【作者】 傅涛；

【导师】 王为忠； 陈冬利；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 硕士

【摘要】 目的:1.建立易行可靠的小鼠颈部异位心脏移植模型,改进小鼠颈部异位心脏移植模型和手术方式。2.培育鉴定T细胞中特异性剔除RBP-J转基因小鼠,通过建立小鼠颈部异位心脏移植模型,探讨Notch信号途径中转录因子RBP-J对小鼠同种异体心脏移植受体T细胞亚群的影响方法:1.在显微镜下对受体手术,游离受体右侧静外静脉和颈总动脉,对供体,经下腔静脉注射肝素钠全身肝素化后,于低温下切取供心;采用供心无名动脉与受体颈总动脉端端间断吻合,供心肺动脉与受体颈外静脉端端吻合,进行小鼠颈部异位心脏移植。2.交配Lck-Cre x RBP-Jflox/+杂合子转基因小鼠,提取其子代小鼠尾巴DNA,通过凝胶电泳,鉴定小鼠基因型,确定基因敲除小鼠。3.选用三种小鼠,即C57BL/6,Balb/c以及RBP-J基因条件性剔除小鼠,采用颈部异位小鼠心脏移植模型,将32只小鼠按随机原则分为2组:A组(n=8)BALB/c→C57BL/6;B组(n=8)BALB/c→RBP-J基因剔除小鼠,移植术后1、3、5、7天留取移植物,观察大体情况及组织病理学变化。4.提取小鼠外周血,行血常规检查。获取受体小鼠胸腺、淋巴结、脾脏,分离T淋巴细胞,抗-CD4-PE、抗-CD8-APC和抗-CD25-FITC染色,行流式细胞仪检测。结果:1.供心总缺血时间40 min,术后复跳率90%,改进后的术式手术成功率有所提高,模型的建立为下一步实验提供了技术基础。2.通过培育繁殖,我们得到转基因小鼠并通过DNA PCR扩增,凝胶电泳鉴定转基因小鼠,为下一步实验提供实验材料。3.建立小鼠颈部异位心脏移植术后, C57小鼠对照组(n=7)移植术后移植心平均存活时间为(6±1.21)d,基因敲除小鼠实验组移植术后移植心平均存活时间为(4±1.8)d,与对照组相比,存活时间明显缩短,(P<0.05)。脾脏细胞的增加主要是Mac1阳性的细胞的绝对数量的增加(P<0.01),以及CD8阳性细胞的增加(P<0.01)。而淋巴结的细胞数的增加主要是T细胞(P<0.01),尤其是CD8阳性的T细胞的增加(P<0.01),以上结果初步提示RBP-J KO受体小鼠相比control受体小鼠,CD8阳性的T细胞对于移植物的反应增强。而脾脏中天然发生的调节性T细胞的绝对数也有所降低(P<0.01)。结论:1.改进后的术式手术成功率高,术中暴露充分、血管处理简单、手术创伤小,污染机率小等,是简单、实用、可靠的手术方式。2.我们培育并鉴定出T细胞中特异性剔除RBP-J转录因子的转基因小鼠,从而为进一步研究转录因子RBP-J在小鼠心脏移植急性排斥反应中对受体T细胞亚群的影响奠定了基础。3.通过建立小鼠颈部异位心脏移植,我们发现基因敲除小鼠的免疫排斥反应有所加强,反应性T细胞增殖加强,调节性T细胞增殖有所减弱,这为研究移植免疫提供新思路。更多还原

【Abstract】 Aim:1. To develop and master the operation methods of heterotopic cervical heart transplantation in mice.2.Select C57BL/6 , Balb/c and treditional gene-knockout mice as the object,and heterotopic transverse part cardiac transplantation was performed to investigate the effect of the transcription factor RBP-J in the Notch/ RBP-J pathway on subgroup T cells after allogene heart transplantation in mice.Mathods:1.Under microscopy , the right external jugular vein and the common carotid artery of the recipient were liberated . After heparinization all over the body of the donor ,the heart was obtained in low temperature. The innominate artery of the donor heart and the right common carotid artery of the recipient were anastomosed using the end-to-end interrupted suture technique.The same method was used in the connection between the pulmonary artery of the donor heart and the right external jugular vein of the recipient.2. Generated Lck-Cre x RBP-J flox/flox mouse in which RBP-J, the key transcription factor of Notch pathway,can be specifically inactivated in T cells.Identified lck-cre mouse lines in which RBP-J can be completely inactivated in T cells by PCR.And select gene-knockou mice as the object.3. A total of 32mice of 3 inbred lines(C57BL/6, Balb/c and conditional knockout C57BL/6) were randomly divided into 2 groups. Group A(n=8)had aniso-strain operation(BALB/c→C57BL/6); group B(n=8)got the same operation (BALB/c→conditional knockout RBP-J C57BL/6). Samples of grafts were taken 6 days after transplantation, then allograft were detected by pathology.4. Peripheral blood was stringed blood routine examination. Lymphoid nodes and spleens were taken after graft stopped work,and the T cells were isolated from spleens,then marked with anti-mouse CD4-PE,anti-mouse CD8-APC, anti-mouse CD25-FITC ,and other colored immune fluorescence antibodies to detect the quantity of sub-T cell with flow cytometerResult:1. We developed the operation methods,the result showed that the the total ischemic time of the graft was 40 min, the rate of the heart re-beat was increased to 90%.2.The mice of conditional knockout transcriptional factor RBP-J is got and identified successfully,the mice is available which lays the foundation for the study of the effect of the transcription factor RBP-J in the Notch/ RBP-J pathway on sub-T cells after allogene heart transplantation in mice.3. Conditional Knockout RBP-J in mice shorted survival time to 4±1.8 days compared to 6±1.21 days in group C57 (p<0.01).Acute rejection pathologically was observed in group A and B.The proliferation of RBP-J deficient T cells,epecially in CD8+ T cells, increased a lot in response to the allograft compared with group C57 (P<0.01),but the Treg cell decreased.Conclusions:1. The advantage of this model lies in easy exposition, short duration of the operation, easy treatment of the blood vessel, low probability of contaminate and high rate of success,so deserves to use in experimental study widely.2. We get the mice in which conditional knockout transcriptional factor RBP-J in T cell, We identified thelck-cre mouse line in which RBP-J can be completely inactivated in T cells by PCR.3.Through established the heterotopic cervical heart transplantation,we found that the the transcription factor RBP-J influenced the acute rejection. The proliferation of RBP-J deficient T cells,epecially in CD8+ T cells, increased a lot,and the Treg cell decreased.These results convinced us to make a deep research on the relationship between the Notch pathway and the transplantation rejection.更多还原