關(guān)向前，劉毛妮

(1安徽省立醫(yī)院，合肥 230001；2安徽中醫(yī)藥大學(xué)第一附屬醫(yī)院)

受體CX3CR1基因敲除對(duì)同種異系小鼠氣管移植術(shù)后排斥反應(yīng)的影響

關(guān)向前1，劉毛妮2

(1安徽省立醫(yī)院，合肥 230001；2安徽中醫(yī)藥大學(xué)第一附屬醫(yī)院)

目的探討趨化因子受體CX3CR1基因敲除對(duì)同種異系小鼠氣管移植后排斥反應(yīng)的影響。方法將24只小鼠分為同系移植對(duì)照組、異系移植對(duì)照組、基因敲除移植組，每組各4對(duì)?；蚯贸浦步M、同系移植對(duì)照組受體和供體均為BABL/c小鼠；異系移植對(duì)照組供體為BABL/c小鼠，受體為C57BL/6小鼠；基因敲除移植組供體為BABL/c小鼠，受體為CX3CR1基因敲除小鼠。各組均行頸背部氣管移植，每只受鼠移植2段氣管。4周后取出各受鼠移植氣管段，石蠟包埋，切片，HE染色，鏡下觀察管腔纖維化阻塞情況；采用ELISA法檢測(cè)受鼠血清高遷移率族蛋白1(HMGB1)水平。結(jié)果同系移植對(duì)照組8個(gè)移植氣管段均無(wú)管腔阻塞，阻塞分?jǐn)?shù)為0；異系移植對(duì)照8個(gè)移植氣管段均出現(xiàn)管腔阻塞，移植氣管管腔內(nèi)充滿(mǎn)黏液，鏡下見(jiàn)管腔內(nèi)有大量增生的成纖維細(xì)胞，阻塞分?jǐn)?shù)為8.0；受體敲除移植組8個(gè)移植氣管段中1個(gè)出現(xiàn)管腔受擠壓、扭曲，1個(gè)阻塞不明顯，其余6個(gè)均未出現(xiàn)較明顯的管腔纖維化阻塞，移植氣管管腔內(nèi)充滿(mǎn)黏液，鏡下見(jiàn)管腔內(nèi)有大量增生的成纖維細(xì)胞，阻塞分?jǐn)?shù)為0.96。移植后4周同系移植對(duì)照組、異系移植對(duì)照組、基因敲除移植組血清HMGB1水平分別為(29.8±1.6)、(83.6±1.4)、(19.8±0.9)ng/mL，基因敲除移植組明顯低于異系移植對(duì)照組(P<0.05)，但與同系移植對(duì)照組比較，P>0.05。結(jié)論受體CX3CR1基因敲除可明顯減輕氣管移植后排斥反應(yīng)。

CX3CR1；基因敲除；氣管移植；排斥反應(yīng)；小鼠

肺移植是終末期肺疾病最有效的治療方式，但目前其移植生存率明顯低于其他實(shí)質(zhì)器官移植[1]。肺移植后排斥反應(yīng)主要是以氣管為靶點(diǎn)的宿主抗移植物免疫排斥反應(yīng)。氣管排斥反應(yīng)主要表現(xiàn)為氣道炎癥、氣道重塑以及氣道上皮細(xì)胞向間充質(zhì)細(xì)胞的轉(zhuǎn)化，氣道上皮細(xì)胞主要轉(zhuǎn)化為成纖維細(xì)胞；成纖維細(xì)胞進(jìn)一步增殖，并分泌大量彈性纖維，阻塞氣管腔，導(dǎo)致閉塞性細(xì)支氣管炎(OB)[2]。OB是影響肺移植受者長(zhǎng)期存活的首要因素[3,4]，抑制OB自然成為提高肺移植存活率的有效方法[5～7]。CX3CR1是趨化因子受體，主要在巨噬細(xì)胞、單核細(xì)胞、中性粒細(xì)胞和T淋巴細(xì)胞表達(dá)。CX3CR1及其配體被證實(shí)具有調(diào)控炎性細(xì)胞向炎癥部位遷移和招募的作用[8]，同時(shí)CX3CR1具有調(diào)節(jié)單核細(xì)胞分化、增殖的作用，且在腫瘤轉(zhuǎn)移及肝臟纖維化形成中有重要的促進(jìn)作用[9～11]。上述提示CX3CR1可能參與了移植后OB的發(fā)生過(guò)程。為驗(yàn)證這一推測(cè)，我們觀察了趨化因子受體CX3CR1基因敲除對(duì)同種異系小鼠氣管移植后排斥反應(yīng)的影響。現(xiàn)報(bào)告如下。

1 材料與方法

1.1 材料 實(shí)驗(yàn)動(dòng)物：24只小鼠均以常規(guī)條件飼養(yǎng)于安徽醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心SPF級(jí)動(dòng)物房。純系BALB/c小鼠和C57BL/6小鼠由華中科技大學(xué)同濟(jì)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供，許可證號(hào)：SCXK(鄂)2004-0007。CX3CR1-基因敲除的C57BL/6小鼠由美國(guó)Jackon實(shí)驗(yàn)室提供。

1.2 頸背部氣管移植 將24只小鼠分為三組，每組各4對(duì)。同系移植對(duì)照組受體和供體均為BABL/c小鼠；異系移植對(duì)照組供體為BABL/c小鼠，受體為C57BL/6小鼠；基因敲除移植組供體為BABL/c小鼠，受體為CX3CR1基因敲除小鼠。三組均行頸背部氣管移植。手術(shù)前將供鼠脫頸處死，放入裝有 75%乙醇的燒杯中 15 min，杯口密封。取出 8 個(gè)軟骨環(huán)長(zhǎng)度的氣管，放入盛有生理鹽水的培養(yǎng)皿中，用 1 mL注射器輕輕沖洗管腔2～3次。受鼠麻醉后，剃去受鼠頸背部毛發(fā)，碘酒消毒皮膚，然后在頸背部?jī)蓚?cè)用消過(guò)毒的眼科剪剪出兩個(gè) 2～3 mm裂口(將皮剪破即可，不要剪過(guò)深)，并沿前爪方向在皮下捅出一條皮下遂道(注意保持皮膚的完整性)。將從供鼠取下的氣管段用消過(guò)毒的彎鑷放入皮下遂道底部，縫合裂口(手術(shù)過(guò)程在超凈臺(tái)內(nèi)進(jìn)行)。手術(shù)后，將受鼠放入清潔級(jí)動(dòng)物房飼養(yǎng)，每 2 天觀察受鼠的精神狀態(tài)和手術(shù)部位感染及毛發(fā)恢復(fù)情況，并適當(dāng)添加繁殖飼料，更換水。

1.3 相關(guān)指標(biāo)觀察

1.3.1 移植氣管阻塞程度 各組均于移植4周取出各移植氣管，石蠟包埋，切片，HE染色，光鏡下觀察氣管腔纖維化閉塞情況，計(jì)算各移植氣管阻塞分?jǐn)?shù)。移植氣管段阻塞分?jǐn)?shù)=管腔阻塞面積/管腔總面積，完全阻塞則阻塞分?jǐn)?shù)為1.0，完全不阻塞則阻塞分?jǐn)?shù)為0.0。各組移植氣管阻塞程度為該組各個(gè)移植氣管段阻塞分?jǐn)?shù)之和。

1.3.2 血清高遷移率族蛋白1(HMGB1)水平 移植4周抽取各組靜脈血，分離血清，采用ELISA法檢測(cè)HMGB1，操作按試劑盒說(shuō)明書(shū)進(jìn)行。

2 結(jié)果

2.1 各組移植氣管阻塞情況比較 同系移植對(duì)照組8個(gè)移植氣管段均無(wú)管腔阻塞，阻塞分?jǐn)?shù)為0；異系移植對(duì)照組8個(gè)移植氣管段均出現(xiàn)管腔阻塞，移植氣管管腔內(nèi)充滿(mǎn)黏液，鏡下見(jiàn)管腔內(nèi)有大量增殖的成纖維細(xì)胞，阻塞分?jǐn)?shù)為8.0；基因敲除移植組8個(gè)移植氣管段中1個(gè)出現(xiàn)管腔受擠壓、扭曲，1個(gè)阻塞不明顯，其余6個(gè)均未出現(xiàn)較明顯的管腔纖維化阻塞，移植氣管管腔內(nèi)充滿(mǎn)黏液，鏡下見(jiàn)管腔內(nèi)有大量增生的成纖維細(xì)胞，阻塞分?jǐn)?shù)為0.96。

2.2 各組血清HMGB1水平比較 移植后4周基因敲除移植組血清HMGB1水平分別為(29.8±1.6)、(83.6±1.4)、(19.8±0.9)ng/mL?；蚯贸浦步M血清HMGB1水平低于異系移植對(duì)照組(P<0.05)，但與同系移植對(duì)照組比較P>0.05。

3 討論

器官移植排斥反應(yīng)的本質(zhì)是受體免疫系統(tǒng)針對(duì)來(lái)自供體的移植物的攻擊反應(yīng)及免疫應(yīng)答[12]。氣管移植后發(fā)生的OB是免疫和/非免疫因素所造成的細(xì)小支氣管炎癥反應(yīng)性損傷及異常修復(fù)過(guò)程，多數(shù)學(xué)者認(rèn)為免疫排斥反應(yīng)是其首要原因[1]。氣管移植排斥的主要靶器官是氣道上皮，即受者針對(duì)移植物氣管上皮發(fā)生相應(yīng)的應(yīng)答反應(yīng)。 OB發(fā)生后氣道上皮膠原沉積和廣泛的纖維組織增生，最終阻塞氣管[13～15]。

OB的發(fā)展進(jìn)程從時(shí)間上可以分為上皮損傷期、炎癥浸潤(rùn)期和纖維化增生期[16,17]。炎癥浸潤(rùn)期屬于過(guò)渡階段，是由于上皮損傷激活固有免疫系統(tǒng)，引起巨噬細(xì)胞、中性粒細(xì)胞等炎性細(xì)胞向特定部位遷移，并分泌、釋放多種炎性因子，促使上皮細(xì)胞間充質(zhì)轉(zhuǎn)化的發(fā)生，為纖維化阻塞的形成奠定基礎(chǔ)[18～20]。

目前氣管移植動(dòng)物模型主要分為原位移植和異位移植。本研究我們建立了小鼠頸背部氣管移植模型，屬于異位移植，此模型穩(wěn)定，操作方便，感染率低。實(shí)驗(yàn)中我們選擇趨化因子受體CX3CR1作為研究靶標(biāo)，試圖通過(guò)在炎癥浸潤(rùn)期干擾炎性細(xì)胞的遷移和浸潤(rùn)來(lái)抑制移植后排斥反應(yīng)。本研究結(jié)果顯示，基因敲除移植組和同系對(duì)照組氣管阻塞情況明顯好于異系對(duì)照組?；蚯贸浦步M和同系對(duì)照組血清HMGB1水平明顯低于異系對(duì)照組。表明受體CX3CR1基因敲除可明顯減輕氣管移植后排斥反應(yīng)。對(duì)此結(jié)果，我們初步的解釋是：CX3CR1基因敲除主要影響了炎癥浸潤(rùn)期巨噬細(xì)胞、中性粒細(xì)胞等炎性細(xì)胞的遷移，從而抑制整個(gè)OB的發(fā)展進(jìn)程。其具體機(jī)制和信號(hào)通路尚有待更進(jìn)一步、更深入的研究。

HMGB1是一種核DNA結(jié)合蛋白，被稱(chēng)為“DNA伴侶”，參與DNA的復(fù)制、轉(zhuǎn)錄及修飾過(guò)程。在細(xì)胞處于炎癥、感染、損傷情況下，HMGB1即從細(xì)胞內(nèi)主動(dòng)釋放至細(xì)胞外，參與細(xì)胞(或機(jī)體)應(yīng)激過(guò)程，如免疫應(yīng)答或移植排斥，故亦被稱(chēng)為損傷式分子蛋白(DAMP)。據(jù)文獻(xiàn)報(bào)道，在同種小鼠氣管移植排斥過(guò)程中，HMGB1分子亦從氣管上皮細(xì)胞內(nèi)轉(zhuǎn)移至細(xì)胞外，通過(guò)RAGE/NF-κB/TGF-β信號(hào)途徑參與移植后閉塞性氣管炎的發(fā)生發(fā)展，其在移植受體血清中的表達(dá)水平也是明顯升高的[21]。本研究采用受鼠血清HMGB1水平來(lái)間接反應(yīng)移植排斥情況，結(jié)果發(fā)現(xiàn)與病理檢測(cè)結(jié)果一致。但HMGB1與CX3CR1在促進(jìn)氣管移植排斥過(guò)程中的內(nèi)在聯(lián)系在本研究中未得以揭示，尚需要更深入的研究。

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EffectofCX3CR1geneknockoutonrejectionofallogeneicmiceaftertrachealtransplantation

GUANXiangqian1,LIUMaoni

(1AnhuiProvincialHospital,Hefei230001,China)

ObjectiveTo investigate the effect of chemokine receptor CX3CR1 gene knockout on rejection of allogeneic mice after tracheal transplantation.MethodsTwenty-four mice were divided into three groups: homologous control group, allogenic control group, and gene knockout transplantation group with 4 pairs in each. In the homologous control group, all donor mice and recipient mice were BALB/c mice. In the allogenic control group, donor mice were BALB/c mice, and recipient mice were C57BL/6 male mice. In the gene knockout transplantation group, donor mice were BALB/c mice, and recipient mice were CX3CR1 knockout mice (C57BL/6 background). Trancheal transplantation operation was performed in all groups, and each recipient mouse was transplanted with 2 tracheas. At week 4 after transplantation, the tracheal grafts were removed and the pathological change was detected with HE staining. Meanwhile, the serum high-mobility group box 1 protein (HMGB1) levels in the recipients of each group were detected with ELISA.ResultsHE staining showed that there was no fibrosis in the tracheal grafts from the homologous control group, and the blocking score was 0. All tracheal grafts from the allogenic control group had fibrosis, and the blocking score was 8.0. In the gene knockout transplantation group, 1 tracheal graft had fibrosis, and the blocking score was only 0.96. At week 4 after transplantation, the HMGB1 levels in the homologous control group, allogenic control group, and gene knockout transplantation group were (29.8±1.6), (83.6±1.4), and (19.8±0.9) ng/mL, and the HMGB1 level was lower in the gene knockout transplantation group than in the allogenic control group (P<0.05), but no significantly difference was found between the homologous control group and the gene knockout transplantation group (P>0.05).ConclusionReceptor CX3CR1 knockout can significantly reduce the rejection after tracheal transplantation.

CX3CR1; gene knockout; trachea transplantation; rejection; mice

國(guó)家自然科學(xué)基金資助項(xiàng)目(30972779)。

關(guān)向前(1983-)，男，碩士，主要研究方向：移植免疫學(xué)和細(xì)胞免疫學(xué)。E-mail: gxq466216@163.com

10.3969/j.issn.1002-266X.2017.40.007

R617

A

1002-266X(2017)40-0026-03

2016-09-04)