【作者】 康振华；

【导师】 王为忠；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 目的：1.制备大鼠抗人RANTES分子单克隆抗体(mAb)，并进行初步鉴定，为研究RANTES分子的组织分布和功能提供实验手段。2.掌握三袖套血管吻合法大鼠节段性异位小肠移植动物模型及静脉营养支持模型的建立方法。3.将制备的大鼠抗人RANTES单克隆抗体应用于大鼠小肠移植模型,验证其在减轻移植排斥中的作用。4.对大鼠抗人RNATES单克隆抗体进行初步的人源化改造。方法：1.应用杂交瘤技术制备大鼠源性抗人RANTES mAb。ELISA鉴定腹水效价；用Western blot鉴定mAb活性；用流式细胞术、免疫细胞化学技术及免疫组织化学技术对抗体进行鉴定。2.选用近交系成年雄性SD和Wistar大鼠进行节段性异位小肠移植,采用三袖套血管吻合法。实验分4组，第1组：非手术对照组（SD）；第2组，同基因移植对照组（SD→SD）；第3组：异基因移植未治疗组(SD→Wistar)；第4组，大鼠抗人RANTES mAb治疗组[SD→Wistar +mAb（3mg·kg-1·d-1，ip）]。移植术后3，5，7天分别取各组大鼠移植肠标本（n=6）进行HE染色，观察大体情况及组织病理学变化。3.应用ELISA试剂盒对术后各组大鼠不同时间点血清IL-2,IL-6,TNF-及RANTES浓度进行检测,并用免疫组化法检测受体大鼠小肠组织内RNATES浓度。4.利用RT-PCR技术，从杂交瘤细胞株中扩增单克隆抗体VH及VL基因，并连接到人源化表达载体中，经转染293细胞后，纯化表达上清中蛋白分子。结果：1.获得2株分泌抗RANTES mAb的杂交瘤细胞株。间接ELISA测定腹水效价均达10-6，一株mAb为IgG1亚类，另一株为IgM；Western blot结果显示两株mAb与人RANTES均有良好的结合活性。在免疫组织化学技术应用中获得较满意效果。2.异基因移植未治疗组大鼠移植肠组织病理学检查显示移植后第3，5，7天分别出现轻、中、重度排斥反应。同基因移植组和单克隆抗体治疗组均未见明显排斥反应征象。3.ELISA试剂盒测试结果显示单克隆抗体治疗组与未治疗组相比,大鼠血清中IL-2,IL-6,TNF-及RANTES浓度均明显减低。而小肠组织中RANTES浓度也明显减低。4.得到2株表达人源化抗体的细胞株,并获得纯化蛋白。结论：1.制备了两株大鼠抗人RANTES单克隆抗体，这2株单克隆抗体均能应用于Western blot、流式细胞术及免疫细胞化学；而只有No.1抗体株能应用于免疫组织化学技术。2.将大鼠抗人RANTES单克隆抗体应用于大鼠小肠移植模型，发现其可减轻术后急性排斥反应的发生。3.改进了大鼠小肠移植模型的制作方法，建立受体大鼠静脉营养支持模型。4.利用嵌合抗体技术对大鼠抗人RNATES单克隆抗体进行了初步的人源化改造，为进一步表达及应用打下了基础。更多还原

【Abstract】 AIM1. To prepare and identify rat monoclonal antibodies against humanRANTES molecule. Provided a new experiment method for research thedistribution and function of RANTES.2．To master the way of setting up the model of segmental small boweltransplantation heterotopically in rats by tri-cuff anastomosis method, andestablishment of rat model of nutrition support.3. To apply the rat mAb against human RANTES in rat small boweltransplantation model, validate the effect of mAb in alleviating the transplant rejection.4. To primary humanize the rat against human RANTES monoclonalantibody.METHODS1. Rat mAbs were prepared by hybridoma technique. Ascites titer wasidentified by ELISA. The activities of mAbs were identified by Western bolt.The characters of mAbs were identified by FCM, immunohistochemistry andimmunocytochemistry.2. Heterotopic small bowel transplant was carried out between inbredmature male SD and Wistar rats, which was used tri-cuff anastomosis method.All recipients were divided into four groups. Group1, SD; Group2, SDSD;Group 3, SDWistar; Group 4, SDWistar+RANTES mAb (3.0mg·kg^-1·d^-1, ip). The grafts were sampled on the postoperative day 3, 5, 7and hematoxylin eosin staining was conducted on the specimens to observepathological change.3. Checked the concentration of IL-2 , IL-6, TNF- and RANTES in ratserum after operation in four groups at different time by ELISA kit. Checked theconcentration of RANTES in tissue of small bowel of rat receptor byimmunohistochemistry .4. We got VH and VL gene of mAb from hybridoma cell lines ByRT-PCR,and connected to the humanization expression vectors. Then wepurified the humanized mAb from transfected 293 cell line.RESULTS1. Two hybridoma cell lines, RANTES No1, RANTES No2, secreting mAbsagainst human RANTES molecule were obtained. The titers of ascitic mAbsreached to 10-6 and the Ig subclasses of RANTES No1 and RANTES No2 were IgG1 and IgM, respectively. Moreover, RANTES No1 could be used inimmunohistochemistry staining.2. The pathological examination of grafts in Group 3 demonstrated the mild,moderate, and severe rejection on postoperative day 3, 5, and 7. While rats inthe Group 2 and 4 showed no obvious indication of rejection.3. The results of ELISA kit detection showed that concentration of IL-2 ,IL-6, TNF- and RANTES in rat serum of mAb therapy group is obviouslydiminution than group 3. Meanwhile, the concentration of RANTES in tissue ofsmall bowel in group 4 is reduced obviously.4. We got two cell lines which can express humanized monoclonalantibody, and got purified protein by affinity chromatography.CONCLUSION1. Prepared two rat mAbs against human RANTES, Both can be used inWestern blot, FCM and immunocytochemistry. Only No.1 can be used inimmunohistochemistry .2. Applied the rat mAb against human RANTES in rat small boweltransplantation model, validated it can alleviate the transplant rejection.3. Improved the techniques in rat small bowel transplantation model andestablished the rat model of vein nutrition support.4. primary humanized the rat against human RANTES monoclonalantibody by chimeric antibody technique, which can provide a useful tool forfurther application.更多还原