【作者】 王素梅；

【导师】 刘树业；

【作者基本信息】 天津医科大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 器官移植是众多终末期器官功能衰竭患者的唯一选择。虽然高效免疫抑制剂的应用明显提高了肝移植患者的生存率,但急性排斥反应(Acute Rejection, AR)仍然是肝移植术后最严重的并发症之一,其发生率仍然高达40%-60%,因此免疫耐受仍然是肝移植研究的重点之一。新兴的蛋白质组学是对基因组所编码的所有蛋白质进行研究的学科。通过蛋白质组学使我们对肝移植免疫耐受的发生、发展有了更详尽的理解。为了观察两种不同品系即近交系Wistar和封闭群SD大鼠原位肝移植术后的急性排斥反应及F蛋白诱导肝移植免疫耐受的情况。我们将健康雄性清洁的近交系Wistar和封闭群SD大鼠随机分为3组,A：同基因对照组：SD→SD；B：急性排斥组：Wistar→SD;C:F蛋白干预组：术前一周胸腺内注射0.4mg。采用Kamada二袖套法,建立上述Wistar和SD组合之间的肝移植模型。A、B和C组受体分别于术后3d、6d、9d及100d,四个时间点各杀死三只大鼠,收集肝组织和血清分别做相应检测,另留取部分新鲜肝组织立即置入液氮中保存备用。剩余的大鼠用于观察肝移植术后长期生存率。实验过程中共完成108例次大鼠原位肝移植(rat orthotopic liver transplantation, ROLT)操作,总的手术成功率为62.96%(68/108),其中模型训练的前期完成45例,24h成活率仅24.44%(11/45)以后随着手术技巧成熟,手术成功率逐渐达到90%以上。大鼠肝移植手术死亡的主要原因为出血、麻醉意外、无肝期太长、血栓形成等。A组同基因对照组,术后组织学检查无排斤反应表现。B组急性排斥组自术后3d开始,出现Ⅰ级排斥反应,6d以后逐渐达到高峰,大多数死于术后12-21d。C组F蛋白干预组亦未见确切排斥反应证据。结果表明：(1)二袖套法操作简便,手术成功率高,可作为肝移植实验的有效手段。(2)成功建立大鼠原位肝移植模型的关键在于完善的手术技术,主要包括高质量的供肝获取、熟练的显微血管吻合技术和尽量短的无肝期。(3)近交系Wistar和封闭群SD大鼠之间建立的原位肝移植模型,能满足急性排斥反应或免疫耐受实验研究的需要。(4)F蛋白可以诱导大鼠肝移植免疫耐受。应用超高效液相色谱串联质谱法(nanoUPLC-MS/MS)研究大鼠原位肝移植术后7天免疫耐受组和同基因对照组差异表达蛋白质的情况,对大鼠原位肝移植后的免疫耐受情况进行初步研究。建立免疫耐受组(Wistar→SD+F蛋白)n=5,建立同基因对照组(SD→SD)n=5.分别于术后7天处死5只受体,取肝组织,提取总蛋白,Trypsin酶解后,使用Waters公司独家专利MSe无标记定量技术,通过利用nanoUPLC-MS/MS技术对酶解后的肽段进行分析,数据库检索鉴定蛋白质,生物信息学工具对所鉴定的蛋白质进行功能分类。结果显示免疫耐受组和同基因对照组相比,两样品中同时存在的大部分蛋白上调,其中9个蛋白点表达水平明显上调,比值变化在2.59倍以上。结合数据库搜索得到鉴定,这些明显上调的蛋白的分子功能主要与细胞骨架、离子结合、氧化应激反应、能量代谢等相关。结果表明本研究建立了利用nanoUPLC-MS/MS技术对大鼠原位肝移植进行蛋白质组学分析的方法,对F蛋白诱导大鼠原位肝移植免疫耐受进行了初步研究,为建立动物模型提供量化的指标,为发现潜在的生物标记物提供方向和数据支持。更多还原

【Abstract】 Organ transplantation is the treatment of choice for patients with end-stage organ failure. The incidence of acute rejection (AR) of liver allograft is reported to be between 40% and 60%. Therefore, the immune tolerance of liver transplantation remains one of the keys. High throughput genomics and proteomics techniques have facilitated a better understanding of immune tolerance.To observe the acute rejection and the immune tolerance after orthotopic liver transplantation in rats between inbred line Wistar and closed colony SD. We established models with Kamada’s two cuff technique and then randomly divided into three groups:Group A:SD→SD;Group B:Wistar→SD; Group C:F protein intervention:intrathymic injection one week before surgery. Three recipients were killed after 3d、6d、9d and 100d. Representative liver allografts tissue and serum specimens were collected for corresponding detection. Remaining recipients were observed for long-term survival. During the experiment 108 models were established, the total success rate was 62.96%(68/108). The success rate was only 24.44%(11/45) during the protophase of model manufacture. Afterward the success rate gradually comes up to more than 90%.The causes of death were hemorrhage, anesthetic accident, long anhepatic phase, thrombus formation and so on. In recipients of A group, rejection was not founded. In recipients of B group, I grade rejection occurred at 3rd day and rejection gradually reached the peak 6 days after transplantation, and most died between 12 to 21 days after operation. In recipients of C group rejection was not founded either. Conclusions:(1) Two cuff technique may provide a practical and stable experimental model.(2)The keys of establishing successful models include high quality donor liver, practiced microsurgical techniques and short anhepatic phase.(3) The model can be used to study acute rejection or immunologic tolerance effectively. (4) F protein can induce immune tolerance in liver transplantation. Application of nanoUPLC-MS/MS technology to research the differentially expressed proteins at 7 days after rat liver orthotopic transplantation between immune tolerance group and syngeneic control group and to have a preliminary study of the immune tolerance. Establish immune tolerance group(Wistar→SD+F protein) n=5, establish syngeneic control group (SD→SD) n=5. After 7 days kill five receptors, obtain liver tissues, extract total protein, after Trypsin hydrolysis, use Waters exclusive patented MSe unmarked quantitative techniques, through the technology of nanoUPLC-MS/MS analyse the peptide hydrolysis, database search identify the protein, bio-informatics tools for the identification of protein functional classification. Comparing the two groups, the two samples simultaneously exist in most of the proteins increases, of which 9 protein spots were significantly up-regulated expression levels, changes in the ratio of 2.59 times. Combination of database search to be identified, these were significantly increased in the main function of the protein molecules with the cytoskeleton, ion binding, oxidative stress response, energy metabolism and other related. This study established a proteomic analysis methods for rat orthotopic liver transplantation with nanoUPLC-MS/MS, carried out a preliminary study for F protein induced immune tolerance after rat liver transplantation, provided quantitative indicators for the establishment of animal models and provided direction and data support for identifying potential biological markers.更多还原