【作者】 叶文学；

【导师】 沈振亚；

【作者基本信息】 苏州大学 ， 胸心血管外科， 2010， 博士

【摘要】 背景:器官移植现在已经成为治疗终末期器官功能衰竭的有效手段,为缓解临床同种供体的不足,异种器官作为最可能的供体来源越来越受到重视。然而,异种器官移植作为远缘移植,其面临的排斥反应较同种移植更为强烈、复杂。异种器官移植排斥反应包括超急排(HAR)、延迟性排斥(DXR)、急性细胞性排斥(ACR)、移植物慢性失功(CGD)四个阶段。目前HAR被大量研究普遍认为已有方法(转基因猪、天然抗体抑制剂等)克服,接下来异种移植研究亟待克服的主要障碍是DXR,在这一过程中,由诱生抗体诱导的移植物Ⅱ型血管内皮细胞激活是异种移植物被排斥、功能丧失的关键。而核转录因子κB(Nuclear Factor-κB,NF-κB)对Ⅱ型血管内皮细胞激活起关键性作用。现已表明NF-κB的功能涉及到免疫反应、胸腺发育、胚胎发生、炎症和急性反应、细胞繁殖、细胞凋亡、病毒感染和其他多种病理过程。但尚未见有应用于研究心脏移植抗排斥方面。小鼠→大鼠心脏移植不发生HAR,而直接表现DXR,是研究DXR的理想动物模型。我们现拟利用RNA干扰(RNAi)技术特异性地抑制小鼠心脏核因子κB(NF-κB)的表达,以期抑制异种移植延迟性排斥反应中Ⅱ型血管内皮细胞的激活,从而克服或减弱DXR,探讨RNA干扰技术在异种器官移植中的应用前景。方法:1.设计并化学合成针对小鼠NF-κB p65的siRNA,合成时对用于转染后示踪的部分siRNA进行荧光素FAM标记。2.小鼠血管内皮细胞(EOMA)的体外转染。分组:(1)空白对照组(细胞内不加任何干扰因素);(2)阴性对照组(细胞转染阴性对照siRNA,即siRNA-NC);(3)实验组(细胞转染NF-κB siRNA)。于转染前一天,接种EOMA细胞,在转染后的24h~72h时提取各组细胞总RNA,行RT-PCR测定细胞内NF-κB p65 mRNA水平;提取各组细胞总蛋白,行Western blot测定NF-κB p65蛋白表达。3.将转染混合物(含2OD阴性对照siRNA)经颈静脉或尾静脉注入实验小鼠。48h后获取心、肺、肝、肾标本,快速冰冻切片荧光显微镜观察体内组织分布,对尾静脉途径与颈静脉途径对组织分布的影响进行比较。4.将转染复合物(含2OD siRNA-NC)经颈静脉注入小鼠,在从1小时到1周的五个时间点摘除心、肝、肺、肾四个器官切片行荧光显微镜检查。将2OD NF-κB p65siRNA和阳离子聚合物in vivo-jetPEI形成的复合物静脉注入成年小鼠,注射后第1、3、5、7天的五个时间点摘取心脏,提取总RNA,行Real-time PCR检查各时间点NF-κB p65mRNA水平动态变化。5.为分析剂量效应关系,实验分五组,每组3只小鼠,四组动物单次注射siRNA和in vivo-jetPEI复合物,siRNA量各组分别为1OD, 2OD, 3OD和4OD,in vivo-jetPEI量按N/P ratio=6和siRNA的量相应配置。其中一假手术组注射同等体积PBS溶液作对照。72h后获取小鼠心脏,行定量RT-PCR测定NF-κB p65 mRNA的表达。6.实验分四组,每组三只小鼠均被注射相同剂量的复合物(含2OD siRNA),实验动物的存活情况被连续观察一周。第1、3、5、7天抽血检测血清主要生化指标。7.分别将siRNA-NC和NF-κB p65 siRNA经in vivo-jetPEI乳化形成的复合物(含2ODsiRNA)一次性经颈静脉注入小鼠,注射后24h摘取心脏,提取总RNA,行Real-time PCR检测NF-κB p65和下游基因ICAM-1、VCAM-1、IL-1a mRNA水平。8.将2ODsiRNA-NC和NF-κB p65 siRNA经in vivo-jetPEI乳化形成的复合物经颈静脉转染小鼠,48h后小鼠心脏移植至大鼠颈部,观察供心存活时间、病理学改变、心脏NF-κB p65的表达和相关粘附分子、细胞因子基因mRNA水平以及大鼠体内诱生抗体移植前后水平变化。结果:1.NF-κB p65 siRNA和阴性对照siRNA均能被阳离子脂质体转染培养细胞,转染效率几近90%。RT-PCR检测发现转染NF-κB p65 siRNA后mRNA的表达水平下调明显,而转染阴性对照siRNA则无干扰效应。2.经尾静脉途径注射,siRNA主要分布于肝脏,心脏和肺分布极少;经颈静脉途径注射则心脏和肺可以获得较多的分布。3.将转染复合物(含2OD siRNA-NC)一次性经颈静脉注入小鼠,结果:在注射后1小时仅肝和肺看到荧光染色,24小时后在心、肺、肝、肾四个器官都看到了荧光染色,并在48h至72h间达到最高,1w时又见减弱。4.将2OD NF-κB p65 siRNA转染复合物经颈静脉注射后第1、3、5、7天的五个时间点监测到NF-κB p65基因表达均受到抑制,沉默效应大于50%,与假手术组和正常小鼠相比P<0.05。5.目的基因NF-κB p65mRNA表达在2OD~4OD剂量组出现明显下调(与1OD组相比较P<0.05),但各组间差别不大(P>0.05),而1OD组表达下降程度轻微(与假手术组相比P>0.05)。6.转染动物均存活,其活力和食欲无任何改变;除了一只小鼠在注射后第一天ALT升高至337U/L,第三天降至正常范围外,所有受试动物的主要生化指标在转染后均未受明显影响。7.剂量为2OD时NF-κB p65 siRNA体内转染小鼠,其心脏NF-κB p65和下游基因ICAM-1、VCAM-1、IL-1a mRNA的表达均下调。8.转染siRNA 48h后,小鼠心脏移植至大鼠颈部。无处理组(单纯将小鼠心脏移植到大鼠颈部)供心平均存活时间为(1.80±0.83)d。对照组(转染siRNA-NC的小鼠心脏移植到大鼠颈部)供心存活时间无延长(与无处理组比较,P>0.05),平均存活时间为(1.60±0.87)d。实验组(转染NF-κB p65 siRNA的小鼠心脏移植到大鼠颈部)见供心存活时间明显延长(与无处理组比较,*P<0.01),移植物平均存活时间为(5.17±1.63)d。9.供心未排斥时,对照组和实验组移植心脏光镜下可见心肌组织排列整齐,部分细胞可见轻度水肿,间质炎症细胞浸润和少量淋巴细胞浸润;电镜下血管内皮肿胀在对照组表现较明显,而实验组表现为部分内皮细胞凋亡。发生排斥时,对照组和实验组供心光镜下见心肌细胞广泛水肿,排列变紊乱,心肌组织间大量的出血,大量炎细胞和少量单个核细胞和淋巴细胞浸润;电镜下实验组和对照组一致呈现部分血管内皮细胞胞质肿胀、粗面内质网增生的激活表现。10.阴性对照组未排斥时供心NF-κB p65 mRNA表达升高,但与正常心肌相比P>0.05;排斥时供心NF-κB p65 mRNA高表达,与正常心肌相比较P<0.05。实验组未排斥时供心NF-κB p65 mRNA表达明显下降,与正常心肌相比较<0.05;排斥时供心NF-κB p65 mRNA仍高表达,但与正常心肌相比较P>0.05。11.阴性对照组供心水平在移植后较正常小鼠心脏明显升高(siNCn VS N, p<0.05; siNCp VS N, p<0.01),且排斥后较排斥前升高更明显(siNCp VS siNCn, P<0.05)。实验组供心VCAM-1 mRNA水平在移植后较正常小鼠心脏明显下降(p<0.01),排斥后表达又明显升高,高于正常水平且与正常心脏相比p<0.01。12.阴性对照组供心移植后ICAM-1 mRNA水平较正常小鼠心脏明显升高(p<0.05),且排斥前后无明显差别(P>0.05)。实验组供心移植后ICAM-1 mRNA水平较正常小鼠心脏明显下降(p<0.05),排斥后表达又升高,但与正常心脏相比别p>0.05。13.阴性对照组供心移植后IL-1a mRNA水平较正常小鼠心脏明显升高(p<0.01),且排斥前后无明显差别(P>0.05)。实验组供心移植后IL-1a mRNA水平较正常小鼠心脏明显下降(p<0.05),但排斥后表达明显升高,与正常心脏相比别p<0.01。14.无处理组(单纯将小鼠心脏移植到大鼠颈部)、对照组(将转染siRNA-NC的小鼠心脏移植到大鼠)、实验组(将转染NF-κB p65 siRNA的小鼠心脏移植到大鼠颈部)三组间受体外周血IgM、IgG水平在移植前、移植后均无明显差异(P>0.05);各组的IgM、IgG水平在移植后较移植前均明显升高(P<0.05)。结论:1.阳离子脂质体Lipofectamine?2000可在小鼠EOMA细胞高效转染化学合成的NF-κB p65 siRNA。2.细胞转染NF-κB p65 siRNA可实现内源性RNA降解,抑制相应的功能蛋白表达;本实验设计针对NF-κB p65的siRNA序列有效,适宜进行体内转染研究。3.颈静脉注射途径是靶向心脏体内转染研究的理想途径。4.颈静脉注射NF-κB p65 siRNA/in vivo-jetPEI复合物能介导ICR小鼠心脏的NF-κB p65基因表达沉默;且获得50%以上沉默效应的siRNA最小剂量为2OD。5. NF-κB p65 siRNA(剂量2OD)转染ICR小鼠后48h至72h心脏siRNA表达最高,是进行心脏移植的最佳时间。6.剂量为2OD时NF-κB p65 siRNA体内转染对ICR鼠是安全的。7.体内转染NF-κB p65 siRNA(剂量2OD)可抑制小鼠心脏NF-κB p65蛋白水平,实现NF-κB下游靶基因转录的有效抑制。8.体内转染NF-κB p65 siRNA(剂量2OD)使小鼠供心存活时间延长。9.体内转染NF-κB p65 siRNA(剂量2OD),小鼠供心未排斥时血管内皮细胞表现为凋亡,而发生排斥时表现为激活。10.体内转染NF-κB p65 siRNA(剂量2OD)可抑制心脏移植后内环境刺激下的供心NF-κB表达和下游基因ICAM-1、VCAM-1、IL-1αmRNA水平。更多还原

【Abstract】 Background:Allotransplantation is nowadays a generally accepted treatment for organ failure, and xenotransplantation, i.e. transplantation of cells, tissues or organs between individuals of different species, is considered a promising, possible solution to the chronic lack of donor organs. Organs of phylogenetically-distant species are subject to much more severe and irreversible rejection reactions than that in allograft. Xenotransplantation rejection includes four stages: hyperacute rejection (HAR), delayed xenograft rejection (DXR), acute cellular rejection (ACR) and chronic graft dysfunction (CGD). It is generally acknowledged that there are methods to overcome the first major barrier of xenotransplantation (HAR), for example, the availability of transgenic pigs and antibody inhibition. The second immune barrier is the delayed or acute vascular xenograft rejection (DXR or AVR) in which the activation of endothelialⅡcells is the key factor of xenograft rejection and graft dysfunction, and the NF-κB takes an important role in the activation of endothelialⅡcells. NF-κB is often associated with immune reaction, thymus gland growth, embryogenesis, inflammation, acute reaction, cells reproduction, apoptosis, viral infection and other diverse pathological processes. Though suppression of the activation of endothelialⅡcells by inhibiting the expression of NF-κB were reported in other fields, there are few studies about NF-κB in the research of heart xenotransplantation by now. Our objective is to delay or weaken delayed xenograft rejection by suppressing the expression of target gene NF-κB using in vivo siRNA in mice. Methods:1. siRNA directed against mouse NF-κB p65 was designed and chemically synthesized based on the oligonucleotide sequences from NCBI. For image identification purpose, a part of the siRNAs were carboxyfluorescein (FAM)-labeled.2. EOMA cell was divided into three groups: (1) blank control group, (2) negative control group, (3) NF-κB siRNA group. One day before transfection, EOMA cell was reseeded in plate. 24h-72h after transfection, total RNA was isolated from EOMA cells using TRIzol Reagent, the expression level of NF-κB p65 mRNA was evaluated by RT-PCR. Total protein was isolated from EOMA cells, and the expression level of NF-κB p65 protein was evaluated by Western blot.3. To determine the tissue distribution of siRNA in mice, FAM-labeling siRNA/in vivo-jetPEI complexes (included 2 OD siRNA-NC) were injected into mice through jugular vein or tail vein, and the epifluorescence activity was examined in various organs 48h later.4. The mice were, via jugular vein, treated with single dose i.v. injections of NF-κB p65 siRNA/in vivo-jetPEI complex (dose: 1OD). Four different organs were dissected at five time points (from 1h to 1w) for the examinations by epifluorescence microscopy. A single dose of NF-κB p65 siRNA/in vivo-jetPEI complex (dose: 2OD) was injected into immune competent mice, then we dissected heart tissue at four time points (from 1d, 3d, 5d, to 7d) for the examination of NF-κBp65 mRNA by RT-PCR and compared the levels of NF-κBp65 mRNA at different time points.5. Correlate the siRNA uptake and distribution with the efficacy of RNAi in particular organs in the following experiments. The mice (3 per cohort) were, via jugular vein, treated with a single dose i.v. injections of NF-κB p65 siRNA/in vivo-jetPEI complex (dose: 1OD, 2OD, 3OD and 4OD siRNA and the volumes of in vivo-jetPEI according with the ratio of N/P =6). An additional group of mice (the sham operated group) were treated in parallel with PBS solution for unspecific effects. RT-PCR was applied to demonstrate the RNAi silencing in the heart after 72h. 6. The mice in the research were divided into 4 groups. Three animals in each group were injected with NF-κB p65 siRNA-/in vivo-jetPEI complex (dose: 2OD). The conditions of the survival, appetite and vigor of each animal had been observed for 7 consecutive days, and a comprehensive clinical biochemistry test was performed 1, 3, 5 and 7days after injection.7. The mice were, via jugular vein, treated with single dose i.v. injections of siRNA/in vivo-jetPEI complex (included 2OD NF-κB p65 siRNA or siRNA-NC). RT-PCR was applied to demonstrate the RNAi silencing in the heart after 24h. The levels of NF-κBp65, ICAM-1, VCAM-1 and IL-1a mRNA were evaluated.8. The mice were treated with siRNA/in vivo-jetPEI complex (included 2OD NF-κB p65 siRNA or siRNA-NC) via jugular vein. 48h after mouse-to-rat cardiac heterotopic xenotransplantation in neck, survival time, pathological changes, and levels of NF-κBp65, ICAM-1, VCAM-1 and IL-1a mRNA in xenografts were investigated. Serum IgM/IgG concentration in rat before operation were compared with those after operation.Results:1. NF-κB p65 siRNA and siRNA-NC were as capable as absorbed in EOMA cells under the help of the vehicle lipofectamine2000, and the transfection efficiency is nearly 90%. Distinct from siRNA-NC, after the NF-κB p65 siRNA transfection, the silencing of gene expression in EOMA cells could be observed.2. Luc activity of siRNA in the liver and kidney was much higher than that in the heart and lung 48 hours after tail vein injection. In animals with jugular vein injection the Luc activity was high in the heart and lung, similar to those in the liver and kidney.3. A single dose of FAM fluorescently labeled siRNA-NC combined with in vivo-jetPEI (included 2OD siRNA-NC) was injected into ICR mice, and four different organs were dissected at five time points (from 1h to 1w) for the examinations by epifluorescence microscopy. An initial microscopic analysis revealed that fluorescence was detectable only in the lungs and livers 60 min post-treatment with siRNA-NC/PEI complex. The FAM-fluorescence appeared in all tissues 24h after complex treatment, though the fluorescence was not strong. The intensity of FAM-fluorescence of the four organs (heart, lung, liver and kidney) increased to the highest point between 48h and 72h after injection,and decreased again 1w after injection.4. The mRNA levels in each group after siRNA treatment demonstrated that the NF-κB p65 mRNA levels decreased significantly (>50%) in 7 days (from 1d, 3d, 5d, to 7d), which were statistically different from the sham operated group (P<0.05).5. Only in the samples derived from the animals treated with 2OD~4OD siRNA, the NF-κB p65 mRNA levels were significantly reduced (>50%, P<0.05), as revealed by the respective mRNA quantification. In these three groups the expression of the target gene were inhibited similarly (P>0.05), whereas in the 1OD group the expression of NF-κB were inhibited slightly (P>0.05).6. Observed for 7 consecutive days after injection, the conditions of the survival, appetite and vigor of each animal had not been impaired till the observation time point. All the biochemical parameters evaluated were in the normal range as compared with those of the normal animals except the ALT value on day 1. There was a transient increase up to 337 of the ALT value on day 1 in the animals injected with complex, and the ALT value falls to a normal range 3 days after the injection.7. The levels of NF-κB p65, ICAM-1, VCAM-1 and IL-1a mRNA decreased markedly after the mice were treated with 2OD NF-κB p65 siRNA.8. The MST of xenograft in group without treatment was (1.80±0.83) d, that in control group was (1.60±0.87) d (vs. group without treatment, P>0.05). The MST of xenograft in experiment group was (5.17±1.63) d (vs. group without treatment, P<0.01).9. Cardiac muscle cells of xenograft before rejection in negative control group and experiment group lined up in order, light edema were seen in some cells, inflammatory cell and lymphocyte infiltration was detected. Endothelium of graft in negative control group exhibited typical swelling cytoplasm, but apoptosis in experiment group by electron microscope. Chaos and severe edema of cardiac muscle cells were seen in xenograft in negative control group and experiment group after rejection. A lot of inflammatory cells and some lymphocytes infiltrated between cardiac muscle cells. Endothelium of graft both in negative control group and experiment group exhibited typical activated appearance (swelling cytoplasm, an expansion of the rough endoplasmic reticulum, indicating that the endothelium is metabolically active and not undergoing cell death by apoptosis) by electron microscope.10. With siRNA-NC transfection, NF-κB p65 mRNA level of graft increased lightly in control group before rejection, vs. normal cardiac muscle, p>0.05. After rejection NF-κB p65 mRNA level of graft increased markedly, vs. normal cardiac muscle, p<0.05. With NF-κB p65 siRNA transfection, NF-κB p65 mRNA level of graft decreased markedly before rejection, vs. normal cardiac muscle, p<0.05. After rejection NF-κB p65 mRNA level of graft was yet high, but vs. normal cardiac muscle, p>0.05.11. The VCAM-1 mRNA level of heart in negative control group increased after transplantation (siNCn VS N, p<0.05; siNCp VS N, p<0.01; siNCp VS siNCn, P<0.05). The VCAM-1 mRNA level of heart in experiment group decreased markedly after transplantation, and increased after rejection (siP65n VS N, p<0.01; siP65p VS N, p<0.01).12. The ICAM-1 mRNA level of heart in negative control group increased after transplantation (siNCn VS N, p<0.05; siNCp VS N, p<0.05; siNCp VS siNCn, P>0.05). The ICAM-1 mRNA level of heart in experiment group decreased markedly after transplantation, and increased after rejection (siP65n VS N, p<0.05; siP65p VS N, p>0.05).13. The IL-1a mRNA level of heart in negative control group increased after transplantation (siNCn VS N, p<0.01; siNCp VS N, p<0.01; siNCp VS siNCn, P>0.05). The IL-1a mRNA level of heart in experiment group decreased markedly after transplantation, and increased after rejection (siP65n VS N, p<0.05; siP65p VS N, p<0.01)14. Serum IgM、IgG concentration in rats had no deference between three groups whether pre- or post-transplantation(P>0.05), and increased markedly after transplantation than pre-transplantation(P<0.05) in each group.Conclusions:1. Cationic liposome Lipofectamine? 2000 could transfect chemically synthesized siRNA to EOMA cells with high effect. 2. Introduction of NF-κB p65 siRNA to EOMA cells could abrogate endogenenous gene expression, the siRNA designed in the experiment was functional in vitro and suitable for in vivo application.3. Jugular vein is an effective transfection route for gene delivery targeting for hearts.4. Administered with NF-κB p65 siRNA/in vivo-jetPEI complex by jugular vein injection leads to successful inhibition of expression of NF-κB p65. The minimum dose of siRNA was 2OD so that the NF-κB p65 mRNA levels decreased significantly (>50%).5. After 2OD NF-κB p65 siRNA administrated, the absorption of siRNA in heart of mouse increased to the highest point between 48h and 72h which was the best time for a mouse to be transplanted to a rat.6. It was safe to a mouse which was transfected a single dose of 2OD NF-κB p65 siRNA in vivo.7. Inhibition of protein transcription of NF-κB p65 by siRNA could efficiently suppress the expression of NF-κB’s target genes.8. Being transfected with 2 OD NF-κB p65 siRNA extended the survival time of cardiac xenograft in mouse-to-rat model.9. Endothelium of graft in the siRNA treated mouse exhibited evidence of apoptosis before rejection and activation after rejection.10. Being administered with 2 OD NF-κB p65 siRNA led to inhibition of expression of NF-κB p65 and its target genes such as ICAM-1、VCAM-1 and IL-1αunder the internal environment after cardiac xenotransplantation.更多还原