賀繼剛 李貝貝 韓金秀 李宏遠(yuǎn) 嚴(yán) 丹

(云南省第一人民醫(yī)院心臟大血管外科,昆明 650032)

過(guò)表達(dá)IDO大鼠骨髓間充質(zhì)干細(xì)胞改善異位移植心臟存活①

賀繼剛 李貝貝 韓金秀 李宏遠(yuǎn) 嚴(yán) 丹②

(云南省第一人民醫(yī)院心臟大血管外科,昆明 650032)

目的探討過(guò)表達(dá)IDO大鼠骨髓間充質(zhì)干細(xì)胞(BMSC)改善大鼠腹腔異位移植心臟的存活機(jī)制。方法通過(guò)慢病毒載體GV308攜帶IDO基因轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞構(gòu)建過(guò)表達(dá)IDO大鼠骨髓間充質(zhì)干細(xì)胞。建立大鼠腹腔異位移植心臟模型，經(jīng)尾靜脈給予相應(yīng)細(xì)胞處理：①利用超聲心動(dòng)圖檢測(cè)移植心臟心功能變化。②采用小動(dòng)物活體成像系統(tǒng)評(píng)估移植心臟局部熒光強(qiáng)度。③再取各組受體大鼠的脾臟，采用流式細(xì)胞技術(shù)檢測(cè)CD40、CD86、CD80、MHCⅡ、CD274、CD45RA、CD45RA+CD45RB、Treg細(xì)胞的表達(dá)情況。④取各組移植心臟，采用HE染色評(píng)估炎性細(xì)胞浸潤(rùn)情況。⑤利用液相芯片檢測(cè)各組血清IL-1ɑ、IL-4、IL-1β、IL-2、IL-10、IFN-γ、IL-18、TGFβ1、TGFβ2、TGFβ3因子的變化情況。結(jié)果①給予相應(yīng)細(xì)胞處理，可見(jiàn)過(guò)表達(dá)IDO-BMSCs處理后2 d移植心臟的EF、FS較其余各組有提高。②采用小動(dòng)物活體評(píng)估可見(jiàn)過(guò)表達(dá)IDO-BMSCs組移植心臟局部熒光強(qiáng)度最強(qiáng)。③給予干預(yù)措施后2 d可見(jiàn)過(guò)表達(dá)IDO-BMSCs組脾臟細(xì)胞CD40、CD86、CD80、MHCⅡ、CD45RA、CD45RA+CD45RB表達(dá)降低，而CD274、Treg細(xì)胞的表達(dá)增高。④采用液相芯片檢測(cè)各組提取血清可見(jiàn)在2 d時(shí)，過(guò)表達(dá)IDO-BMSCs組血清中IL-1α、IL-4、IL-1β、IL-2、IFN-γ、IL-18是降低的；IL-10、TGFβ1、TGFβ2、TGFβ3表達(dá)量是升高的。HE染色證實(shí)過(guò)表達(dá)IDO-BMSCs組炎性細(xì)胞浸潤(rùn)少于其他組。結(jié)論過(guò)表達(dá)IDO的BMSCs可以通過(guò)有效調(diào)節(jié)免疫DC、T細(xì)胞及細(xì)胞因子，從多個(gè)免疫層面改善移植心臟存活。

IDO；大鼠骨髓間充質(zhì)干細(xì)胞；免疫抑制

20世紀(jì)以來(lái)，由于器官移植技術(shù)、移植免疫基礎(chǔ)研究以及各種免疫抑制劑的進(jìn)展，器官移植已成為治療器官功能衰竭的有效治療手段。到2000年底，全球統(tǒng)計(jì)，共施行近80萬(wàn)例次各種器官移植。但不可否認(rèn)目前器官移植已經(jīng)進(jìn)入了一個(gè)尷尬的境地。一方面是隨著醫(yī)療生活水平改善，越來(lái)越多的器官終末期患者期盼器官移植。另一方面由于器官來(lái)源緊張及移植后的免疫排斥反應(yīng)或免疫抑制藥物嚴(yán)重副作用使患者得不到有效治療、治療效果不佳、生活質(zhì)量無(wú)法改善。而供體來(lái)源的緊張短期內(nèi)無(wú)法有效緩解。本研究正是基于上述社會(huì)背景下，結(jié)合骨髓間充質(zhì)干細(xì)胞免疫抑制調(diào)節(jié)特性聯(lián)合吲哚胺2,3-雙加氧酶(IDO)，試圖尋找到一種有效改善心臟移植存活的生物免疫抑制方法。

1 材料與方法

1.1材料

1.1.1實(shí)驗(yàn)動(dòng)物 健康4周齡SPF級(jí)SD大鼠及Wistar大鼠各60只，均為雄性，由成都達(dá)碩實(shí)驗(yàn)動(dòng)物中心提供，動(dòng)物許可證號(hào)為SCXK(川)2015-030。所有動(dòng)物的喂養(yǎng)、觀察均按照GLP(非臨床研究管理規(guī)范)規(guī)定執(zhí)行。

1.1.2主要試劑和儀器 TGFBMAG-64K-03試劑盒及RECYTMAG-65K-07試劑盒購(gòu)自美國(guó)Merck millipore。 CD40-FITC、CD80-PE、CD86-PE、MH-CLASS Ⅱ-FITC、CD45RA-APC、CD3-FITC、CD4-APC、CD8a-PE、CD4-FITC、CD25-APC、FOXP3-PE、Foxp3 Staining Buffer 均購(gòu)自eBioscience(美國(guó))；CD29、CD44、CD90、CD73、CD45、CD11b、CD34均購(gòu)自BioLegend(美國(guó))；CD274-FITC購(gòu)自Biorbyt(美國(guó))；CD45RA+CD45RB-FITC購(gòu)自Thermo(美國(guó))；LS-C62680(ox62)FITC購(gòu)自LSBio(美國(guó))；流式細(xì)胞儀BD Accuri C6 購(gòu)自美國(guó)BD公司；MAGPIX檢測(cè)儀購(gòu)自美國(guó)Luminex公司；小動(dòng)物活體成像儀購(gòu)自美國(guó)Caliper IVIS Lumina LT公司；心臟彩超儀購(gòu)自美國(guó)PHILIPS EPIQ 7C。

1.2方法

1.2.1大鼠BMSC的分離、培養(yǎng)及鑒定 全骨髓培養(yǎng)大鼠BMSC，胰酶消化傳代至第7代。取生長(zhǎng)至P7代BMSCs，待細(xì)胞融合達(dá)80%～90%時(shí)。制備單細(xì)胞懸液，采用流式細(xì)胞術(shù)檢測(cè)CD29、CD44、CD90、CD73、CD45、CD11b、CD34( CD29單抗5 μl做1∶ 20稀釋至100 μl，CD44單抗5 μl做1∶ 40稀釋至100 μl，CD90 5 μl作 1∶ 20稀釋至100 μl，CD73 5 μl 作1∶ 20稀釋至100 μl，CD45、CD11b、CD34單抗5 μl做1∶40稀釋至100 μl)。

1.2.2慢病毒載體及基因開(kāi)啟技術(shù)構(gòu)建過(guò)表達(dá)IDO大鼠骨髓間充質(zhì)干細(xì)胞 ①將已構(gòu)建成功的IDO基因插入慢病毒包裝質(zhì)粒GV308中，構(gòu)建GV308-IDO重組慢病毒包裝質(zhì)粒。②將構(gòu)建成功的GV308-IDO重組慢病毒包裝質(zhì)粒轉(zhuǎn)染入大鼠骨髓間充質(zhì)干細(xì)胞并加入基因開(kāi)啟劑DOX(強(qiáng)力霉素)。③轉(zhuǎn)染成功后利用Q-PCR方法檢測(cè)轉(zhuǎn)染48 h IDO基因量的表達(dá)。

1.2.3大鼠腹腔異位移植心臟模型建立 ①取供體(Wistar大鼠)心臟開(kāi)腹后將50 U/L的4℃冰肝素鹽水5 ml經(jīng)下腔靜脈注射，2 min后剪開(kāi)腹主動(dòng)脈及下腔靜脈充分放血，剪開(kāi)胸腔放入鹽水冰削，結(jié)扎下腔、左右上腔后剪斷升主動(dòng)脈，游離肺動(dòng)脈后剪斷肺動(dòng)脈，結(jié)扎心臟后的肺靜脈。取下心臟。②供體心臟吻合到受體(SD大鼠)腹腔主動(dòng)脈及下腔靜脈,將受體開(kāi)腹后，分離腹主動(dòng)脈及下腔靜脈，套3-0絲線，阻斷下腔靜脈及腹主動(dòng)脈，剪開(kāi)腹主動(dòng)脈及下腔靜脈，采用連續(xù)單點(diǎn)吻合方法將供體心臟的主動(dòng)脈及肺動(dòng)脈與受體心臟的腹主動(dòng)脈及下腔靜脈吻合。

1.2.4采用心臟彩超評(píng)估移植心臟心功能變化和采用小動(dòng)物活體成像評(píng)估移植心臟熒光強(qiáng)度 收集過(guò)表達(dá)IDO-BMSCs、空載體-BMSCs、BMSCs細(xì)胞懸液。每106細(xì)胞中加入5 μl Dir(2.5 mmol/L)。在37℃培養(yǎng)40 min。利用PBS洗脫3次后應(yīng)用。經(jīng)尾靜脈注射到大鼠腹腔異位移植心臟術(shù)后3 d的模型中，每只400 μl，106個(gè)細(xì)胞。并將建模未處理組及正常大鼠給予喂養(yǎng)DOX(0.25 mg/L)作為對(duì)照組1及對(duì)照組3。另外一組建模未處理的大鼠按40 mg/kg的標(biāo)準(zhǔn)喂養(yǎng)嗎替麥考，作為對(duì)照組2。注射后分別喂養(yǎng)2 d，進(jìn)行心臟彩超評(píng)估移植心臟心功能和小動(dòng)物活體成像。

1.2.5體內(nèi)流式檢測(cè) ①完成小動(dòng)物活體成像后，取其大鼠脾臟組織，制備成單細(xì)胞懸液。取100 μl細(xì)胞懸液分裝到1.5 ml EP管中，加相應(yīng)抗體，避光孵育15 min。每支100 μl的細(xì)胞懸液加相應(yīng)抗體量為CD40 0.5 mg/ml 1 μl、 CD86 0.2 mg/ml 0.8 μl、CD80 0.2 mg/ml 1.4 μl、 MHCⅡ 0.5 mg/ml 2 μl、CD274 0.5 mg/ml 1 μl、CD45RA 0.2 mg/ml 0.5 μl、CD45RA+CD45RB 0.1 mg/ml 1.4 μl、OX62 0.1 mg/ml 8 μl、CD3 0.5 mg/ml 0.5 μl、CD4 0.2 mg/ml 1 μl、CD8 0.2 mg/ml 0.5 μl。②Treg細(xì)胞染色步驟：取100 μl細(xì)胞懸液到離心管中，加細(xì)胞表面抗原CD4 0.5 mg/ml 0.5 μl和CD25 0.2 mg/ml 0.5 μl，避光孵育20 min。加1 ml Foxp3 Fixation/Permeabilization工作液，渦旋，室溫避光孵育40 min。加2 ml 1×Permeabilization緩沖液，1 000 r/min離心5 min，棄上清。用100 μl Permeabilization緩沖液重懸細(xì)胞，1 000 r/min離心5 min棄上清使剩余體積約為100 μl。加3 μl細(xì)胞內(nèi)染色抗體0.2 mg/ml Foxp3，室溫避光孵育40 min。加 2 ml 1×Permeabilization緩沖液，1 000 r/min離心5 min，棄上清。加300 μl PBS重懸細(xì)胞后，采用BD Accuri C6流式細(xì)胞儀進(jìn)行檢測(cè)。

1.2.6液相檢測(cè) 待小動(dòng)物活體成像后，取其新鮮全血，制備成血清用于液相檢測(cè)，按RECYTMAG-65K-07試劑盒及TGFBMAG-64K-03試劑盒檢測(cè)說(shuō)明書(shū)完成細(xì)胞因子濃度監(jiān)測(cè)。

1.2.7心臟石蠟切片與HE染色 將心臟組織切成約1 cm×1 cm×1 cm放入包埋盒中，固定心臟組織并進(jìn)行脫水，浸蠟，包埋，連續(xù)切片，貼片，烤片。切片進(jìn)行常規(guī)脫蠟、水化；伊紅染色15 s；中性樹(shù)膠封片，顯微鏡下觀察顯色結(jié)果并拍照。

2 結(jié)果

2.1大鼠BMSC流式細(xì)胞儀鑒定結(jié)果 結(jié)果顯示超過(guò)90%大鼠骨髓間充質(zhì)干細(xì)胞表達(dá)CD29、CD44、CD90、CD73。但不表達(dá)CD45、CD11b、CD34。

2.2慢病毒轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞并加入DOX(強(qiáng)力霉素) 慢病毒轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞加入DOX后48 h RT-PCR結(jié)果可見(jiàn)過(guò)表達(dá)組IDO表達(dá)量為62 906.27±4.36；對(duì)照組的表達(dá)量為0.05±0.02，見(jiàn)圖1。

2.3大鼠腹腔異位移植模型建立 異位心臟移植48 h，心臟彩超結(jié)果，見(jiàn)圖2。

2.4采用心臟彩超評(píng)估移植心臟心功能改變 大鼠腹腔異位心臟移植模型建立3 d，經(jīng)大鼠尾靜脈給予各組BMSCs，采用心臟彩超評(píng)估移植心臟心功能改變，可見(jiàn)過(guò)表達(dá)IDO-BMSCs組其射血分?jǐn)?shù)前后差值為18.367±1.32較其余各組高。環(huán)比收縮前后差值為14.5±1.26較其余各組仍有較大改善，見(jiàn)圖3。

2.5采用小動(dòng)物活體成像評(píng)估移植心臟熒光強(qiáng)度 大鼠心臟移植模型建立3 d后給予注射過(guò)表達(dá)IDO-BMSCs、空載體-BMSCs、BMSCs后2 d完成小動(dòng)物活體成像，給予注射各組干細(xì)胞后，可見(jiàn)過(guò)表達(dá)IDO-BMSCs組平均熒光強(qiáng)度為8.224±1.56，較其余各組熒光強(qiáng)度高，見(jiàn)圖4。

圖1 慢病毒轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞后48 h IDO-PCRFig.1 Result of IDO-PCR that 48 hours after using lentiviral vector transfect rats bone marrow mesenchymal stem cellsNote:RT-PCR results showed that in the rats bone marrow mesenchymal stem cells and comparison of NC(Negative control group),OE(overexpressing IDO group) expression level of IDO gene was 62 906.27 times of NC (P<0.05).

圖2 腹腔異位移植心臟48 h后復(fù)查心臟彩超結(jié)果Fig.2 After rat abdominal heterotopic heart transplan-tation model established 48 h that color Doppler examination resultNote:The white arrow points to the formation of the thrombus；The black arrow points to the transplanted heart.

圖3 大鼠移植模型建立3 d后給予IDO-BMSCs、空載體-BMSCs、BMSCs處理后2 d移植心臟心功能Fig.3 Using color Doppler examination assessment of transplanted cardiac function after intervention 2 daysNote:EF and FS were significantly improved in the transplant heart after 3 days of rat abdominal heterotopic heart transplantation model intervention by IDO-BMSCs 2 days, and there was significant difference between the other groups (P<0.05).

圖4 給予干預(yù)措施2 d后完成移植心臟局部熒光強(qiáng)度評(píng)價(jià)Fig.4 Assessing cardiac local average fluorescence intensity after intervention 2 daysNote:The group of IDO-BMSCs the cardiac local average fluorescence intensity was the strongest,and there were significant difference between the other groups (P<0.05).

2.6采用流式細(xì)胞術(shù)檢測(cè)受體大鼠脾臟細(xì)胞免疫抗原分子變化 提取給予干預(yù)措施(IDO-BMSCs、空載體-BMSCs、BMSCs、嗎替麥考)2 d后受體大鼠脾臟細(xì)胞完成CD40、CD86、CD80、MHCⅡ、CD274、CD45RA、CD45RA+CD45RB、Treg表達(dá)檢測(cè)，將未給予干預(yù)措施組(未處理組)及正常大鼠的脾臟細(xì)胞作為對(duì)照：可見(jiàn)過(guò)表達(dá)IDO-BMSCs組CD40表達(dá)為3.24±0.23；CD86表達(dá)為9.24±0.74；CD80表達(dá)為8.54±0.26；MHCⅡ表達(dá)為24.76±0.36；CD45RA表達(dá)為47.23±1.27；CD45RB+CD45RA表達(dá)為26.15±1.46其表達(dá)較其余組低。過(guò)表達(dá)IDO-BMSCs組CD247表達(dá)為10.37±1.42；Treg細(xì)胞表達(dá)為6.54±2.35，其表達(dá)較其余組高，如圖5。

圖5 給予干預(yù)措施2 d后流式檢測(cè)脾臟細(xì)胞抗原分子表達(dá)Fig.5 Using flow cytometry detection of splenic cell antigen molecules expression after intervention 2 daysNote:Overexpression IDO-BMSCs group CD40，CD86，CD80，MHCⅡ，CD45RA，CD45RA+CD45RB expression are the lowest，and no significant difference compared with the normal group(P>0.05),and there was significant difference between the other groups (P<0.05).Overexpressing IDO-BMSCs group CD274 expression was the highest，and there was significant difference between the other groups (P<0.05).Overexpressing IDO-BMSCs group Treg expression was the highest，and there was significant difference between the other groups (P<0.05).

圖6 給予干預(yù)措施后2 d提取受體大鼠血清檢測(cè)細(xì)胞因子表達(dá)Fig.6 Detection of cytokine expression in serum of recipient rats after intervention 2 daysNote:Overexpress IDO-BMSCs group the expression of IL-1α,IL-4,IL-1β,IL-2,IFN-γ,IL-18 were reduced,and there was significant difference between the other groups (P<0.05),but IL-10 were rised and had significant difference between the other groups(P<0.05).

2.7采用液相芯片檢測(cè)受體大鼠血清體液免疫分子變化 取腹腔異位移植術(shù)后2 d受體大鼠血清，使用RECYTMAG-65K-07試劑盒檢測(cè)IL-1ɑ、IL-4、IL-1β、IL-2、IL-10、IFN-γ、IL-18及使用TGFBMAG-64K-03試劑盒檢測(cè)TGFβ1、TGFβ2、TGFβ3，可見(jiàn)過(guò)表達(dá)IDO-BMSCs組IL-1ɑ、IL-4、IL-1β、IL-2、IFN-γ、IL-18表達(dá)分別為(78.341±2.45、3.455±0.24、134.563±7.21、45.761±4.81、38.273±1.67、178.346±2.34)較其余組低但較正常組對(duì)照組高。IL-10的表達(dá)為(1 003.245±3.56 )較其余組表達(dá)增高，見(jiàn)圖6。

圖8 大鼠心臟移植模型建立3 d后給予干預(yù)措施2 d后HE染色結(jié)果Fig.8 After 3 days of rat abdominal heterotopic heart transplantation model, and after intervention 2 days,result of HE stainingNote:A.Overexpressing IDO-BMSCs group；B.Empty vector-BMSCs group；C.BMSCs group；D.Mycophenolate mofetil；E.Untreated model group；F.Normal heart.Untreated model group the infiltration of inflammatory cells were higher than others, but overexpressing IDO-BMSCs the infiltration of inflammatory cells are lesser than others.

圖7 給予干預(yù)措施后2 d提取受體大鼠血清檢測(cè)細(xì)胞因子表達(dá)Fig.7 Detection of TGFβ1,TGFβ2,TGFβ3 expression in serum of recipoient rats after intervantion 2 daysNote:Overexpressing IDO-BMSCs group the expression of TGFβ1,TGFβ2,TGFβ3 were rised, and there was significant difference between the other groups (P<0.05).

2.8采用液相芯片檢測(cè)受體大鼠血清TGFβ1、TGFβ2、TGFβ3的變化 給予干預(yù)措施后2 d提取受體大鼠血清，檢測(cè)TGFβ1、TGFβ2、TGFβ3的量，可見(jiàn)過(guò)表達(dá)IDO-BMSCs組TGFβ1、TGFβ2、TGFβ3表達(dá)量為(80 006.982±4.23、0.321±0.12、7.346±1.12)較其余各組表達(dá)量高，見(jiàn)圖7。

2.9HE染色結(jié)果 大鼠心臟移植模型建立3 d后，給予注射相應(yīng)BMSCs細(xì)胞后2 d取其腹腔心臟，制備石蠟切片，完成HE染色(圖8A～F)。可見(jiàn)過(guò)表達(dá)IDO-BMSCs組其炎癥細(xì)胞浸潤(rùn)較正常組多但較其余組少。

3 討論

20世紀(jì)以來(lái)，由于器官移植技術(shù)、移植免疫基礎(chǔ)研究以及各種免疫抑制劑的進(jìn)展，器官移植已成為臨床治療器官功能衰竭的有效治療手段。但不可否認(rèn)的是免疫排斥一直是阻礙器官移植進(jìn)一步發(fā)展的瓶頸。本實(shí)驗(yàn)正是基于上述社會(huì)背景下，結(jié)合最新免疫調(diào)節(jié)研究結(jié)果，試圖尋找到一種有效的生物免疫抑制方法。

MSCs具有免疫調(diào)節(jié)功能，目前認(rèn)為其可能涉及以下途徑：①M(fèi)SCs持續(xù)不斷表達(dá)低水平的MHCⅠ分子但不表達(dá)MHCⅡ及協(xié)同刺激分子，包括CD80、CD86或者CD40[1]。因此，MSCs將不會(huì)激活異種或異體淋巴細(xì)胞而其本身缺乏免疫原性。②MSCs能夠抑制T、B淋巴細(xì)胞的激活和增生，部分是通過(guò)將細(xì)胞抑制于G0/G1細(xì)胞周期階段[2,3]。MSCs也可能通過(guò)可溶性細(xì)胞因子(如：IL-6、M-CSF)干涉樹(shù)突細(xì)胞的分化、成熟及功能[4]。③MSCs能夠通過(guò)釋放抗炎及抗凋亡分子修飾損傷組織的微環(huán)境并且保護(hù)損傷組織[5,6]。但目前MSCs臨床應(yīng)用免疫排斥仍不成熟，歸根還是由于其調(diào)節(jié)免疫耐受的效果不確切。

IDO主要通過(guò)三條途徑完成免疫調(diào)節(jié)：①通過(guò)產(chǎn)生犬尿氨酸(KYN)[其是芳基羥化物(AhR)受體的一個(gè)天然配體]。IDO催化分解色氨酸并產(chǎn)生毒性代謝產(chǎn)物犬尿氨酸，其被淋巴細(xì)胞攝取可以阻止細(xì)胞進(jìn)入G1期，而凋亡[6]。犬尿氨酸途徑，包括(犬尿氨酸、3OH-犬尿氨酸)，可以誘導(dǎo)Tregs產(chǎn)生或抑制DCs[7，8]。治療上，應(yīng)用天然及合成的犬尿氨酸產(chǎn)物可以促進(jìn)免疫耐受，保護(hù)移植器官，減少由于病原感染而引起的組織損傷[9]。②通過(guò)消耗色氨酸以觸發(fā)細(xì)胞敏感轉(zhuǎn)錄信號(hào)途徑。色氨酸的消耗可以經(jīng)由分子應(yīng)激反應(yīng)途徑，諸如：GCN2激酶和在哺乳動(dòng)物中對(duì)色氨酸缺失反應(yīng)的帕雷霉素樣受體(mTOR)兩者的激活可以發(fā)揮免疫調(diào)節(jié)作用。任何氨基酸的缺失都可以激活GCN2激酶而導(dǎo)致下游目標(biāo)的磷酸化，真核細(xì)胞啟動(dòng)因子(eIF)-2α的磷酸化可以阻斷大多數(shù)mRNA物種核糖體的轉(zhuǎn)錄，但是它可以選擇性增強(qiáng)少量轉(zhuǎn)錄因子轉(zhuǎn)錄[10]。色氨酸的缺失也可影響到營(yíng)養(yǎng)敏感性mTOR途徑[11]。mTOR激活后可通過(guò)IDO、精氨酸酶、色氨酸羥化酶和其他酶抑制炎性位點(diǎn)色氨酸的分解代謝而影響Tregs細(xì)胞和效應(yīng)性T細(xì)胞的功能[11]。③在DCs上表達(dá)的IDO可以直接作為細(xì)胞間信號(hào)分子[12]。IDO信號(hào)分子的功能是不依賴(lài)IDO酶的活性的。它主要是通過(guò)IDO和Src同族的第2域(SHP-1/SHP-2)與磷酸酯酶相接觸，觸發(fā)IDO的免疫調(diào)節(jié)域發(fā)揮作用。有研究表明，IDO-SHP復(fù)合體是維持TGF-β對(duì)老鼠血漿DCs發(fā)揮作用的關(guān)鍵因素[13]。

本實(shí)驗(yàn)正是在上述研究基礎(chǔ)上通過(guò)建立大鼠異位心臟移植模型，采用心臟彩超、小動(dòng)物活體成像、流式細(xì)胞、液相芯片、HE染色技術(shù)證實(shí)：①大鼠異位心臟移植模型建立后，給予相應(yīng)細(xì)胞處理，可見(jiàn)過(guò)表達(dá)IDO-BMSCs處理后2 d移植心臟的EF、FS較其余各組有所提高。②進(jìn)一步采用小動(dòng)物活體評(píng)估也可見(jiàn)過(guò)表達(dá)IDO-BMSCs組移植心臟局部熒光強(qiáng)度最強(qiáng)。③給予干預(yù)措施后2 d可見(jiàn)過(guò)表達(dá)IDO-BMSCs組脾臟細(xì)胞CD40、CD86、CD80、MHCⅡ、CD45RA、CD45RA+CD45RB表達(dá)降低，而CD274、Treg細(xì)胞的表達(dá)增高。④采用液相芯片檢測(cè)各組提取血清可見(jiàn)，2 d時(shí)過(guò)表達(dá)IDO-BMSCs組血清中IL-1ɑ、IL-4、IL-1β、IL-2、IFNγ、IL-18是降低的。IL-10、TGFβ1、TGFβ2、TGFβ3表達(dá)量是升高的。HE染色證實(shí)過(guò)表達(dá)IDO-BMSCs組炎性細(xì)胞浸潤(rùn)少于其他組。

綜上所述，過(guò)表達(dá)IDO的BMSCs可以通過(guò)有效調(diào)節(jié)免疫DC、T細(xì)胞及細(xì)胞因子。從多個(gè)免疫層面改善移植心臟存活。

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[收稿2017-05-28]

(編輯 倪 鵬)

ImprovedsurvivalofheterotopichearttransplantoverexpressingIDOratbonemesenchymalstemcells

HEJi-Gang,LIBei-Bei,HANJin-Xiu,LIHong-Yuan,YANDan.
CardiovascularSurgery,theFirstPeople′sHospitalofYunnanProvince,Kunming650032,China

Objective:To explores the improvement in survival mechanism of a rat model of enterocoelia heterotopic heart transplant with rat bone marrow mesenchymal stem cells(BMSCs) IDO-overexpressing.MethodsIDO-overexpressing rat BMSCs were produced through transfection of rat BMSCs with IDO gene carried by the lentiviral vector GV308.A rat model of enterocoelia heterotopic heart transplantation was established.This rat model

a cell treatment via its tail veins,as follows:①Echocardiography was employed to detect the functional changes in the transplanted heart.②The fluorescence intensity of the different parts of the transplanted heart was evaluated using a body imaging system for small living animals.③Receptors rat spleens cells were obtained and used for a flow cytometric detection of the expression levels of CD40,CD86,CD80,MHCⅡ,CD274,CD45RA,CD45RA+CD45RB,and Treg cells.④A transplanted heart was obtained after injection to evaluate inflammatory cell infiltration through HE staining.⑤Liquid phase chips were used to detect changes in the serum factors IL-1ɑ,IL-4,IL-1β,IL-2,IL-10,IFN-γ,IL-18,TGFβ1,TGFβ2 and TGFβ3 in after injection cells.Results①After the rat heterotopic heart transplantation model and the corresponding cell treatment were established,after over-expressed IDO-BMSCs treatment 2 days the EF and FS were higher in the transplanted heart than other groups.②The fluorescence intensity of the parts of the transplanted heart was highest in the IDO-BMSC overexpression group as revealed by small animal living body evaluation.③Two days after the interventions,spleen cells in the over-expressed IDO-BMSCs group showed reduced expression levels of CD40,CD86,CD80,MHCⅡ,CD45RA,CD45RA+CD45RB and increased expression levels of CD274 and Treg cells as revealed by flow cytometry.④Liquid phase chips were used to examine the serum obtained from each group 2 days after the intervention,and the results showed that the expression levels of IL-1α,IL-4,IL-1β,IL-2,IFN-γ and IL-18 in the IDO-BMSC overexpression group decrease.By contrast,the expression levels of IL-10,TGFβ1,TGFβ2 and TGFβ3 increase.HE staining results demonstrate that inflammatory cell infiltration was lower in IDO-BMSC overexpression group than in other groups.ConclusionIDO-overexpressing BMSCs improve the survival of a transplanted heart through effective adjustment of immune DC and T cells,as well as cell factors.

IDO;Rat bone mesenchymal stem cell;Immunosuppression

①本文為國(guó)家自然科學(xué)基金(81460073)、云南省科技廳-昆明醫(yī)科大學(xué)應(yīng)用基礎(chǔ)研究聯(lián)合專(zhuān)項(xiàng)(2014FB089)、云南省教育廳科學(xué)研究基金(2015Z051)、中國(guó)博士后科學(xué)基金(2015M582764XB)、成都醫(yī)學(xué)院2015年度科研項(xiàng)目(CYZ15-18)和云南省醫(yī)學(xué)后備人才(H-201607)項(xiàng)目。

②通訊作者，E-mail：jiganghe999@163.com。

R392.4

A

1000-484X(2017)10-1447-06

10.3969/j.issn.1000-484X.2017.10.002

賀繼剛(1980年-)，男，副主任醫(yī)師，碩士生導(dǎo)師，主要從事干細(xì)胞在終末重癥心臟疾病中的應(yīng)用研究。