大鼠肝移植急性排斥模型的建立

发布时间：2018-11-15 06:57

【摘要】： 背景随着外科技术的发展和强有效的免疫抑制剂的临床应用,肝脏移植已经成为治疗慢性终末期肝病、急性肝功能衰竭等疾病的主要治疗方式。临床肝脏移植技术的发展有赖于大量动物实验研究,实验大鼠由于其价格低廉、遗传背景清晰、饲养管理的简捷可控性使其成为众多实验动物的首选。大鼠肝脏移植模型是研究肝脏移植器官保存、缺血—再灌注损伤、血流动力学、免疫抑制剂、移植排斥反应及免疫耐受等方面的理想动物模型。自1973年Lee[1、2]首次建立大鼠肝脏移植模型,经过30多年众多中外学者的改进,现在改良的Kamada“二袖套法”已成为广泛采用的经典术式。在大鼠的品系选择方面,国外大多选用近交系大鼠,如:Lewis—BN、DA—Lewis、PVG—Lewis等,国内由于鼠种、价格等诸多因素,广泛选用封闭群SD与近交系Wistar大鼠进行移植。目的建立大鼠原位肝脏移植急性排斥模型,验证以Lewis大鼠为供体、BN大鼠为受体的组合的排斥强度,为肝脏移植排斥反应或免疫耐受的实验研究提供模型及实验依据。方法⑴.按照改良Kamada法建立近交系大鼠Lewis—BN的肝脏移植模型;⑵.将术后大鼠随机分成两组:实验组( n=15) ,术后未进行特殊的干预;对照组( n=15) ,术后1～7天用FK506 0. 2mg/( kg·d)灌胃。术后4、7和10天各组随机处死3只,收集血清标本与肝脏标本,其余血清标本经尾静脉取血获得;⑶.观察各组剩余6只BN大鼠的生存情况;⑷.将各组的肝脏组织标本HE染色后在光镜下观察病理改变;⑸.将各组所采集的血清样本行肝功能检测; (6).ELISA法检测血清γ-干扰素(IFN-γ)、白介素-10(IL-10)的含量;⑺免疫组化SP法检测肝脏组织内IFN-γ、IL-10的表达;⑻.TUNEL法检测肝细胞凋亡; 结果⑴经过对实验准备期100例大鼠肝脏移植手术的熟悉和相互配合,我们施行了共32例Lewis—BN大鼠的肝脏移植手术,手术成功率为93.8%(30/32);⑵术后实验组BN大鼠在7天以后相继死亡,中位生存期为9天,相应的出现了肝功能的恶化:转氨酶、胆红素的升高、白蛋白的下降,移植肝病理提示中-重度的免疫排斥;而FK506处理后的BN大鼠术后中位生存期为51天,较BN大鼠明显延长(P0.05),肝功能亦好于实验组大鼠,病理提示不明显或轻度排斥反应;⑶术后第7、10天,实验组BN大鼠血清IFN-γ的含量明显高于术前和对照组(P0.05),移植肝内亦呈阳性表达,而对照组出现了IL-10的高表达,显著高于术前和实验组(P0.05)。⑷实验组肝细胞凋亡较FK506干预组显著。结论⑴以近交系Lewis大鼠为供体、BN大鼠为受体的组合发生的急性排斥反应强烈而又稳定,是理想的大鼠肝脏移植急性排斥模型;⑵熟练的手术操作、精细的显微外科技术、有效的麻醉以及良好的围手术期管理是成功建立大鼠肝脏移植模型的关键;⑶血清IFN-γ、IL-10的不同水平表达有助于大鼠肝移植后发生急性排斥的诊断。
[Abstract]:Background with the development of surgical techniques and the clinical application of powerful immunosuppressants, liver transplantation has become the main treatment for chronic end-stage liver disease, acute liver failure and other diseases. The development of clinical liver transplantation technology depends on a large number of animal experiments. Experimental rats are the first choice for many experimental animals because of their low price, clear genetic background, simple and controllable feeding and management. Rat liver transplantation model is an ideal animal model to study organ preservation, ischemia-reperfusion injury, hemodynamics, immunosuppressant, graft rejection and immune tolerance. The rat liver transplantation model was established for the first time by Lee in 1973. After more than 30 years' improvement, the modified Kamada "two-cuff method" has become a classical procedure widely used. In the selection of rat strains, inbred rats are mostly used in foreign countries, such as Lewis-BN,DA-Lewis,PVG-Lewis and so on. Because of many factors, such as rat species, price and so on, closed group SD and inbred Wistar rats are widely used for transplantation in China. Objective to establish an acute rejection model of orthotopic liver transplantation (OLT) in rats, and to verify the rejection intensity of the combination of Lewis rats as donors and BN rats as recipients, and to provide a model and experimental basis for the experimental study of liver transplantation rejection or immune tolerance. Method 1. Liver transplantation model of inbred rat Lewis-BN was established by modified Kamada method. 2. The rats were randomly divided into two groups: experimental group (n = 15) without special intervention and control group (n = 15) with FK506 0. 2mg/ (kg d). On the 7th and 10th day after operation, 3 rats were randomly killed in each group. The serum and liver samples were collected, and the other serum samples were collected from caudal vein. The survival status of the remaining 6 BN rats in each group was observed. The pathological changes were observed by HE staining under light microscope. The serum samples of each group were tested for liver function. (6) the levels of serum interferon 纬 (IFN- 纬) and interleukin-10 (IL-10) were detected by). ELISA method. (7) the expression of IFN- 纬 and IL-10 in liver tissue was detected by immunohistochemical SP method, and apoptosis of hepatocytes was detected by 8.TUNEL method. Results (1) after being familiar with and cooperating with each other in 100 cases of liver transplantation, we performed liver transplantation in 32 cases of Lewis-BN rats. The success rate of the operation was 93.8% (30 / 32). 2After 7 days, BN rats in the experimental group died successively, and the median survival time was 9 days. The corresponding deterioration of liver function appeared: the elevation of transaminase, bilirubin, the decrease of albumin, and the pathological indication of moderate to severe immunological rejection in the transplanted liver. The median survival time of BN rats treated with FK506 was 51 days, which was significantly longer than that of BN rats (P0.05). The liver function of BN rats was better than that of experimental rats. 3 on the 7th day after operation, the levels of IFN- 纬 in serum of BN rats in the experimental group were significantly higher than those in the preoperative and control groups (P0.05), and the expression of IL-10 was also positive in the transplanted liver, while the high expression of IL-10 was found in the control group. 4 the hepatocyte apoptosis in experimental group was significantly higher than that in FK506 intervention group (P0.05). Conclusion 1Acute rejection induced by the combination of inbred Lewis rats and BN rats is strong and stable. It is an ideal model for acute rejection of liver transplantation in rats. 2 skilled operation, fine microsurgical techniques, effective anesthesia and good perioperative management are the key to the successful establishment of rat liver transplantation model; 3 the expressions of IFN- 纬 and IL-10 in serum were helpful for the diagnosis of acute rejection after liver transplantation in rats.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R657.3;R-332

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