連晶瑤，丁苗慧，秦國(guó)慧，張 毅，王純耀*

(1.鄭州大學(xué)第一附屬醫(yī)院，鄭州 450052； 2.鄭州大學(xué)臨床醫(yī)學(xué)系，鄭州 450052； 3.鄭州大學(xué)，鄭州 450052)

研究進(jìn)展

免疫系統(tǒng)人源化小鼠模型的研究進(jìn)展

連晶瑤1,3，丁苗慧2，秦國(guó)慧1，張 毅1，王純耀1,3*

(1.鄭州大學(xué)第一附屬醫(yī)院，鄭州 450052； 2.鄭州大學(xué)臨床醫(yī)學(xué)系，鄭州 450052； 3.鄭州大學(xué)，鄭州 450052)

動(dòng)物模型是生物醫(yī)學(xué)科學(xué)研究中所建立的具有人類(lèi)模擬性表現(xiàn)的動(dòng)物材料，作為實(shí)驗(yàn)假說(shuō)和臨床假說(shuō)的實(shí)驗(yàn)基礎(chǔ)，可以縮短研究時(shí)間，觀察疾病的發(fā)生、發(fā)展或預(yù)防與治療的全過(guò)程，并可在人為控制條件下進(jìn)行各種實(shí)驗(yàn)研究，對(duì)各種疾病的相關(guān)機(jī)制研究有著重要意義。人類(lèi)的生物醫(yī)學(xué)研究主要受限于生物體復(fù)雜性，為了克服這個(gè)限制，基于可接受異種移植物的嚴(yán)重聯(lián)合免疫缺陷(SCID)或重組激活基因(Ragnull)無(wú)效小鼠的免疫缺陷特征開(kāi)發(fā)人源化小鼠模型，這些具有人體免疫力的小鼠模型已被廣泛用于研究人類(lèi)免疫生物學(xué)的基本原理以及人類(lèi)疾病的復(fù)雜病理的潛在機(jī)制。這種方法是促進(jìn)醫(yī)學(xué)科學(xué)發(fā)展的重要途徑之一，具有實(shí)用性和前瞻性。本文將對(duì)人源化小鼠模型的應(yīng)用及研究進(jìn)展進(jìn)行綜述。

動(dòng)物模型；人源化小鼠；治療；進(jìn)展

人源化小鼠應(yīng)用于研究人類(lèi)免疫細(xì)胞、人類(lèi)自身免疫性疾病、病毒感染、移植生物學(xué)和腫瘤生物學(xué)的發(fā)展和應(yīng)用。通過(guò)移植成熟的人類(lèi)免疫細(xì)胞、胎兒人類(lèi)胸腺、骨髓、肝組織等，構(gòu)建具有人體免疫力的小鼠模型。具有人源化免疫系統(tǒng)的動(dòng)物模型將顯著促進(jìn)我們對(duì)人類(lèi)免疫生物學(xué)和免疫相關(guān)疾病如自身免疫性疾病、病毒感染以及腫瘤和移植排斥的認(rèn)識(shí)。這些動(dòng)物模型是研究人淋巴細(xì)胞生物學(xué)和免疫應(yīng)答的重要研究工具[1,2]。現(xiàn)已經(jīng)有探究人源化小鼠人源細(xì)胞的檢測(cè)方法[3]。人源化小鼠由人類(lèi)細(xì)胞、組織、器官甚至人類(lèi)基因構(gòu)成的小鼠[4]，包括用人的肺、腎、胰腺、胃、肝、卵巢、子宮內(nèi)膜、神經(jīng)和皮膚組織移植的模型[5-8]。嚴(yán)重聯(lián)合免疫缺陷(SCID)或重組激活基因(Rag)小鼠缺乏T和B細(xì)胞，最初被用作重建人體免疫的接受者[9]。最近，越來(lái)越多的基因修飾的SCID或Rag小鼠模型被使用，包括SCID[10]，NOD/SCID[11]，Ragnull[12]和NOD/LtSz-Rag1nullPfpnull[13]等，這些小鼠是先天免疫缺陷的。人源化小鼠模型作為研究人類(lèi)疾病的活體模型，在闡明發(fā)病機(jī)制、藥物篩選等方面具有巨大的優(yōu)勢(shì)和廣泛的應(yīng)用前景[14]。本綜述主要介紹免疫系統(tǒng)人源化小鼠模型的現(xiàn)狀及研究進(jìn)展。

1 人源化小鼠模型的建立

1.1接種小鼠選擇用于移植異種人造血和免疫細(xì)胞

為了提高人類(lèi)免疫細(xì)胞或組織的移植效率，要求不同的條件治療方案和移植手段，包括宿主先天免疫細(xì)胞的清除以及植入成熟的人類(lèi)免疫細(xì)胞、胎兒胸腺、肝組織、骨髓和CD34+血液干細(xì)胞(HSC)[15]等的小鼠模型的構(gòu)建。SCID小鼠缺乏T細(xì)胞受體和免疫球蛋白基因的重排，導(dǎo)致T細(xì)胞和B細(xì)胞的缺失[16]，SCID小鼠中功能性T和B細(xì)胞的缺失有助于同種異體移植物或異種移植物的接受，沒(méi)有嚴(yán)重的排斥反應(yīng)[17]。Rag1或Rag2通過(guò)產(chǎn)生DNA雙鏈斷裂引發(fā)TCR和免疫球蛋白基因的VDJ重排，因此小鼠中的純合突變體導(dǎo)致不能產(chǎn)生成熟的T和B細(xì)胞，導(dǎo)致SCID樣表型[18]。具有Rag1和穿孔素基因的靶向突變的NOD小鼠，命名為NOD/LtSz-Rag1nullPfpnull小鼠，其缺乏成熟的T、B和NK細(xì)胞[19]?，F(xiàn)在越來(lái)越多的科學(xué)家來(lái)開(kāi)發(fā)具有更多缺陷先天免疫修飾的SCID小鼠或其他人源化小鼠模型。NOD/Shi-SCID小鼠也具有類(lèi)似于NOD/LtSz-SCID小鼠的嚴(yán)重免疫功能障礙[20]。NOD/LtSz-SCID或NOD/Shi-SCID小鼠的先天免疫缺陷可能很好地解釋了體內(nèi)人異種移植物存活增加的問(wèn)題[21]。CD47可以通過(guò)與巨噬細(xì)胞上表達(dá)的信號(hào)調(diào)節(jié)蛋白(SIRP)反應(yīng)，有效地保護(hù)靶細(xì)胞免受巨噬細(xì)胞吞噬作用[22]。

1.2敲除受體小鼠中的先天免疫細(xì)胞

SCID或Ragnull小鼠的先天免疫力成為限制人免疫細(xì)胞重建的主要因素。在受體小鼠中敲除NK細(xì)胞、單核細(xì)胞和巨噬細(xì)胞等某些亞群的方法可以改善人類(lèi)HSCs或免疫細(xì)胞移植[23,24]。CD122抗體或IL-2R抗體等用于敲除NK細(xì)胞，脂質(zhì)體包封的二氯亞甲基雙膦酸鹽敲除巨噬細(xì)胞[25]，這會(huì)嚴(yán)重影響小鼠嗜中性粒細(xì)胞[26]和粒細(xì)胞[27]，這可能控制SCID或Ragnull小鼠對(duì)人類(lèi)移植物的天然免疫應(yīng)答，并與人類(lèi)移植物的增加顯著相關(guān)。

1.3人類(lèi)生長(zhǎng)因子的影響

人類(lèi)免疫細(xì)胞在異種免疫缺陷型受體小鼠中的低移植效率可能通過(guò)提供人類(lèi)生長(zhǎng)因子而有所改善。在小鼠中，CD4和CD8單陽(yáng)性胸腺細(xì)胞的成熟需要TNF-α的誘導(dǎo)[28]。T細(xì)胞的發(fā)育、增殖和存活對(duì)上皮衍生的IL-7具有重要的依賴(lài)性[29]，人Fc-IL7融合蛋白的使用大大提高了NOD/SCID小鼠的脾臟和外周血中人T細(xì)胞的存活[30]。IL-15對(duì)造血功能有促進(jìn)作用，包括T細(xì)胞的增殖，B細(xì)胞的成熟和NK細(xì)胞的發(fā)育[31,32]。rhIL-15可以改善轉(zhuǎn)染人類(lèi)PBL后NOD/SCID小鼠人T細(xì)胞的移植和重建[33]。此外，IL-12或IL-18的使用增強(qiáng)了小鼠中人CD4+和CD8+T細(xì)胞的移植[34]。在用人PBL或骨髓細(xì)胞(BMC)接種的SCID小鼠中，注射重組人生長(zhǎng)激素(rhGH)或重組人催乳素(rhPRL)強(qiáng)烈促進(jìn)胸腺和脾臟中的人T細(xì)胞移植，IgG/M血清水平增強(qiáng)[35]。

1.4通過(guò)輻射或化學(xué)藥劑為供體細(xì)胞制造“空間”

盡管通過(guò)NK細(xì)胞或巨噬細(xì)胞的抗體進(jìn)行預(yù)處理可以清除NOD/SCID小鼠的殘留免疫力，但是可能需要亞致死輻射或化學(xué)試劑的制備方案來(lái)制造用于接種異種人類(lèi)HSCs或免疫細(xì)胞的“空間”。這些預(yù)處理可能導(dǎo)致生長(zhǎng)因子和化學(xué)引誘物濃度的增加，并為受體小鼠中人HSC和免疫細(xì)胞的發(fā)育和再輻射保留一定量的空間，一些免疫抑制劑和烷化劑已經(jīng)顯示出與輻射相似的效果。例如，與3.5 Gy照射相比，單次劑量(35 mg/kg)的Busilvex能夠進(jìn)行人類(lèi)細(xì)胞的等同移植[36]。通常，人類(lèi)HSC移植在NOD/SCID小鼠中需要2～3 Gy預(yù)輻射，并且人類(lèi)免疫細(xì)胞可以在受照射的受體中很好的生存[37]。

1.5直接植入成熟的人免疫細(xì)胞獲得人源化小鼠

通過(guò)用針對(duì)受體小鼠NK細(xì)胞、巨噬細(xì)胞或粒細(xì)胞的抗體處理，人免疫細(xì)胞的移植顯著改善[38]。與SCID小鼠相比，用人PBLs腹膜內(nèi)移植的Rag2null小鼠顯示有限的人類(lèi)移植率和較低水平的人免疫球蛋白[39]。600Gy照射可以提高Rag2null小鼠人類(lèi)植入的效率[40]。為了提供適當(dāng)?shù)奈h(huán)境，移植到SCID小鼠中，包含必需的細(xì)胞組分有T細(xì)胞、B細(xì)胞和抗原呈遞細(xì)胞(APC)。在這些小鼠中，人免疫缺陷病毒(HIV)可以正常復(fù)制[41]，分化成產(chǎn)生人IgM或IgG。分析抗原特異性細(xì)胞免疫應(yīng)答是非常困難的，人T和B細(xì)胞介導(dǎo)的這種異種反應(yīng)不僅引起了致命的移植物抗宿主病(GVHD)[42]，而且嚴(yán)重限制了人類(lèi)PBL對(duì)外源性抗原的反應(yīng)能力[43]。因此，這種方法在生物醫(yī)學(xué)研究中的應(yīng)用很有限。當(dāng)人類(lèi)臍帶血CD34+細(xì)胞注射到NOD/SCID胎兒中時(shí)，人類(lèi)免疫細(xì)胞在胎兒小鼠環(huán)境中不能有效地自我更新和分化[44]。

1.6移植人類(lèi)胸腺和HSC獲得人源化小鼠

建立人源化小鼠模型的另一個(gè)重要方法是在SCID小鼠的腎膠囊下移植胎兒人胸腺和肝組織，導(dǎo)致良好的血管胸腺樣器官[45]。觀察到來(lái)自胎兒人肝組織移植物的人類(lèi)HSCs遷移到胸腺移植物中[46]。這些人源化小鼠為體內(nèi)研究人體免疫功能提供了強(qiáng)大的模型。NOD/SCID或Rag2null小鼠沒(méi)有T和B細(xì)胞，沒(méi)有NK活性，并且缺失DC功能[47]，因此是比較好的小鼠模型。由于人類(lèi)抗小鼠異種免疫反應(yīng)引起的GVHD的高發(fā)生率或可能性，直接移植高劑量的成熟人類(lèi)T細(xì)胞和其他免疫細(xì)胞可能不是理想的選擇。因此，在免疫缺陷小鼠中植入胎兒人胸腺組織和HSC以重建人類(lèi)免疫可能是一種最佳方法。胎兒人胸腺或肝組織和CD34+HSCs的共移植策略可以維持人類(lèi)免疫細(xì)胞多譜系的發(fā)育，包括T細(xì)胞，B細(xì)胞和DC，提供更強(qiáng)大的適應(yīng)性和先天免疫力。

2 人源化小鼠模型在生物醫(yī)學(xué)研究中的應(yīng)用

人源化小鼠廣泛應(yīng)用于人類(lèi)HSCs的自我更新和多能分化能力的研究，以及人類(lèi)對(duì)病毒感染，腫瘤和移植的免疫力。人臍帶血(UCB)、骨髓和外周血被用作移植人HSCs的來(lái)源。從人UCB移植CD34+細(xì)胞與來(lái)自骨髓或外周血的CD34+細(xì)胞相比，NOD/SCID小鼠的移植水平更高[48]。人類(lèi)CD34+群體可以分為兩個(gè)獨(dú)特特征的亞群(CD34+CD38+和CD34+CD38-)。CD34+CD38+細(xì)胞早期重新產(chǎn)生，而CD34+CD38-細(xì)胞的增殖在晚期代表了更原始的群體和更高的T細(xì)胞前體[49]。人類(lèi)CD34+Lin-Thy-1+祖細(xì)胞的群體可以重新填充胸腺移植物。此外，人類(lèi)CD34+細(xì)胞或CD34+Lin-Thy-1+CD10+群體在SCID小鼠中產(chǎn)生T、B、NK和DC群體。當(dāng)將人CD34+細(xì)胞移植到NOD/SCID/IL2R無(wú)效小鼠中時(shí)，重構(gòu)免疫細(xì)胞的重組，包括人T細(xì)胞、B細(xì)胞、單核細(xì)胞、巨噬細(xì)胞和DC[50,51]。人源化小鼠中的人DC在發(fā)育，表現(xiàn)和功能上類(lèi)似于人類(lèi)發(fā)現(xiàn)的DC亞群[52]。自身免疫性疾病從具有器官特異性和多系統(tǒng)自身免疫疾病的患者獲得的人PBL在SCID小鼠中存活數(shù)月，并產(chǎn)生具有與供體相同特異性的IgG和自身抗體[53]。因此，這提供了一種可能的方法來(lái)研究人體自身免疫性疾病在體內(nèi)模型中的發(fā)病機(jī)制和效應(yīng)階段。這些結(jié)果表明人源化小鼠可以作為抗體介導(dǎo)的人自身免疫性皮膚病的模型。

2.1病毒感染

人造模型被用于研究病毒與人類(lèi)免疫系統(tǒng)之間的相互作用，并評(píng)估疫苗和治療劑對(duì)人類(lèi)病毒等的作用。艾滋病毒感染主要限于體外或臨床研究，人源化小鼠已廣泛用于研究HIV發(fā)病機(jī)制和體內(nèi)治療[54,55]。在接種人類(lèi)PBL的SCID小鼠中，HIV感染被限制在短時(shí)間內(nèi)，因?yàn)镃D4+T細(xì)胞迅速耗盡，缺乏補(bǔ)充來(lái)源[56]。在將未經(jīng)治療的HIV感染患者的PBLs誘導(dǎo)轉(zhuǎn)移到NOD/SCID小鼠后，觀察到強(qiáng)烈的HIV特異性抗體反應(yīng)[57]。在接種人胸腺和肝組織的人源化小鼠中，艾滋病毒感染對(duì)于一些胸腺CD3-CD4+CD8-T細(xì)胞的細(xì)胞呈現(xiàn)出先天性趨向[58]。在移植人類(lèi)CD34+細(xì)胞后，在NOD/SCID/IL-2小鼠的脾臟、骨髓或胸腺中檢測(cè)到感染CCR5和CXCR4-嗜性HIV-1分離物后的長(zhǎng)期病毒血癥。CXCR4-嗜性HIV病毒感染所有淋巴器官，而CCR5-嗜性HIV病毒感染主要限于胸外組織。兩種病毒株都導(dǎo)致人類(lèi)長(zhǎng)期淋巴器官傳播感染與HIV感染密切相似。已經(jīng)開(kāi)發(fā)了人源化小鼠模型，用于對(duì)HIV抗病毒化合物進(jìn)行臨床前評(píng)估，包括疊氮胸苷[59]，雙脫氧肌苷，雙脫氧胞嘧啶，奈韋拉平，蛋白酶抑制劑[60]。SARS病毒導(dǎo)致了亞洲的致命疫情[61]。人PBLs構(gòu)建的人源化小鼠被用于研究針對(duì)SARS-CoV的新型候選疫苗。SARS DNA疫苗誘導(dǎo)特異于SARS抗原的人細(xì)胞毒性T淋巴細(xì)胞和針對(duì)SARS-CoV的人中和抗體[62]，證明血管緊張素轉(zhuǎn)換酶2是SARS-CoV的功能受體[63]。對(duì)登革熱病毒發(fā)病機(jī)理和免疫力的理解的一個(gè)主要限制是缺乏理想的人源化動(dòng)物模型。用人臍帶血造血祖細(xì)胞或胎兒肝衍生的CD34+細(xì)胞移植的照射的NOD/SCID小鼠在生理環(huán)境中實(shí)現(xiàn)登革熱病毒感染的復(fù)制。發(fā)現(xiàn)這些模型易感染登革病毒感染，發(fā)現(xiàn)典型的發(fā)燒和血小板減少癥狀，并用于評(píng)估登革病毒的發(fā)病機(jī)制[64]。此外，人源化小鼠也已經(jīng)用于研究EBV[65]，巨細(xì)胞病毒(CMV)[66]和流感感染[67]的病理和治療等。

2.2對(duì)同種異體移植物或異種移植物的免疫應(yīng)答或耐受性

在接種人胸腺和肝組織或PBL的SCID小鼠中移植同種異體HLA錯(cuò)配的胎兒胰腺導(dǎo)致人單核細(xì)胞浸潤(rùn)胰腺和隨后的排斥反應(yīng)[68]。人類(lèi)T細(xì)胞對(duì)SCID小鼠中同種異體供體的皮膚移植物的排斥負(fù)責(zé)[69,70]。像人類(lèi)皮膚移植一樣，皮膚微血管被破壞，皮膚壞死[71]。人類(lèi)PBL移植的SCID小鼠被建立為延遲型超敏反應(yīng)(DTH)的模型[72]。從供體受體的腎移植受者分離出的適應(yīng)性CD4+CD25+Treg細(xì)胞介導(dǎo)人源化SCID小鼠中供體特異性DTH的抑制[73]。從供體A和來(lái)自供體B的胎兒胸腺移植人胎肝的SCID小鼠開(kāi)發(fā)了混合嵌合人胸腺[74]。在該模型中，與供體A反應(yīng)的人T細(xì)胞通過(guò)在胸腺中的選擇被克隆缺失，而與供體B的同種異體胸腺上皮細(xì)胞相互作用的T細(xì)胞可能對(duì)供體B有潛在的響應(yīng)[75]。人體小鼠模型用于研究體內(nèi)異種豬移植物的免疫應(yīng)答[76]。用胎兒豬胸腺和人肝組織移植的SCID小鼠中的豬胸腺移植物支持由人類(lèi)胎兒肝細(xì)胞提供的造血前體的多克隆功能性人T細(xì)胞的正常發(fā)育[77]。這些人類(lèi)T細(xì)胞對(duì)供體豬抗原具有特異性的耐受性，但對(duì)非供體豬異種抗原和同種異體抗原反應(yīng)正常。外源IL-2沒(méi)有消除耐受性，表明中樞克隆缺失而不是無(wú)反應(yīng)是可能的耐受機(jī)制[77]。最近，研究結(jié)果表明，人類(lèi)功能性CD4+CD25+Foxp3+Treg細(xì)胞可以在NOD/SCID小鼠的異種豬胸腺移植物中發(fā)育。嵌合體由人胸腺肝組織和豬HSC在SCID小鼠中構(gòu)建[78]。在這種嵌合體中發(fā)育的人類(lèi)T細(xì)胞顯示出對(duì)豬供體的特異性無(wú)反應(yīng)性，因?yàn)樗狈ωi血液細(xì)胞和抗供體豬反應(yīng)的排斥，以及供體豬白細(xì)胞抗原(SLA)匹配皮膚的接受移植物，而混合嵌合小鼠中的人T細(xì)胞拒絕了第三方豬皮膚移植物，并測(cè)定對(duì)第三方豬和同種異體人類(lèi)抗原作出反應(yīng)[78]。造血嵌合體誘導(dǎo)供體細(xì)胞T細(xì)胞耐受性的能力主要是由成熟供體反應(yīng)性胸腺細(xì)胞的胸腺內(nèi)克隆缺失引起的[79]。

2.3抗腫瘤免疫反應(yīng)

惡性腫瘤可以無(wú)限制地生長(zhǎng)，逃避人類(lèi)免疫監(jiān)視。SCID或Rag小鼠可以成功地植入異種人類(lèi)腫瘤，包括各種各樣的實(shí)體人類(lèi)腫瘤和血液腫瘤[80]，其中人類(lèi)腫瘤生物學(xué)、生長(zhǎng)、血管發(fā)生和轉(zhuǎn)移已被評(píng)估。在免疫缺陷小鼠中成功移植人類(lèi)腫瘤和人類(lèi)免疫細(xì)胞的能力已經(jīng)促成了人源化小鼠模型的開(kāi)發(fā)和使用，以評(píng)估抗腫瘤治療。事實(shí)上，理想的模型是具有完整人類(lèi)免疫系統(tǒng)的人源化小鼠，這將允許在完整的人免疫微環(huán)境的背景下評(píng)估腫瘤免疫生物學(xué)的機(jī)制。人源化小鼠可用于評(píng)估抑制人腫瘤生長(zhǎng)的治療方法，包括使用血管生成抑制劑[81]、基于細(xì)胞的療法[82]、人源化抗體[83]、傳統(tǒng)的免疫抑制和免疫治療方案[84]和腫瘤生長(zhǎng)抑制劑[85]。人源化小鼠提供了評(píng)估人細(xì)胞因子和趨化因子的機(jī)會(huì)，其增強(qiáng)人白細(xì)胞的先天和適應(yīng)性抗腫瘤免疫應(yīng)答，從而提供臨床相關(guān)的模型。

3 展望

人源化小鼠模型的建立為疾病的病因?qū)W、發(fā)展過(guò)程和治療研究提供了極大的幫助,尤其是近來(lái)分子生物學(xué)技術(shù)的提高及在人源化小鼠模型中的應(yīng)用,為各種疾病有關(guān)基因研究提供了依據(jù),為進(jìn)一步研究疾病發(fā)生機(jī)制及靶向治療提供了廣闊的前景。隨著研究的深入以及各種技術(shù)的進(jìn)步,人們將建立更為完善的人源化小鼠模型,在疾病研究及治療方面取得更大的突破。

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Researchprogressofhumanizedmousemodelsinimmunesystem

LIAN Jing-yao1,3， DING Miao-hui2， QIN Guo-hui1， ZHANG yi1， WANG Chun-yao1,3*

(1.The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China； 2.Department of Clinical Medicine, Zhengzhou University, Zhengzhou 450052; 3.College of Life Science, Zhengzhou University, Zhengzhou 450052))

Animal model is an animal material with human mimic performance established in biomedical scientific research. It can be used as experimental basis for studies of experimental hypothesis and clinical hypothesis. It can shorten the research time and observe the whole process of disease occurrence, development or prevention and treatment.Human biomedical research is largely limited by the biological complexity. In order to overcome this limitation, based on the immunosuppressive characteristics of a severely immunodeficient (SCID) or recombinant activated gene (Ragnull) in mice, humanized mouse models of human diseases can be established and have been widely used to study the underlying principles of human immunobiology and complex pathological mechanisms of human diseases. This approach has become one of the important ways to promote the development of medical sciences, with practicality and foresight. In this paper, the application and research progress of humanized mouse models are reviewed.

Humanized mouse models; Human diseases

R-33

A

1671-7856(2017) 10-0113-07

10.3969.j.issn.1671-7856. 2017.10.022

2017-04-16

連晶瑤(1990-)女，碩士生，研究方向：腫瘤免疫。E-mail: jingyao725@163.com

王純耀(1962-)男，研究方向：實(shí)驗(yàn)動(dòng)物相關(guān)研究。E-mail: chunyao@zzu.edu.cn