巨噬细胞在混合嵌合体诱导异种免疫耐受中作用的实验研究

发布时间：2018-04-21 21:19

  本文选题：巨噬细胞 + 嵌合体　； 参考：《苏州大学》2007年硕士论文

【摘要】： 目的:在豚鼠到大鼠实验动物模型中,利用脂质体包裹clodronate清除受体巨噬细胞后,采用非清髓性的预处理方案诱导建立混合嵌合体,研究巨噬细胞在建立非协调性异种混合嵌合体中的作用。并在此基础上,行豚鼠到大鼠的腹部心脏移植,研究混合嵌合体的形成对受体的免疫功能影响,探讨异种免疫耐受的机制。 方法:将SD大鼠(受体)30只随机分成3组:所有大鼠受体接受60Coγ射线全身照射,经环磷酰胺预处理,照射后4h内经尾静脉输注BMC 2x108/ml×0.8ml。A组仅输注豚鼠BMC;B组输注豚鼠BMC前输注不包含clodronate空脂质体;C组输注豚鼠BMC前输注包裹clodronate空脂质体。BMC移植后24小时用免疫组化法检查受体大鼠肝脏、脾脏中巨噬细胞清除情况;用流式细胞仪法测定BMC移植后24小时内不同时间点受体外周血中豚鼠细胞比例、24小时时受体脾、骨髓中豚鼠细胞比例,以及移植后21天、35天嵌合率;于BMC移植后21天,行MLR测定,并用流式细胞仪法测定T淋巴亚群的变化。 在成功建立混合嵌合体的基础上,行豚鼠到大鼠的心脏移植。实验随机分为六组:I组(空白组,n=5)、II组(CVF组,n=5)III组(CVF+clodronate脂质体组,n=5)、IV组(CVF+豚鼠BMC移植组,n=5)、V组(CVF+豚鼠BMC移植+空脂质体组,n=5)、VI组(CVF+豚鼠BMC移植+包裹clodronate脂质体组,n=5)。流式细胞仪法检测移植前后大鼠抗豚鼠IgG水平;记录移植物平均存活时间、检测移植物病理组织学。 结果:输注包裹clodronate脂质体后24小时受体大鼠肝脾(红髓及边缘带)中巨噬细胞消失,21天后与正常对照相比无差异。BMC移植后24小时内各时间点,C组大鼠外周血中豚鼠细胞比例均高于A、B组; 24小时时大鼠脾、骨髓中豚鼠细胞比例,C组亦明显高于A、B两组;比较21天及35天嵌合水平,C组明显高于A、B两组。21天时CD3+、CD4+和CD8+T细胞比例均下降,各组CD4/CD8比值与对照组相比无差异。MLR显示耐受大鼠的免疫应答反应性降低,C组较其他组比最低。 心脏移植后,I组移植心表现为超急性排斥反应,II、III、IV、V、VI各组移植心存活时间分别为:306±45分钟、314±51分钟、366±35分钟、372±25分钟、424±32分钟,病理均表现为急性血管排斥反应改变。移植后抗豚鼠IgG水平均有轻微升高,VI组上升幅度最小。 结论:1、利用脂质体包裹clodronate的方法可建立清除巨噬细胞动物模型。2、巨噬细胞在BMC移植后迅速减少中起重要作用,在BMC移植前去除巨噬细胞可提高嵌合率。3、建立混合嵌合体虽然不能影响T细胞亚群的分布,但能使受者T细胞对供者抗原反应性降低,也可延缓与T细胞活化相关的IgG抗体升高。4、运用CVF克服超急性排斥反应的情况下,混合嵌合体能使豚鼠到大鼠心脏移植供心存活时间延长,并与嵌合水平成正相关。
[Abstract]:Objective: in guinea pig to rat model, the mixed chimerism was induced by non-myeloablative pretreatment after clodronate was encapsulated with liposome to clear the macrophages. To study the role of macrophages in the establishment of discordant heterologous chimerism. On this basis, the abdominal heart transplantation from guinea pigs to rats was performed to study the effect of mixed chimerism on the immune function of the receptors, and to explore the mechanism of xenogeneic tolerance. Methods: thirty SD rats were randomly divided into three groups: all the receptors were irradiated by 60Co 纬 -ray and pretreated with cyclophosphamide. Within 4 hours after irradiation, BMC 2x108/ml 脳 0.8ml.A group was injected via caudal vein. The liver of recipient rats was examined by immunohistochemistry at 24 hours after transplantation of clodronate empty liposome, clodronate empty liposome, and clodronate empty liposome, without clodronate empty liposome in group C, without clodronate empty liposome. The clearance of macrophages in spleen, the percentage of guinea pig cells in peripheral blood of recipients at 24 hours after BMC transplantation and the percentage of guinea pig cells in bone marrow at 24 hours after BMC transplantation were determined by flow cytometry. The changes of T lymphoid subsets were measured by flow cytometry and MLR were measured 21 days after BMC transplantation. On the basis of successful establishment of mixed chimerism, cardiac transplantation from guinea pig to rat was performed. The experiment was randomly divided into six groups: group 鈪，

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