HLA-G基因与移植免疫耐受实验性研究

发布时间：2018-06-08 06:00

  本文选题：HLA-G + 肾移植模型　； 参考：《安徽医科大学》2017年硕士论文

【摘要】：【背景】肾移植后排斥反应是影响移植受者存活的重要因素,而长期应用免疫抑制剂有其不可避免的缺点,因此使移植肾产生免疫耐受是移植界的重要课题。人类白细胞抗原G(human leukocyte antigen-G,HLA-G)作为一种免疫耐受分子,对母胎耐受及正常妊娠有着极其重要的作用,而其在器官移植中的作用也逐渐被发现,移植术后患者体内HLA-G的表达已被证实可以降低排斥反应的发生率。通过向大鼠移植肾转染HLA-G基因使其在移植肾内高表达,有望成为诱导移植肾免疫耐受的新途径。【目的】本实验拟向大鼠移植肾中转染HLA-G基因并将转染过基因的移植肾移植到受体大鼠上,通过检测大鼠移植肾的病理变化以及HLA-G基因的表达情况,反应出移植肾通过转染HLA-G基因对移植术后移植肾免疫排斥反应的影响,为HLA-G基因诱导移植肾免疫耐受的基础实验性研究提供有力的研究工具。【方法】分别建立SD大鼠移植实验组和对照组,实验组为转染过含有HLA-G基因的慢病毒,阴性对照组为转染过不含有HLA-G基因的慢病毒,空白对照组为灌注液处理的移植肾,然后分别将三组移植肾通过建立动静脉袖套的方法将移植肾移植到受体肾动静脉上,同时将移植肾膀胱瓣膜缝合到受体膀胱壁上完成大鼠肾移植模型的建立。建模成功后分别于造模后一周开始,每周三组随机选取三只大鼠,麻醉后沿腹正中切开观察移植肾变化,处死并取出移植肾,部分放-80℃保存,其余甲醛固定。然后分别对三组移植肾组织进行HE染色分析移植肾病理变化,免疫组化以及Western blot分析HLA-G基因的表达情况,分析出转染HLA-G基因对大鼠移植肾的免疫排斥反应的影响。【结果】共建立大鼠肾移植模型30只,死亡3只,成功率90%,其中2只死于出血,一只死于尿瘘。通过对三组移植肾的病理分析发现,对照组排斥反应明显,而实验组排斥反应明显低于对照组,同时免疫组化及Western blot分析HLA-G基因的表达情况发现,对照组均不表达HLA-G基因,而实验组均表达HLA-G基因,而且随着实践的延长,表达量增加。【结论】本实验通过建立肾动静脉袖套方法建立大鼠肾移植模型,有效的减少手术时间,简化手术复杂程度,为肾移植模型的普通化提供新的思路。同时,实验还发现转染HLA-G基因可以有效减少移植后急性免疫排斥反应的发生,延缓慢性免疫排斥反应,为转染HLA-G基因诱导移植肾免疫耐受的实验性研究奠定了基础。
[Abstract]:[background] rejection after renal transplantation is an important factor affecting the survival of recipients, and the long-term application of immunosuppressants has its inevitable shortcomings. As an immunological tolerance molecule, human leukocyte antigen-GG plays an extremely important role in maternal and fetal tolerance and normal pregnancy, and its role in organ transplantation has been gradually discovered. The expression of HLA-G has been shown to reduce the incidence of rejection after transplantation. The high expression of HLA-G gene was induced by transfection of HLA-G gene into the transplanted kidney of rats. [objective] to transfect HLA-G gene into the transplanted kidney of rats and transplant the transfected kidney to the recipient rats. By detecting the pathological changes and the expression of HLA-G gene in the transplanted kidney of rats, the effect of HLA-G gene transfection on the immune rejection of the transplanted kidney was revealed. [methods] A SD rat experimental group and a control group were established respectively. The experimental group was transfected with lentivirus containing HLA-G gene. The negative control group was infected with lentivirus which did not contain HLA-G gene, while the blank control group was treated with perfusion solution. The three groups of transplanted kidneys were transplanted to the recipient renal arteriovenous by establishing an arteriovenous cuff. At the same time, the graft bladder valve was sutured to the recipient bladder wall to complete the establishment of rat renal transplantation model. Three rats were randomly selected each week after modeling. After anesthesia, the allografts were observed by incision along the middle of the abdomen, then killed and removed, some of them were preserved at -80 鈩，

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