàn)精神委靡,4 h后飲食欲下降,右乳區(qū)比左乳區(qū)明顯發(fā)熱,乳汁LMT檢測(cè)已呈強(qiáng)陽性。24 h后山羊更加委靡,個(gè)別羊全身顫抖甚至無法站立并出現(xiàn)拉稀,飲食欲幾近廢絕,右乳區(qū)明顯腫大發(fā)熱變硬,乳汁發(fā)黃、pH升高,產(chǎn)奶量嚴(yán)重下降。48 h后山羊躺臥,飲食欲廢絕,右乳區(qū)腫大變硬發(fā)涼,乳汁已成血乳。大腸埃希菌感染組山羊在灌注細(xì)菌后4 h開始精神不振,右乳區(qū)稍有發(fā)熱,乳汁LMT也是強(qiáng)陽性。但24 h后精神狀況出現(xiàn)好轉(zhuǎn),少量飲食,右乳區(qū)仍較左乳區(qū)發(fā)熱,產(chǎn)奶量下降,乳汁pH亦有升高;體溫在感染后升高明顯,感染后72 h時(shí)仍未恢復(fù)正常。

2.2 促炎性細(xì)胞因子 IL-1β、IL-6 、IL-8、IL-12 、IL-17和TNF-α的變化

金黃色葡萄球菌感染組山羊乳樣中IL-1β、IL-6、IL-8、IL-12、IL-17 和 TNF-α水平呈持續(xù)上升趨勢(shì),血清中細(xì)胞因子水平則表現(xiàn)為先上升,并在感染后4h達(dá)到峰值,然后下降的趨勢(shì)。大腸埃希菌感染山羊乳樣中的細(xì)胞因子濃度則呈先上升后下降趨勢(shì),并且在灌注后24h水平最高,血清中 IL-1β,IL-6和IL-17表現(xiàn)為先上升后下降趨勢(shì),分別在感染后4、24、8 h細(xì)胞因子水平最高,IL-8和 TNF-α濃度始終低于感染前,IL-12變化規(guī)律不明顯。除IL-6外,血清中其他檢測(cè)細(xì)胞因子水平始終高于乳樣(圖1～圖6)。

圖1～圖6乳樣中細(xì)胞因子水平的變化趨勢(shì)明顯:金黃色葡萄球菌感染山羊細(xì)胞因子呈持續(xù)上升特點(diǎn),大腸埃希菌感染山羊則呈先上升后下降特點(diǎn);血清中細(xì)胞因子水平變化規(guī)律不明顯。除IL-6外,血清中細(xì)胞因子水平始終高于乳樣中。

圖1 感染后72 h內(nèi) IL-1β濃度變化Fig.1 Concentration of IL-1βin 72 hours after infection

圖2 感染后 72 h內(nèi)IL-6濃度變化Fig.2 Concentration of IL-6 in 72 hours after infection

圖3 感染后72 h內(nèi)IL-8濃度變化Fig.3 Concentration of IL-8 in 72 hours after infection

圖4 感染后72 h內(nèi)IL-12濃度變化Fig.4 Concentration of IL-12 in 72 hours after infection

圖5 72 h內(nèi)IL-17濃度變化Fig.5 Concentration of IL-17 in 72 hours after infection

圖6 感染后72 h內(nèi) TNF-α濃度變化Fig.6 Concentration of TNF-α in 72 hours after infection

2.3 乳腺組織病理學(xué)觀察

乳腺組織石蠟切片形態(tài)學(xué)觀察顯示,正常健康奶山羊乳腺腺泡腔緊密排列,腺泡內(nèi)無其他細(xì)胞出現(xiàn)(圖7)。感染葡萄球菌的奶山羊乳腺腺小葉的腺泡腔中可見腺泡細(xì)胞、淋巴細(xì)胞、中性粒細(xì)胞及巨噬細(xì)胞浸潤;間質(zhì)水腫,結(jié)締組織間隙可見大量淋巴細(xì)胞、中性粒細(xì)胞浸潤(圖8)。

圖7 健康泌乳期奶山羊乳腺組織切片(10×40)腺泡緊密排列,腺泡內(nèi)無其他細(xì)胞浸潤Fig.7 Mammary histology pathological section of healthy lactation dairy goat(10×40).Acinus were closely arranged,and there were no other cells in acinus

圖8 實(shí)驗(yàn)性乳腺炎奶山羊乳腺組織切片(10×40)腺泡腔內(nèi)炎性細(xì)胞浸潤,間質(zhì)水腫Fig.8 Mammary histology pathological section of experimental mastitis dairy goat(10×40).Inflammato ry cells infiltrating in acinar lumina,interstitial edema

3 討 論

臨床型乳房炎動(dòng)物,通常都會(huì)表現(xiàn)出肉眼可見的臨床癥狀,表現(xiàn)為動(dòng)物精神委靡,表現(xiàn)為食欲不振,體溫升高,乳房病變,乳汁變色,增稠或者呈水樣、乳中有凝塊等。有些動(dòng)物伴隨著顫栗或長臥不起等。實(shí)驗(yàn)性乳房炎病理模型描述中,患乳房炎乳腺腺泡腔內(nèi)充滿炎性細(xì)胞、變性脫落上皮細(xì)胞和空泡性滲出物(脂肪),間質(zhì)水腫、間質(zhì)組織增生,上皮萎縮。本試驗(yàn)中奶山羊臨床癥狀及乳腺組織形態(tài)學(xué)觀察顯示實(shí)驗(yàn)性乳房炎模型成功建立,這也進(jìn)一步為我們鑒別乳房炎提供了可參考的臨床資料。

IL-1可誘導(dǎo)肝細(xì)胞合成急性期蛋白和一些補(bǔ)體組分促進(jìn)中性粒細(xì)胞釋放炎性介質(zhì)和炎癥蛋白直接參與炎性反應(yīng);IL-6促進(jìn)Ig的分泌,促進(jìn)單核細(xì)胞及中性粒細(xì)胞產(chǎn)生過氧化物,加速肝細(xì)胞APP合成從而介導(dǎo)機(jī)體抗炎癥的自身穩(wěn)定反應(yīng);IL-8又稱中性粒細(xì)胞激活蛋白,能夠趨化中性粒細(xì)胞向炎癥部位遷移,并激活中性粒細(xì)胞,促使其釋放溶酶體酶及超氧化物實(shí)現(xiàn)對(duì)病原體的殺傷作用;IL-12可以促進(jìn)NK細(xì)胞的殺傷水平和黏附分子的表達(dá),同時(shí)還可以上調(diào)腫瘤壞死因子(TNF);IL-17具有強(qiáng)大的招募中性粒細(xì)胞的作用,調(diào)節(jié)并促進(jìn)多種炎性介質(zhì)的產(chǎn)生,通過不同機(jī)制發(fā)揮致炎作用,主要介導(dǎo)防御胞外病原微生物感染,參與炎癥反應(yīng)和自身免疫,與免疫調(diào)節(jié)、免疫病理和宿主免疫關(guān)系密切;TNF-α位于促炎癥細(xì)胞因子相互連接形成的網(wǎng)絡(luò)頂點(diǎn),作為誘導(dǎo)細(xì)胞反應(yīng)中的最初物質(zhì),在細(xì)胞和亞細(xì)胞水平上激發(fā)一系列級(jí)聯(lián)反應(yīng)[10],可以誘導(dǎo)IL-1、IL-6等細(xì)胞因子的瀑布樣釋放。TNF-α一方面能增強(qiáng)嗜中性粒細(xì)胞的殺菌活性,另一方面還可以加速嗜中性粒細(xì)胞通過內(nèi)皮細(xì)胞壁的滲出[11-12]。乳房炎病原菌釋放的毒素可以募集白細(xì)胞和乳腺上皮細(xì)胞分泌細(xì)胞因子,這些細(xì)胞因子在乳房炎發(fā)生過程中是重要的促炎性因子。促炎因子一旦產(chǎn)生,不但可以自身激活,還能促進(jìn)其他促炎因子的產(chǎn)生,引起連鎖和放大效應(yīng),即“瀑布”效應(yīng)。本實(shí)驗(yàn)中兩個(gè)不同組乳樣細(xì)胞因子濃度呈不同規(guī)律變化,我們也得到了部分與以前報(bào)道相似的結(jié)論[6,13],奶山羊感染病原菌后乳樣中各種促炎性細(xì)胞因子水平都有明顯升高。結(jié)合奶山羊臨床癥狀表現(xiàn)可以知道伴隨著各種細(xì)胞因子濃度的升高,金黃色葡萄球菌在乳腺中繁殖迅速并且釋放大量毒素,炎癥反應(yīng)和乳腺組織損傷呈進(jìn)行性加重。而大腸埃希菌對(duì)奶山羊乳房炎的致病力是有限的,其感染通常引起急性乳房炎但易于治愈。本研究為闡明乳腺的免疫機(jī)制及防御體系并通過人工干預(yù)提高乳腺的健康水平提供了理論依據(jù)。

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The Histopathological Observation and Detection of Inflammatory Cytokines of Experimental Mastitis in Dairy Goat

HAN Yan-sen1,MOU Shan2,CHEN De-kun2

(1.College of Li fe Science,Northwest A&F University,Yang ling,Shaanxi,712100,China;2.College of veterinary medicine,Northwest A&F University,Yang ling,Shaanxi,712100,China)

Choosed 8 healthy Guanzhong dairy goats which were in the lactation period and divided intoE.coli)group and S.aureus group.The teat on the right side of mammary glands ofE.coli)group and S.aureus group was inoculated respectively with pathogenicity E.coli(3 103CFU)and S.aureus(3 102CFU),and the contrary one with phosphate buffer solution(PBS).The blood and milk samples were collected at different times and the inflammatory cytokines levels in sera and milk was detected before and after the infection,then,collected the mammary tissue at 72 h post-infection and made histology pathological section.The results showed that the cytokines IL-1β,IL-6,IL-8,IL-12,IL-17 and TNF-αof S.aureus group in milk were rising,the cytokines levels of Saureus group in sera rose first and then declined;the cytokines levels ofE.coli)group in milk rose first and then declined,the trends on cytokines levels in sera were not obvious.Histology pathological section observation showed there were deciduous acinar cells,lymphocytes,polymorphonuclear(PMN)and macrophages in acinar lumina whose mammary gland was infected by S.aureus;there were interstitial edema,and a lot of lymphocytes,polymorphonuclear(PMN)and macrophages in connective tissue clearance.

mastitis;histopathology;cytokine

S852.31;S858.27

A

1007-5038(2011)08-0053-04

2011-04-27

公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)(201103038)

韓炎森 (1985-),男,山西長子人,碩士研究生,主要從事生物信息學(xué)分子設(shè)計(jì)及免疫學(xué)研究。