杜蕾，孫靜，葛良鵬*，劉作華*

(1. 西南大學(xué)榮昌校區(qū)，重慶 402460；2. 重慶市畜牧科學(xué)院，重慶 402460；3.農(nóng)業(yè)部養(yǎng)豬科學(xué)重點(diǎn)實(shí)驗(yàn)室，重慶 402460；4.重慶市養(yǎng)豬科學(xué)重點(diǎn)實(shí)驗(yàn)室，重慶 402460)

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無(wú)菌豬的研究進(jìn)展

杜蕾1,2，孫靜2,3,4，葛良鵬2,3,4*，劉作華2,3,4*

(1. 西南大學(xué)榮昌校區(qū)，重慶 402460；2. 重慶市畜牧科學(xué)院，重慶 402460；3.農(nóng)業(yè)部養(yǎng)豬科學(xué)重點(diǎn)實(shí)驗(yàn)室，重慶 402460；4.重慶市養(yǎng)豬科學(xué)重點(diǎn)實(shí)驗(yàn)室，重慶 402460)

無(wú)菌豬是采用現(xiàn)有微生物檢測(cè)技術(shù)，檢測(cè)不出活的微生物(包括細(xì)菌、真菌、寄生蟲和病毒等)的一種悉生物，在現(xiàn)代畜牧生產(chǎn)和醫(yī)學(xué)生物學(xué)研究中具有重要的科學(xué)價(jià)值。無(wú)菌豬最早用于畜牧生產(chǎn)的疫病凈化，研究腸道菌群、動(dòng)物疫病之間的關(guān)系。無(wú)菌豬和人在解剖、生理及遺傳上具相似性、無(wú)微生物背景干擾，目前在醫(yī)學(xué)生物學(xué)研究腸道菌群與生長(zhǎng)發(fā)育與疾病發(fā)生發(fā)展等方面發(fā)揮著重要作用，也作為以腸道菌群為靶點(diǎn)的預(yù)防、診斷及治療新技術(shù)研究的特殊動(dòng)物模型。本文主要綜述無(wú)菌豬的特性、研究進(jìn)展及未來(lái)的發(fā)展方向。

無(wú)菌豬；動(dòng)物模型；研究進(jìn)展

1959年,Trexler等[1]最早開(kāi)始研究軟塑料無(wú)菌隔離裝置，1960年,Landy等[2]在此基礎(chǔ)上構(gòu)建無(wú)菌實(shí)驗(yàn)室，通過(guò)子宮切除術(shù)獲得第一頭無(wú)菌豬(germ-free pigs,GF pigs)。最早的GF豬培育是為了凈化畜牧生產(chǎn)過(guò)程中的重大疫病，研究腸道菌群與動(dòng)物疫病之間的關(guān)系，用于提高畜牧生產(chǎn)成績(jī)。由于GF豬的微生物背景清晰，保證了科學(xué)研究不受背景微生物的干擾；同時(shí)豬在解剖、生理、遺傳學(xué)、營(yíng)養(yǎng)代謝等[3-5]方面與人極其相似，結(jié)合異種菌群移植技術(shù)，可用于腸道菌群與人類健康關(guān)系的研究[6]。近年來(lái)，隨著腸道菌群移植(fecal microbiota transplant，F(xiàn)MT)技術(shù)和宏基因組測(cè)序技術(shù)的進(jìn)步，以GF豬為基礎(chǔ)，基于腸道菌群與動(dòng)物生長(zhǎng)發(fā)育，疾病發(fā)生等已成為研究熱點(diǎn)。無(wú)菌豬在現(xiàn)代畜牧生產(chǎn)和醫(yī)學(xué)、生物學(xué)研究中顯示出重要的科學(xué)價(jià)值和廣泛的應(yīng)用前景。本文就GF豬的特性、研究進(jìn)展及未來(lái)發(fā)展做一綜述。

1 無(wú)菌豬的特性

GF豬是通過(guò)無(wú)菌剖腹產(chǎn)手術(shù)、飼養(yǎng)于無(wú)菌隔離器，經(jīng)現(xiàn)有檢測(cè)手段，檢測(cè)不出任何微生物的特殊動(dòng)物[7]。由于缺乏腸道菌群，GF動(dòng)物與普通級(jí)動(dòng)物(conventional animals,CV animals)在血液、生長(zhǎng)發(fā)育等方面存在多種差異[8]。血常規(guī)指標(biāo)中，GF豬的WBC、MON與MON%、EOS與EOS%、BAS與BAS%、HGB、HCT、RBC、PCT、PLT，血清生化指標(biāo)TP、ALB、GLO、BUN、UA、CHOL、TG、HDL含量均顯著低于CV豬，且腸內(nèi)未發(fā)現(xiàn)腸系膜淋巴結(jié)[9]。另有研究顯示：GF豬的小腸重量，小腸壁厚度和小腸/體重系數(shù)也均要小于CV豬[10]。GF豬有小腸絨毛更長(zhǎng)、隱窩更淺和固有層多孔性減少等特性[11]。腸道菌群與消化酶關(guān)系密切，GF豬腸道內(nèi)的氨肽酶N和乳糖酶的活性高于CV豬，這可能與微生物引起的酶滅活密切相關(guān)[12]。在免疫系統(tǒng)發(fā)育方面，GF豬血液中免疫球蛋白濃度偏低，次級(jí)免疫器官發(fā)育受阻，在空腸的黏膜固有層中，樹(shù)突狀細(xì)胞和T細(xì)胞的數(shù)量較少，小腸中前炎癥細(xì)胞因子IL-1β和 IL-6的表達(dá)量高[11]。

盡管GF豬與CV豬在胃腸道結(jié)構(gòu)和免疫系統(tǒng)方面有許多差異，但GF豬仍是腸道菌群功能研究的理想動(dòng)物模型。與無(wú)菌嚙齒類動(dòng)物相比，豬的生理和遺傳特性與人更為接近，特別是豬的腸道系統(tǒng)和雜食特性[4]，與人高度相似。此外豬體型大、方便開(kāi)展復(fù)雜的外科手術(shù)，可頻繁地采集血液或其他體液[13]；GF豬微生物背景清晰，無(wú)多余微生物干擾[11]；豬的胎盤結(jié)構(gòu)決定GF豬出生時(shí)無(wú)母源抗體的干擾[14, 15]，作為研究環(huán)境因素對(duì)免疫影響的特殊模型。特定菌群接種GF豬構(gòu)建的悉生豬(gnotobiotic, GN)模型具有高度轉(zhuǎn)化性[16]，因此GF豬和悉生豬廣泛應(yīng)用于輪狀病毒、出血性大腸桿菌、艱難梭菌和志賀氏痢疾菌的研究，以及益生菌和疫苗效果評(píng)價(jià)。是目前研究腸道菌群與器官發(fā)育、腸道形態(tài)、生理性能及免疫調(diào)節(jié)之間關(guān)系的獨(dú)特動(dòng)物模型。

2 無(wú)菌豬目前的研究應(yīng)用進(jìn)展

2.1 畜牧生產(chǎn)方面

無(wú)菌豬最早用于重大疾病凈化，以降低家畜生產(chǎn)的疾病風(fēng)險(xiǎn)[17]。產(chǎn)腸毒素性大腸桿菌病(ETEC)是幼齡小豬最易患的疾病[18]，極易引起仔豬腹瀉甚至導(dǎo)致死亡。Kohler等利用GF豬成功鑒定造成仔豬腹瀉的產(chǎn)腸毒素性大腸桿菌病致病亞型[18]，Lin[19]利用斷奶GF豬構(gòu)建ETEC腹瀉仔豬模型，驗(yàn)證了K88疫苗對(duì)ETEC病的防治效果。Yamada等[20]利用GF豬模型觀察豬傳染性腦膜炎(PTV)的臨床癥狀，發(fā)現(xiàn)其病理機(jī)制。為了驗(yàn)證豬新型冠狀病毒的致病性，Jung等用兩株冠狀病毒OH-FD22和OH-FD100接種GF豬，發(fā)現(xiàn)這兩株病毒均會(huì)導(dǎo)致豬傳染性腹瀉(PEDV)[21]。隨著研究的發(fā)展，GF豬為模型用于研究飼料的轉(zhuǎn)化和營(yíng)養(yǎng)吸收，研發(fā)或評(píng)估新型飼料產(chǎn)品，益生素等飼料添加劑。

2.2 醫(yī)學(xué)生物學(xué)研究

利用GF豬構(gòu)建人源菌群豬模型，重現(xiàn)供體腸道微生物特性，已用于腸道微生物與人類健康及疾病發(fā)生的研究。Shen等[22]構(gòu)建人源菌群悉生豬模型研究短鏈低聚果糖的益生作用，向接種成年糞便懸液的悉生豬連續(xù)飼喂每千克體重0.5 g的低聚果糖37 d之后，能有效增加腸道雙歧桿菌的數(shù)量。A組輪狀病毒(HRV)是全世界嬰幼兒感染脫水性腹瀉的主要原因[23]，鑒于豬對(duì)HRV病毒的長(zhǎng)期敏感性，Saif等[24]利用GF豬構(gòu)建人輪狀病毒的豬模型，進(jìn)行免疫動(dòng)態(tài)分析。Hulst等[25]利用微陣列分析輪狀病毒進(jìn)入腸道后的轉(zhuǎn)錄反應(yīng)，發(fā)現(xiàn)鳥(niǎo)苷結(jié)合蛋白2(GBP2)利于機(jī)體形成對(duì)抗輪狀病毒等腸道性疾病的先天性屏障。Yang等[16]進(jìn)一步通過(guò)GF豬模型研究發(fā)現(xiàn)米糠可以促進(jìn)益生菌增長(zhǎng)、加強(qiáng)免疫屏障功能、激發(fā)先天性免疫，進(jìn)而對(duì)抗HRV引起的腹瀉。

2.3 免疫機(jī)制相關(guān)研究

無(wú)菌動(dòng)物的腸道相關(guān)淋巴組織(GALT)缺乏，腸內(nèi)的淋巴細(xì)胞數(shù)量、分泌性IgA漿細(xì)胞[26]、上皮內(nèi)淋巴細(xì)胞[27]或固有層的CD4+T淋巴細(xì)胞均低于CV豬[28]。豬回腸黏膜由共生菌群的胞外信號(hào)控制形成大量B細(xì)胞，影響腸道免疫蛋白的形成[29]。Potockova等[30]發(fā)現(xiàn)B細(xì)胞雖未在回腸內(nèi)發(fā)育但卻大量存在，表明菌群定植能促進(jìn)回腸內(nèi)B細(xì)胞的存在以及小腸淋巴細(xì)胞表達(dá)。共生菌對(duì)宿主免疫結(jié)構(gòu)有深遠(yuǎn)影響，定植將導(dǎo)致固有層的DC細(xì)胞和T細(xì)胞廣泛增加[31]。免疫細(xì)胞表面的一些受體能限制其殺傷自體組織，腫瘤組織同樣能夠激活這些受體，導(dǎo)致特異性免疫細(xì)胞無(wú)法對(duì)其進(jìn)行識(shí)別與殺傷。此外，廣泛認(rèn)為是腸道微生物種群的差異影響腫瘤生長(zhǎng)，微生物能夠影響藥物治療效果[32, 33]。Sinkora等[34]發(fā)現(xiàn)回腸派爾淋巴結(jié)(IPP)是小腸內(nèi)B細(xì)胞優(yōu)先積累的區(qū)域，猜測(cè)B細(xì)胞的積累可能是為使細(xì)菌在無(wú)菌動(dòng)物上定植，而不是通過(guò)積累細(xì)菌定植而促進(jìn)B細(xì)胞增殖。Haverson等[15]讓GF豬分別定植血清型O83和O86的兩株大腸桿菌菌株20 d后，研究?jī)芍甏竽c桿菌對(duì)免疫結(jié)構(gòu)發(fā)育的影響，發(fā)現(xiàn)固有層的DC細(xì)胞、上皮組織和固有層的T細(xì)胞廣泛增加，最早遷移的細(xì)胞是單核細(xì)胞，T細(xì)胞遷移的速度稍慢。

2.4 食品科學(xué)及營(yíng)養(yǎng)學(xué)

無(wú)菌動(dòng)物模型適用于研究和評(píng)價(jià)功能性食品對(duì)腸道微生物群落代謝的影響，在功能性食物消化率、生物利用度等研究上提供更可靠的反饋[35]。研究表明，補(bǔ)充益生元或益生菌可有效改善腸道微生物紊亂造成的哮喘[36]、濕疹[37]、炎癥性疾病[38]、壞死性小腸結(jié)腸炎[39]和肥胖[40]等。Veiga等[41]研究發(fā)現(xiàn)益生菌產(chǎn)生大量的丁酸鹽，減少沃氏嗜膽菌(Bilophilawadsworthia)的數(shù)量，對(duì)腸道健康有益。Liu等[42]發(fā)現(xiàn)益生菌鼠李糖乳桿菌GG株(Lactobacillusrhamnosusstrain GG，LGG)對(duì)于治療輪狀病毒引起的回腸上皮損傷有效。營(yíng)養(yǎng)代謝方面，Thompson等[43]發(fā)現(xiàn)新霉素可以在不破壞腸黏膜的情況下，增加GF豬糞便中的中性固醇類物質(zhì)和脂肪酸的排泄。

2.5 疫苗及生物制劑

腸道菌群與免疫系統(tǒng)發(fā)育密切相關(guān)，因此無(wú)菌動(dòng)物是進(jìn)行疫苗和生物制劑評(píng)價(jià)的理想動(dòng)物模型。Jeong等[44]對(duì)宋內(nèi)志賀菌(Shigellasonnei)活菌疫苗的毒性進(jìn)行評(píng)估，發(fā)現(xiàn)初生GF豬對(duì)不同的口服志賀菌菌株毒性敏感。Foster[45]發(fā)現(xiàn)GF豬口服去毒的嬰兒沙門氏菌(Salmonellainfantis)突變體后，動(dòng)物斷奶前體重明顯升高，且能阻止鼠傷寒沙門氏菌(Salmonellatyphimurium)的感染。Wen等[46]研究了LGG的不同劑量對(duì)接種HRV疫苗免疫效果的影響，發(fā)現(xiàn)LGG接種6次到14次，每次濃度106～109CFU/mL之間時(shí)可誘導(dǎo)HRV特異性IFN-γ因子產(chǎn)生T細(xì)胞，而口服LGG也能阻止HRV感染引起的腸道微生物組成的改變[47]。此外，無(wú)菌動(dòng)物也已用于治療類風(fēng)濕性關(guān)節(jié)炎[48]、抗乙肝新途徑[49]等的研究。

3 未來(lái)的研究趨勢(shì)

3.1 腸道菌群相關(guān)的健康研究

腸道微生物的數(shù)量是宿主細(xì)胞數(shù)量的十倍以上，所含基因是宿主基因的數(shù)百倍，被稱為機(jī)體的最大免疫器官[50]。與宿主免疫、營(yíng)養(yǎng)和發(fā)病機(jī)理等健康相關(guān)研究息息相關(guān)[51]。Man等[52]利用化合物處理小鼠模擬結(jié)腸癌的發(fā)生，發(fā)現(xiàn)AIM2的缺失會(huì)導(dǎo)致小腸干細(xì)胞異常增殖，改變腸道菌群的組成，進(jìn)而導(dǎo)致結(jié)腸癌的發(fā)生。克利夫蘭診所研究人員首次在腸道中發(fā)現(xiàn)一種在動(dòng)物脂肪消化過(guò)程中產(chǎn)生的物質(zhì)—氧化三甲胺(TMAO)，它與動(dòng)脈粥樣硬化及心臟疾病的發(fā)生直接相關(guān)，靶向抑制TMAO作用腸道微生物可以幫助抑制某些心臟疾病的發(fā)生[53]。Suárez等[54]研究發(fā)現(xiàn)，無(wú)菌小鼠機(jī)體中存在高水平的特定細(xì)胞因子，而抑制這種特殊信號(hào)或許會(huì)損傷抗生素誘導(dǎo)的皮下脂肪褐變過(guò)程、抑制腸道菌群剔除小鼠的葡萄糖表型，有助于開(kāi)發(fā)治療肥胖等疾病的新型療法。還有眾多疾病如：壞死性結(jié)腸炎(NEC)[39]、炎癥性腸病(IBD)[38]，心臟疾病[53]、糖尿病[55]、肥胖[56]，帕金森[57]、兒童自閉癥[58]、風(fēng)濕性關(guān)節(jié)炎[59]和自身免疫疾病、癌癥發(fā)病機(jī)理[60]等均與腸道菌群相關(guān)。因此，可預(yù)見(jiàn)未來(lái)GF豬和悉生豬模型將在動(dòng)物健康養(yǎng)殖和人類健康研究中發(fā)揮重要作用。

3.2 人類器官的潛在供體

利用無(wú)菌豬作為自體器官體外培養(yǎng)工廠將是未來(lái)器官移植供體的重要研究方向。2010年Kobayashi等[61]通過(guò)種間囊胚注射多能干細(xì)胞，在胰腺缺陷型小鼠體內(nèi)形成胰腺器官，并能成功傳代表達(dá)。2013年Matsunari等[62]開(kāi)始利用體細(xì)胞克隆技術(shù)形成的囊胚導(dǎo)入腎臟或胰腺缺陷型豬上，獲得缺陷型器官并穩(wěn)定傳代。利用GF豬與上述器官缺陷豬技術(shù)相結(jié)合的技術(shù)，將病人組織、細(xì)胞或體外構(gòu)建的組織工程器官導(dǎo)入缺陷型GF豬，經(jīng)培養(yǎng)后移植，可有效解決器官移植供體不足的問(wèn)題，并有效降低移植的免疫排斥[63]。

3.3 微生物遺傳學(xué)

特殊微生物的存在或缺乏則對(duì)宿主的特征影響顯著，而無(wú)菌動(dòng)物則是研究微生物遺傳學(xué)的獨(dú)特模型。Bercik等[64]研究發(fā)現(xiàn)攜帶更富冒險(xiǎn)精神的小鼠腸道內(nèi)的微生物后，原來(lái)“相對(duì)害羞”的小鼠表現(xiàn)出更具探索性的行為，說(shuō)明腸道微生物能驅(qū)動(dòng)著宿主行為，并表現(xiàn)出明顯差異；Yano等[65]發(fā)現(xiàn)一個(gè)由20種產(chǎn)芽孢細(xì)菌組成的微生物組可以增加無(wú)菌小鼠機(jī)體血清素的產(chǎn)生水平，而這種外周血清素的水平和多種疾病的發(fā)生有關(guān)，如腸易激綜合征、心血管疾病及骨質(zhì)疏松癥；微生物可實(shí)現(xiàn)物種間的DNA轉(zhuǎn)移，并入宿主基因組中，Turnbaugh等[66]發(fā)現(xiàn)家庭成員有相似的腸道菌群結(jié)構(gòu)，同卵比異卵雙胞胎糞便菌群差異小，說(shuō)明微生物可實(shí)現(xiàn)相互轉(zhuǎn)移。利用GF豬進(jìn)行微生物遺傳學(xué)研究，將豐富傳統(tǒng)遺傳學(xué)的內(nèi)容，有利于促進(jìn)遺傳學(xué)中一些基本理論的闡明。

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Advances in research on germ-free pig models

DU Lei1,2, SUN Jing2,3,4, GE Liang-peng2,3,4*, LIU Zuo-hua2,3,4*

(1. Department of Animal Science, Rongchang Campus, Southwest University, Chongqing 402460,China；2. Chongqing Academy of Sciences, Chongqing 402460； 3.Key Laboratory of Pig Industry Science, Ministry of Agriculture,Chongqing 402460； 4. Chongqing Key Laboratory of Pig Industry Sciences, Chongqing 402460)

Germ-free (GF) pigs are a special and adaptable experimental animal model for biomedical studies and animal productions, which are negative for bacteria, viruses, yeast and fungi tested by current microbiological examination. GF pigs were initially used in cleanse of epidemic diseases in animal production and in a bid to study the relationship between animal disease and intestinal flora. Because of the similarities to humans in anatomy, physiology and hematology, and the clear microbiological background, GF pigs have been playing an important role in detecting the relationship between intestinal flora with growth and the development of diseases in medical biology, and also providing a special medical animal model for intestinal flora targeted prevention, diagnosis and treatment for update technology research in the clinic. This paper reviews the characteristics, advancements and research tendency of GF piglets.

Germ-free pigs; Animal Model; Research advancesCorresponding author: LIU Zuo-hua. E-mail: liuzuohua66@163.com; GE Liang-peng, E-mail: geliangpeng1982@163.com

國(guó)家“863”計(jì)劃(2014AA021602)；重慶市國(guó)際合作項(xiàng)目(CSTC2013gjhz80002)；重慶市基礎(chǔ)與前沿研究(cstc2013jcyjC80001)；重慶市農(nóng)發(fā)資金(12402)資助項(xiàng)目；無(wú)菌動(dòng)物應(yīng)用示范平臺(tái)(cstc2015pt-kjyfsf0024)。

杜蕾，碩士研究生，專業(yè)：動(dòng)物營(yíng)養(yǎng)與飼料科學(xué)，E-mail： dulei127899@163.com

劉作華，研究員，研究方向：地方豬資源保護(hù)與開(kāi)發(fā)利用，E-mail： liuzuohua66@163.com；葛良鵬，研究員，研究方向：動(dòng)物資源創(chuàng)新開(kāi)發(fā)利用。E-mail: geliangpeng1982@163.com

研究進(jìn)展

Q95-33

A

1005-4847(2016)05-0546-05

10.3969/j.issn.1005-4847.2016.05.020

2016-01-30