腺相关病毒介导的PD-L1基因转染对大鼠移植肝的保护作用及其机制
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摘要
研究背景
器官移植后器官功能维持的主要障碍是术后排斥反应和免疫抑制剂的毒性。基因治疗为肝移植领域供肝抵抗移植后排斥反应和损伤提供了新的治疗思路。将免疫调节基因导入移植肝内,使其表达致耐受分子,产生能抑制受体免疫反应的细胞因子,可有效地抑制排斥反应的发生和增强移植肝的保护功能,能在不用或少量使用免疫抑制剂的情况下延长移植肝存活,甚至长期存活。
程序性死亡配体-1(Programmed Death Ligand-1, PD-L1)是一种重要的抑制性共刺激分子,可通过激活初始T细胞、抑制活化的效应T细胞及调节细胞因子的分泌等参与多种免疫过程。体外研究发现:PD-L1与其受体程序性死亡-1(Programmed Death-1, PD-1)结合后,能抑制活化T细胞增殖及细胞因子的产生。动物实验也证实,在心脏、胰岛、角膜、皮肤等多种器官移植模型中,PD-L1均能抑制排斥反应及延长移植物的存活;这强烈提示了PD-L1可能具有保护移植肝免受排斥反应损伤的治疗潜能。PD-L1可通过多种机制产生免疫抑制效应,与肝移植联系起来考虑,它可能通过抑制活化T细胞的增殖及细胞因子的合成、分泌,从而阻断宿主对移植肝的攻击。此外,免疫抑制分子的局部表达有可能减少其全身性副作用、增加其生物利用度及治疗效应,因此是减轻排斥反应、促进移植物长期存活的很有前景的方法。腺相关病毒(Adeno-associated Virus, AAV)是移植物保护分子转染和表达的理想载体。报告基因红色荧光蛋白(Red Fluorescent Protein, RFP)因其具有灵敏度高、信号清楚等特点亦日益受到关注。
研究目的
基于以上分析,本研究拟通过建立近交系大鼠原位肝移植模型,以RFP为报告基因,将AAV作为PD-L1的载体,采用冷保存期门静脉灌注夹闭法转染供肝,观察其安全性和有效性。在此基础上,于活体内观察PD-L1基因转染对大鼠异基因肝移植术后肝脏的保护作用及其与亚剂量CsA的协同效果,并通过检测PD-L1对移植肝CD4+、CD8+、CD25+淋巴细胞浸润及细胞因子INF-γ、IL-17、IL-10、TGF-β1表达的影响,试图阐明PD-L1在抑制大鼠肝移植急性排斥反应中的可能机制。
方法与结果
第一部分近交系大鼠肝移植急性排斥模型的建立及排斥反应观察
大鼠随机分为3组:①G1(同基因肝移植组);②G2(异基因肝移植组);③G3(CsA组,移植后0～7d腹腔注射CsA 2.5mg/Kg.d,余同G2)。采用改良“二袖套法”建立大鼠肝移植急性排斥模型。术后观察一般情况、平均存活时间(Median Survival Time, MST),分别在术后3、7、14及21d采用全自动生化分析仪检测肝功能,光镜下观察移植肝组织学变化,根据Banff标准判断排斥反应强度。结果表明:在无外界干预的情况下,G1大鼠无急性排斥表现;G2受体多在术后7d出现耳廓黄染、尿黄等,至14d进行性加重伴血性腹水等;G3用药期间大鼠精神差,停药后逐渐恢复,至14d出现轻度急排表现;三组大鼠MST分别为:G1(106.7±0.24)d,G2(18.3±1.15)d和G3(28.7±0.98)d,G3较G2明显延长,差异显著(P<0.05);肝功及肝组织病理学改变早于上述表现,术后3d急排大鼠ALT、TBIL明显升高伴轻度的排斥反应病理改变,7d后上述指标进一步恶化,至14d最为典型,与G1、G3相比差异显著(P<0.05);各时段排斥分级与病理变化趋势一致:术后3d G3与G2急性排斥活动指数(Rejection Activity Index, RAI)评分无明显差异,而7d、14d及21d G2 RAI评分明显高于G1、G3 (P<0.05)。
第二部分重组PD-L1-AAV2-RFP转染大鼠移植肝的安全性和有效性
1.重组PD-L1-AAV2-RFP载体PCR鉴定:以重组载体为模板,在PCR仪上进行扩增反应,取PCR产物行1%琼脂糖凝胶电泳,紫外灯下观察发现扩增条带与预期的891bp大小的特异性条带相吻合,表明PD-L1基因构建正确。
2.重组PD-L1-AAV2-RFP载体感染活性检测:重组病毒感染BHK细胞后荧光显微镜下观察RFP基因表达。结果表明PD-L1-AAV2-RFP感染后24h即可观察到红色荧光,48h后转染率可达30%,表明其具有较强的感染活性,可用于体内外转染研究。
3.大鼠随机分为3组:①G1(同基因肝移植组);②G2(AAV空载体组,供肝切取后经门静脉灌注AAV2-RFP空病毒液夹闭冷转染供肝2h,移植前乳酸林格氏液经门静脉冲洗供肝,余同G1);③G3(PD-L1转染组,所用灌注液为PD-L1-AAV2-RFP,余同G2)。改良“二袖套法”建立大鼠肝移植基因转染模型,于荧光显微镜下观察移植肝RFP的表达,分别采用实时荧光定量RT-PCR、IHC从mRNA及蛋白水平检测移植肝PD-L1的表达,肝功能及肝组织病理检测同第一部分。结果发现:ALT与TBIL分别于术后3d、7d达到峰值,14d基本恢复正常,各组间无显著性差异;与未转染组相比,PD-L1转染后肝组织病理未见明显差异;G2、G3术后7d即可在荧光显微镜下观察到微弱的红色荧光,14、21d逐渐增强,呈高亮度的红色荧光,而三组大鼠肝外器官中始终无红色荧光;RT-PCR及IHC检测显示未转染组PD-L1mRNA基因和蛋白均微弱表达;PD-L1转染7d后随时间延长靶基因表达逐渐增强,21d达峰值,主要位于汇管区周围;除3d外,余各时段PD-L1基因转录及蛋白表达G3较G1、G2明显增强(P<0.05)。
第三部分腺相关病毒介导的PD-L1表达对大鼠移植肝的保护作用及其机制
大鼠随机分为6组:①G 1(同基因肝移植组);②G2(异基因肝移植组);③G3(AAV空载体组,供肝切取后经门静脉灌注AAV2-RFP空病毒液夹闭冷转染供肝2h,移植前乳酸林格氏液经门静脉冲洗供肝,余同G2);④G4(CsA组,移植后0～7d予腹腔注射CsA 2.5mg/kg.d,余同G2);⑤G5(PD-L1转染组,所用灌注液为PD-L1-AAV2-RFP,余同G3);⑥G6(联合治疗组,所用灌注液为PD-L1-AAV2-RFP,术后0～7d予腹腔注射CsA 2.5mg/kg.d,余同G2)。改良“二袖套法”建立大鼠肝移植模型,利用组织病理、IHC、ELISA等技术,观察术后移植肝病理改变、PD-L1蛋白表达及其对移植肝CD4+、CD8+、CD25+细胞浸润和细胞因子INF-γ、IL-17、IL-10、TGF-β1表达的影响。结果显示:G1大鼠无急性排斥表现;肝功能损害较轻,ALT与TBIL分别于术后3d、7d达到峰值,14d基本恢复正常;肝脏病理学呈Banff 0级排斥反应;各时段PD-L1蛋白微弱表达;汇管区未见明显淋巴细胞浸润;IFN-γ无明显表达,而IL-17、IL-10和TGF-β1呈低表达;大鼠MST为(106.7±0.24)d。G2、G3 7d开始出现急排表现,14d症状加重;肝功能指标明显恶化;肝脏病理学呈BanffⅡ～Ⅲ级排斥反应,RAI评分随时间推移逐渐升高;各时段PD-L1蛋白微弱表达;移植肝大量CD4+、CD8+、CD25+细胞浸润;细胞因子IFN-γ、IL-17上调表达,而IL-10、TGF-β1表达下降;大鼠MST为(18.3±1.15)d、(18.3±0.72)d。与G2、G3相比,G4、G5及G6大鼠发生急性排斥反应的时间均明显延迟,直至14d才出现轻度排斥表现;肝功能部分改善,术后出现ALT、TBIL再次升高及肝功能衰竭的时间明显推迟;肝脏病理学呈BanffⅠ～Ⅱ级排斥反应;G5、G6各时段移植肝PD-L1蛋白的表达随时间逐渐增强,G4无明显变化;移植肝CD4+、CD8+、CD25+细胞浸润明显减少,尤以CD8+细胞为著(P<0.05);移植肝细胞因子IFN-γ、IL-17含量显著下降,而IL-10、TGF-β1含量明显升高,差异显著(P<0.05);受体MST明显延长,但未获得长期存活,仅为(28.7±0.98)d、(29.3±1.47)d。PD-L1基因转染联合亚剂量CsA治疗的大鼠各时段肝功能损害进一步减轻,RAI评分显著降低,受体MST明显延长至(34.0±1.49)d (P<0.05)。
四、结论
1.采用改良“二袖套法”成功建立了LEW-BN组合大鼠肝移植急性排斥模型。
2.冷保存期经门静脉灌注PD-L1-AAV2-RFP夹闭法转染供肝,可安全、有效地将目的基因转染至大鼠移植肝内并分泌功能性蛋白。
3.在本实验条件下,采用PD-L1-AAV2-RFP(1×1011v.g./只)经门静脉灌注夹闭冷保存2h是介导足量蛋白表达的理想方案。
4.腺相关病毒介导的PD-L1基因转染对移植肝具有显著的保护作用,可延长移植肝存活,且与CsA有协同效果。
5. PD-L1基因可抑制肝移植急性排斥反应,其机制可能是PD-L1基因转染增强了PD-L1/PD-1信号通路,通过抑制移植肝CD4+、CD8+、CD25+T细胞浸润及其功能,在上调细胞因子IL-10、TGF-β1表达的同时,下调INF-γ、IL-17的表达,从而发挥对移植肝的保护作用。
Background
Liver transplantation is an effective or even only approach of treatment to some end stage liver diseases, but acute rejection remains to be the primary clinical problem and challenge of transplantation. The outcome of allogeneic organ transplants is, in part, dependent on the balance between co-stimulatory and modulatory signals that accompany TCR-mediated recognition by recipient’s T cells. The local expression of a gene encoding an immunosuppressive protein within a graft can generate local immunosuppression, which enhanced co-inhibitory signals in recipients, making gene therapy a viable approach for facilitating transplantation.
Programmed death ligand-1 (PD-L1), a newer member of the CD28 family, has been implicated as a negative regulator of T cell activation. In previous research, engagement of programmed death-1 (PD-1) by PD-L1 inhibited proliferation and cytokine production by activated T cells. Conversely, blockade of PD-L1 using specific monoclonal antibodies stimulated T cell proliferation and cytokine production. Meanwhile, recent studies showed that interfering with the PD-L1/PD-1 co-stimulatory pathway prolonged allograft survival in various organ transplantation models, suggesting that they might play an important role in maternal acceptance of liver allograft. Thus, the recombinant PD-L1 gene was chosen as the immunomodulator.
This study focused on establishing acute rejection model of orthotopic liver transplantation in inbred rats. The effective way of transferring the target gene into the liver allograft with PD-L1-AAV2-RFP was also explored. Furthermore, we investigated the protective effects and mechanisms of modification of liver allograft with target gene on the survival in vivo. The main results and conclusions were as follows:
Part 1 Establishment of acute rejection model of orthotopic liver transplantation in inbred rats
Objective To establish acute rejection model of orthotopic liver transplantation in inbred rats.
Methods We established LEW-BN rat orthotopic liver transplantation model using“the innovated two-cuff technique”. Experimental groups included:①G1: Isogenic (ISO) group.②G2: Allogeneic (ALLO) group.③G3: CsA group injected CsA (2.5mg/kg.d) intraperitoneally daily from day 0 to day 7 postoperatively. Recipients were sacrificed on POD 3, 7, 14, and 21 respectively. Liver tissues and blood samples were collected. The general states, median survival time (MST), and histopathological characteristic of recipients were observed. Serum levels of ALT and TBIL were measured with automatic biochemical analyser.
Results In G2, typical mild, moderate and severe acute rejection occurred on POD 3, 7 and 14 respectively, and the delayed occurrence of graft rejection were observed in G3. The MST of G3 (28.7±0.98)d was prolonged significantly compared with that of G2 (18.3±1.15)d (P<0.05). Serum ALT and TBIL levels of G2 increased obviously on POD 3 and has been booming ever since. Serum ALT and TBIL levels of G2 were significantly higher than those of G1 and G3. Typical histological change occurred on POD 14 in G2 and the light rejection in G3. There was no significant difference in rejection activity index (RAI) scores between G2 and G3 on POD 3. Whereas on d7, d14 and d21 after operation, RAI scores of G2 were marked higher than that of G3 and G1 (P<0.05).
Conclusions
1. Rat orthotopic liver transplantation of LEW to BN strain is an ideal acute rejection model.
2. The model has stable and typical allograft rejection. It can be used in the study of liver transplantation immunity.
Part 2 The security and effectiveness of recombinant adeno-associated virus transfecting PD-L1 gene into livers of donor
Objective To find the effective way of transfecting PD-L1 gene into livers of donor with recombinant adeno-associated virus.
Methods The PD-L1-AAV2-RFP was identified by PCR. The transfection efficiency of PD-L1-AAV2-RFP was determined by observing the expression of RFP in BHK cell with fluorescence microscope. We established the PD-L1-AAV2-RFP gene transfer model and recipients were randomly divided into three groups:①G1: ISO group.②G2: AAV group introduced through back-table portal vein perfusion with AAV2-RFP for 2 hours, and the graft was flushed with Ringer’s lactate solution to remove any free virus before implantation.③G3: PD-L1 group introduced with PD-L1-AAV2-RFP. Liver allografts harvested on POD 3, 7, 14, and 21 were fixed in 10% buffered formalin and embedded in paraffin for histological study by hematoxylin-eosin staining. Part of the grafts were frozen for RFP expression analysis. PD-L1 mRNA and protein expression in grafts were detected by using Realtime RT-PCR and IHC respectively. The function of liver was also measured. Results The fragment was verified by PCR analysis to make sure that the cDNA is just the one we expected. Evident red fluorescence was observed and the transfection rate increased with the prolongation of time, suggesting that the donor liver might be transfected efficiently by PD-L1-AAV2-RFP. Serum levels of ALT and TBIL increased obviously on POD 3 and 7 respectively, then gradually returned back to normal with time. Pathological changes of the specimens showed only mild ischemia-reperfusion injury on POD 7, with no statistical difference among three groups (P>0.05). Red fluorescence was not observed in sections of heart, lung, kidney, and spleen after transplantation. However, evident red fluorescence was observed in sections of livers after operation except day 3 and increased in the later. The expressions of PD-L1mRNA and protein were both upregulated in the liver of donor at 7th day after transfection, and reached its peak at 21st day in G3, but still lower in G1 and G2.
Conclusions
1. PD-L1-AAV2-RFP is effective and safe for gene delivery in rat liver transplantation.
2. Back-table perfusion through the portal vein with PD-L1-AAV2-RFP (1×1011v.g./animal) and preservation of grafts for 2 hours is the optimal protocol to induce sufficient protein expression.
Part 3 The protective effects of PD-L1 gene transfer mediated by AAV-2 on liver allograft of rat and its mechanisms
Objective To evaluate the protective effects and mechanisms of the liver allograft transfected with PD-L1 gene in vivo.
Methods Recipients were randomized into six groups:①G1: ISO group.②G2: ALLO group.③G3: AAV group introduced with AAV2-RFP.④G4: CsA group injected CsA (2.5mg/kg.d) intraperitoneally daily from day 0 to day 7 postoperatively.⑤G5: PD-L1 group introduced with PD-L1-AAV2-RFP.⑥G6: Combination therapy group introduced with PD-L1-AAV2-RFP plus a subtherapeutic regimen of CsA. We established ROLT model using“the innovated two-cuff technique”. Gene delivery to donor liver was done by intracoronary instillation of virus containing solution (1×1011v.g./animal) through the portal vein. On POD 3, 7, 14 and 21, grafts were taken for IHC to assess the expression of PD-L1.Ig, CD4+, CD8+ and CD25+ lymphocytes, for ELISA to analysis the concentration of IFN-γ, IL-17, IL-10, and TGF-β1 respectively. The MST, liver function and histopathological changes were also observed.
Results In G1, no acute rejection was found, biochemical datas showed that the levels of ALT and TBIL increased during the first weeks after operation and decreased to normal with time. Fewer PD-L1-positive cells in the graft were detected. The expression of IFN-γ, CD4+, CD8+, and CD25+ lymphocytes were not observed in various period of time. Otherwise, IL-17, IL-10, and TGF-β1 were all low-expressed. The MST was (106.7±0.24)d. In G2 and G3, typical mild, moderate and severe acute rejection occurred on POD 3, 7 and 14 respectively. The function of liver were markedly higher than those of other groups (P<0.05). Fewer PD-L1-positive cells in the graft were detected. A lot of CD4+, CD8+ and CD25+ lymphocytes infiltrated in portal area of grafts, especially CD8+ T cells (P<0.05). On POD 3, the concentration of IFN-γand IL-17 in grafts increased continuously and even decreased on POD 21, while the expression of IL-10 and TGF-β1 were down-regulated gradually. The MST were (18.3±1.15)d and (18.3±0.72)d. In G4, G5 and G6, acute rejection still occurred after operation with light rejection sign in histopathological change. The function of gene transfected liver was inferior to that of G2 and G3 significantly (P<0.05). Although fewer PD-L1-positive cells were detected in G4, a certain number of PD-L1-positive cells were found in G5 and G6. Compared with G2 and G3, those allografts treated with PD-L1 or CsA had a significantly reduced cellular infiltrate, especially CD8+ cell. The concentration of IFN-γand IL-17 in grafts were greatly inhibited, accompanying with greatly increased secretion of IL-10 and TGF-β1. Allografts transduced with the PD-L1 survived for longer periods of time (P<0.05). PD-L1 gene transfer combined with a subtherapeutic regimen of CsA was superior to CsA alone: (34.0±1.49)d vs. (28.7±0.98)d.
Conclusions
1. Adeno-associated virus mediated PD-L1 gene delivery significantly prolongs liver allograft survival in a rat model.
2. Enhanced expression of PD-L1 protein in donor liver attenuates acute allograft rejection. The effect is additive to that of a subtherapeutic regimen of CsA.
3. PD-L1 gene may take the effect in liver transplantation via reducing intragraft infiltrates of T lymphocytes, suppression of INF-γand IL-17 production as well as stimulation of IL-10 and TGF-β1 production.
器官移植后器官功能维持的主要障碍是术后排斥反应和免疫抑制剂的毒性。基因治疗为肝移植领域供肝抵抗移植后排斥反应和损伤提供了新的治疗思路。将免疫调节基因导入移植肝内,使其表达致耐受分子,产生能抑制受体免疫反应的细胞因子,可有效地抑制排斥反应的发生和增强移植肝的保护功能,能在不用或少量使用免疫抑制剂的情况下延长移植肝存活,甚至长期存活。
程序性死亡配体-1(Programmed Death Ligand-1, PD-L1)是一种重要的抑制性共刺激分子,可通过激活初始T细胞、抑制活化的效应T细胞及调节细胞因子的分泌等参与多种免疫过程。体外研究发现:PD-L1与其受体程序性死亡-1(Programmed Death-1, PD-1)结合后,能抑制活化T细胞增殖及细胞因子的产生。动物实验也证实,在心脏、胰岛、角膜、皮肤等多种器官移植模型中,PD-L1均能抑制排斥反应及延长移植物的存活;这强烈提示了PD-L1可能具有保护移植肝免受排斥反应损伤的治疗潜能。PD-L1可通过多种机制产生免疫抑制效应,与肝移植联系起来考虑,它可能通过抑制活化T细胞的增殖及细胞因子的合成、分泌,从而阻断宿主对移植肝的攻击。此外,免疫抑制分子的局部表达有可能减少其全身性副作用、增加其生物利用度及治疗效应,因此是减轻排斥反应、促进移植物长期存活的很有前景的方法。腺相关病毒(Adeno-associated Virus, AAV)是移植物保护分子转染和表达的理想载体。报告基因红色荧光蛋白(Red Fluorescent Protein, RFP)因其具有灵敏度高、信号清楚等特点亦日益受到关注。
研究目的
基于以上分析,本研究拟通过建立近交系大鼠原位肝移植模型,以RFP为报告基因,将AAV作为PD-L1的载体,采用冷保存期门静脉灌注夹闭法转染供肝,观察其安全性和有效性。在此基础上,于活体内观察PD-L1基因转染对大鼠异基因肝移植术后肝脏的保护作用及其与亚剂量CsA的协同效果,并通过检测PD-L1对移植肝CD4+、CD8+、CD25+淋巴细胞浸润及细胞因子INF-γ、IL-17、IL-10、TGF-β1表达的影响,试图阐明PD-L1在抑制大鼠肝移植急性排斥反应中的可能机制。
方法与结果
第一部分近交系大鼠肝移植急性排斥模型的建立及排斥反应观察
大鼠随机分为3组:①G1(同基因肝移植组);②G2(异基因肝移植组);③G3(CsA组,移植后0～7d腹腔注射CsA 2.5mg/Kg.d,余同G2)。采用改良“二袖套法”建立大鼠肝移植急性排斥模型。术后观察一般情况、平均存活时间(Median Survival Time, MST),分别在术后3、7、14及21d采用全自动生化分析仪检测肝功能,光镜下观察移植肝组织学变化,根据Banff标准判断排斥反应强度。结果表明:在无外界干预的情况下,G1大鼠无急性排斥表现;G2受体多在术后7d出现耳廓黄染、尿黄等,至14d进行性加重伴血性腹水等;G3用药期间大鼠精神差,停药后逐渐恢复,至14d出现轻度急排表现;三组大鼠MST分别为:G1(106.7±0.24)d,G2(18.3±1.15)d和G3(28.7±0.98)d,G3较G2明显延长,差异显著(P<0.05);肝功及肝组织病理学改变早于上述表现,术后3d急排大鼠ALT、TBIL明显升高伴轻度的排斥反应病理改变,7d后上述指标进一步恶化,至14d最为典型,与G1、G3相比差异显著(P<0.05);各时段排斥分级与病理变化趋势一致:术后3d G3与G2急性排斥活动指数(Rejection Activity Index, RAI)评分无明显差异,而7d、14d及21d G2 RAI评分明显高于G1、G3 (P<0.05)。
第二部分重组PD-L1-AAV2-RFP转染大鼠移植肝的安全性和有效性
1.重组PD-L1-AAV2-RFP载体PCR鉴定:以重组载体为模板,在PCR仪上进行扩增反应,取PCR产物行1%琼脂糖凝胶电泳,紫外灯下观察发现扩增条带与预期的891bp大小的特异性条带相吻合,表明PD-L1基因构建正确。
2.重组PD-L1-AAV2-RFP载体感染活性检测:重组病毒感染BHK细胞后荧光显微镜下观察RFP基因表达。结果表明PD-L1-AAV2-RFP感染后24h即可观察到红色荧光,48h后转染率可达30%,表明其具有较强的感染活性,可用于体内外转染研究。
3.大鼠随机分为3组:①G1(同基因肝移植组);②G2(AAV空载体组,供肝切取后经门静脉灌注AAV2-RFP空病毒液夹闭冷转染供肝2h,移植前乳酸林格氏液经门静脉冲洗供肝,余同G1);③G3(PD-L1转染组,所用灌注液为PD-L1-AAV2-RFP,余同G2)。改良“二袖套法”建立大鼠肝移植基因转染模型,于荧光显微镜下观察移植肝RFP的表达,分别采用实时荧光定量RT-PCR、IHC从mRNA及蛋白水平检测移植肝PD-L1的表达,肝功能及肝组织病理检测同第一部分。结果发现:ALT与TBIL分别于术后3d、7d达到峰值,14d基本恢复正常,各组间无显著性差异;与未转染组相比,PD-L1转染后肝组织病理未见明显差异;G2、G3术后7d即可在荧光显微镜下观察到微弱的红色荧光,14、21d逐渐增强,呈高亮度的红色荧光,而三组大鼠肝外器官中始终无红色荧光;RT-PCR及IHC检测显示未转染组PD-L1mRNA基因和蛋白均微弱表达;PD-L1转染7d后随时间延长靶基因表达逐渐增强,21d达峰值,主要位于汇管区周围;除3d外,余各时段PD-L1基因转录及蛋白表达G3较G1、G2明显增强(P<0.05)。
第三部分腺相关病毒介导的PD-L1表达对大鼠移植肝的保护作用及其机制
大鼠随机分为6组:①G 1(同基因肝移植组);②G2(异基因肝移植组);③G3(AAV空载体组,供肝切取后经门静脉灌注AAV2-RFP空病毒液夹闭冷转染供肝2h,移植前乳酸林格氏液经门静脉冲洗供肝,余同G2);④G4(CsA组,移植后0～7d予腹腔注射CsA 2.5mg/kg.d,余同G2);⑤G5(PD-L1转染组,所用灌注液为PD-L1-AAV2-RFP,余同G3);⑥G6(联合治疗组,所用灌注液为PD-L1-AAV2-RFP,术后0～7d予腹腔注射CsA 2.5mg/kg.d,余同G2)。改良“二袖套法”建立大鼠肝移植模型,利用组织病理、IHC、ELISA等技术,观察术后移植肝病理改变、PD-L1蛋白表达及其对移植肝CD4+、CD8+、CD25+细胞浸润和细胞因子INF-γ、IL-17、IL-10、TGF-β1表达的影响。结果显示:G1大鼠无急性排斥表现;肝功能损害较轻,ALT与TBIL分别于术后3d、7d达到峰值,14d基本恢复正常;肝脏病理学呈Banff 0级排斥反应;各时段PD-L1蛋白微弱表达;汇管区未见明显淋巴细胞浸润;IFN-γ无明显表达,而IL-17、IL-10和TGF-β1呈低表达;大鼠MST为(106.7±0.24)d。G2、G3 7d开始出现急排表现,14d症状加重;肝功能指标明显恶化;肝脏病理学呈BanffⅡ～Ⅲ级排斥反应,RAI评分随时间推移逐渐升高;各时段PD-L1蛋白微弱表达;移植肝大量CD4+、CD8+、CD25+细胞浸润;细胞因子IFN-γ、IL-17上调表达,而IL-10、TGF-β1表达下降;大鼠MST为(18.3±1.15)d、(18.3±0.72)d。与G2、G3相比,G4、G5及G6大鼠发生急性排斥反应的时间均明显延迟,直至14d才出现轻度排斥表现;肝功能部分改善,术后出现ALT、TBIL再次升高及肝功能衰竭的时间明显推迟;肝脏病理学呈BanffⅠ～Ⅱ级排斥反应;G5、G6各时段移植肝PD-L1蛋白的表达随时间逐渐增强,G4无明显变化;移植肝CD4+、CD8+、CD25+细胞浸润明显减少,尤以CD8+细胞为著(P<0.05);移植肝细胞因子IFN-γ、IL-17含量显著下降,而IL-10、TGF-β1含量明显升高,差异显著(P<0.05);受体MST明显延长,但未获得长期存活,仅为(28.7±0.98)d、(29.3±1.47)d。PD-L1基因转染联合亚剂量CsA治疗的大鼠各时段肝功能损害进一步减轻,RAI评分显著降低,受体MST明显延长至(34.0±1.49)d (P<0.05)。
四、结论
1.采用改良“二袖套法”成功建立了LEW-BN组合大鼠肝移植急性排斥模型。
2.冷保存期经门静脉灌注PD-L1-AAV2-RFP夹闭法转染供肝,可安全、有效地将目的基因转染至大鼠移植肝内并分泌功能性蛋白。
3.在本实验条件下,采用PD-L1-AAV2-RFP(1×1011v.g./只)经门静脉灌注夹闭冷保存2h是介导足量蛋白表达的理想方案。
4.腺相关病毒介导的PD-L1基因转染对移植肝具有显著的保护作用,可延长移植肝存活,且与CsA有协同效果。
5. PD-L1基因可抑制肝移植急性排斥反应,其机制可能是PD-L1基因转染增强了PD-L1/PD-1信号通路,通过抑制移植肝CD4+、CD8+、CD25+T细胞浸润及其功能,在上调细胞因子IL-10、TGF-β1表达的同时,下调INF-γ、IL-17的表达,从而发挥对移植肝的保护作用。
Background
Liver transplantation is an effective or even only approach of treatment to some end stage liver diseases, but acute rejection remains to be the primary clinical problem and challenge of transplantation. The outcome of allogeneic organ transplants is, in part, dependent on the balance between co-stimulatory and modulatory signals that accompany TCR-mediated recognition by recipient’s T cells. The local expression of a gene encoding an immunosuppressive protein within a graft can generate local immunosuppression, which enhanced co-inhibitory signals in recipients, making gene therapy a viable approach for facilitating transplantation.
Programmed death ligand-1 (PD-L1), a newer member of the CD28 family, has been implicated as a negative regulator of T cell activation. In previous research, engagement of programmed death-1 (PD-1) by PD-L1 inhibited proliferation and cytokine production by activated T cells. Conversely, blockade of PD-L1 using specific monoclonal antibodies stimulated T cell proliferation and cytokine production. Meanwhile, recent studies showed that interfering with the PD-L1/PD-1 co-stimulatory pathway prolonged allograft survival in various organ transplantation models, suggesting that they might play an important role in maternal acceptance of liver allograft. Thus, the recombinant PD-L1 gene was chosen as the immunomodulator.
This study focused on establishing acute rejection model of orthotopic liver transplantation in inbred rats. The effective way of transferring the target gene into the liver allograft with PD-L1-AAV2-RFP was also explored. Furthermore, we investigated the protective effects and mechanisms of modification of liver allograft with target gene on the survival in vivo. The main results and conclusions were as follows:
Part 1 Establishment of acute rejection model of orthotopic liver transplantation in inbred rats
Objective To establish acute rejection model of orthotopic liver transplantation in inbred rats.
Methods We established LEW-BN rat orthotopic liver transplantation model using“the innovated two-cuff technique”. Experimental groups included:①G1: Isogenic (ISO) group.②G2: Allogeneic (ALLO) group.③G3: CsA group injected CsA (2.5mg/kg.d) intraperitoneally daily from day 0 to day 7 postoperatively. Recipients were sacrificed on POD 3, 7, 14, and 21 respectively. Liver tissues and blood samples were collected. The general states, median survival time (MST), and histopathological characteristic of recipients were observed. Serum levels of ALT and TBIL were measured with automatic biochemical analyser.
Results In G2, typical mild, moderate and severe acute rejection occurred on POD 3, 7 and 14 respectively, and the delayed occurrence of graft rejection were observed in G3. The MST of G3 (28.7±0.98)d was prolonged significantly compared with that of G2 (18.3±1.15)d (P<0.05). Serum ALT and TBIL levels of G2 increased obviously on POD 3 and has been booming ever since. Serum ALT and TBIL levels of G2 were significantly higher than those of G1 and G3. Typical histological change occurred on POD 14 in G2 and the light rejection in G3. There was no significant difference in rejection activity index (RAI) scores between G2 and G3 on POD 3. Whereas on d7, d14 and d21 after operation, RAI scores of G2 were marked higher than that of G3 and G1 (P<0.05).
Conclusions
1. Rat orthotopic liver transplantation of LEW to BN strain is an ideal acute rejection model.
2. The model has stable and typical allograft rejection. It can be used in the study of liver transplantation immunity.
Part 2 The security and effectiveness of recombinant adeno-associated virus transfecting PD-L1 gene into livers of donor
Objective To find the effective way of transfecting PD-L1 gene into livers of donor with recombinant adeno-associated virus.
Methods The PD-L1-AAV2-RFP was identified by PCR. The transfection efficiency of PD-L1-AAV2-RFP was determined by observing the expression of RFP in BHK cell with fluorescence microscope. We established the PD-L1-AAV2-RFP gene transfer model and recipients were randomly divided into three groups:①G1: ISO group.②G2: AAV group introduced through back-table portal vein perfusion with AAV2-RFP for 2 hours, and the graft was flushed with Ringer’s lactate solution to remove any free virus before implantation.③G3: PD-L1 group introduced with PD-L1-AAV2-RFP. Liver allografts harvested on POD 3, 7, 14, and 21 were fixed in 10% buffered formalin and embedded in paraffin for histological study by hematoxylin-eosin staining. Part of the grafts were frozen for RFP expression analysis. PD-L1 mRNA and protein expression in grafts were detected by using Realtime RT-PCR and IHC respectively. The function of liver was also measured. Results The fragment was verified by PCR analysis to make sure that the cDNA is just the one we expected. Evident red fluorescence was observed and the transfection rate increased with the prolongation of time, suggesting that the donor liver might be transfected efficiently by PD-L1-AAV2-RFP. Serum levels of ALT and TBIL increased obviously on POD 3 and 7 respectively, then gradually returned back to normal with time. Pathological changes of the specimens showed only mild ischemia-reperfusion injury on POD 7, with no statistical difference among three groups (P>0.05). Red fluorescence was not observed in sections of heart, lung, kidney, and spleen after transplantation. However, evident red fluorescence was observed in sections of livers after operation except day 3 and increased in the later. The expressions of PD-L1mRNA and protein were both upregulated in the liver of donor at 7th day after transfection, and reached its peak at 21st day in G3, but still lower in G1 and G2.
Conclusions
1. PD-L1-AAV2-RFP is effective and safe for gene delivery in rat liver transplantation.
2. Back-table perfusion through the portal vein with PD-L1-AAV2-RFP (1×1011v.g./animal) and preservation of grafts for 2 hours is the optimal protocol to induce sufficient protein expression.
Part 3 The protective effects of PD-L1 gene transfer mediated by AAV-2 on liver allograft of rat and its mechanisms
Objective To evaluate the protective effects and mechanisms of the liver allograft transfected with PD-L1 gene in vivo.
Methods Recipients were randomized into six groups:①G1: ISO group.②G2: ALLO group.③G3: AAV group introduced with AAV2-RFP.④G4: CsA group injected CsA (2.5mg/kg.d) intraperitoneally daily from day 0 to day 7 postoperatively.⑤G5: PD-L1 group introduced with PD-L1-AAV2-RFP.⑥G6: Combination therapy group introduced with PD-L1-AAV2-RFP plus a subtherapeutic regimen of CsA. We established ROLT model using“the innovated two-cuff technique”. Gene delivery to donor liver was done by intracoronary instillation of virus containing solution (1×1011v.g./animal) through the portal vein. On POD 3, 7, 14 and 21, grafts were taken for IHC to assess the expression of PD-L1.Ig, CD4+, CD8+ and CD25+ lymphocytes, for ELISA to analysis the concentration of IFN-γ, IL-17, IL-10, and TGF-β1 respectively. The MST, liver function and histopathological changes were also observed.
Results In G1, no acute rejection was found, biochemical datas showed that the levels of ALT and TBIL increased during the first weeks after operation and decreased to normal with time. Fewer PD-L1-positive cells in the graft were detected. The expression of IFN-γ, CD4+, CD8+, and CD25+ lymphocytes were not observed in various period of time. Otherwise, IL-17, IL-10, and TGF-β1 were all low-expressed. The MST was (106.7±0.24)d. In G2 and G3, typical mild, moderate and severe acute rejection occurred on POD 3, 7 and 14 respectively. The function of liver were markedly higher than those of other groups (P<0.05). Fewer PD-L1-positive cells in the graft were detected. A lot of CD4+, CD8+ and CD25+ lymphocytes infiltrated in portal area of grafts, especially CD8+ T cells (P<0.05). On POD 3, the concentration of IFN-γand IL-17 in grafts increased continuously and even decreased on POD 21, while the expression of IL-10 and TGF-β1 were down-regulated gradually. The MST were (18.3±1.15)d and (18.3±0.72)d. In G4, G5 and G6, acute rejection still occurred after operation with light rejection sign in histopathological change. The function of gene transfected liver was inferior to that of G2 and G3 significantly (P<0.05). Although fewer PD-L1-positive cells were detected in G4, a certain number of PD-L1-positive cells were found in G5 and G6. Compared with G2 and G3, those allografts treated with PD-L1 or CsA had a significantly reduced cellular infiltrate, especially CD8+ cell. The concentration of IFN-γand IL-17 in grafts were greatly inhibited, accompanying with greatly increased secretion of IL-10 and TGF-β1. Allografts transduced with the PD-L1 survived for longer periods of time (P<0.05). PD-L1 gene transfer combined with a subtherapeutic regimen of CsA was superior to CsA alone: (34.0±1.49)d vs. (28.7±0.98)d.
Conclusions
1. Adeno-associated virus mediated PD-L1 gene delivery significantly prolongs liver allograft survival in a rat model.
2. Enhanced expression of PD-L1 protein in donor liver attenuates acute allograft rejection. The effect is additive to that of a subtherapeutic regimen of CsA.
3. PD-L1 gene may take the effect in liver transplantation via reducing intragraft infiltrates of T lymphocytes, suppression of INF-γand IL-17 production as well as stimulation of IL-10 and TGF-β1 production.
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