【作者】 张静；

【导师】 蒋凡； 王建丽；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2022， 硕士

【摘要】 研究背景:器官移植是临床上器官衰竭终末期有效的治疗手段,但因为主要组织相容性抗原(Major Histocompatibility Antigen,MHA)存在个体差异,同种异体移植手术后早期会启动受体的固有免疫和适应性免疫应答,导致急性排斥反应,其中CD4+T细胞在介导同种异体组织损伤和随后的炎症反应方面发挥关键作用。因此,移植术后应用免疫抑制剂抑制T细胞的过度增殖、活化具有重要意义。CX-5461是特异性的RNA聚合酶Ⅰ抑制剂,通过抑制核糖体DNA的转录进而影响核糖体的生物合成,其作为抗肿瘤药物已经进入Ⅱ期临床试验。我们课题组以往的研究发现,CX-5461可有效治疗多种增殖性血管疾病,特别是我们观察到CX-5461具有显著的抗免疫炎症活性。基于以上结果,在本研究中我们使用体外培养的人原代T细胞及小鼠同种异体皮肤移植模型,探索CX-5461潜在的免疫抑制作用,阐明其对器官移植后的急性排斥反应是否有抑制作用。研究目的:探索CX-5461对T细胞功能及急性移植排斥反应的药理作用研究内容:1.体外检测CX-5461对人原代T细胞功能的影响2.明确CX-5461在体内是否可以有效抑制急性同种异体免疫排斥反应研究方法:1.用不同浓度的CX-5461处理人原代T细胞,EDU掺入试验加流式细胞术检测CX-5461对T细胞增殖的影响;qRT-PCR检测CX-5461对T细胞活化的影响。2.建立小鼠背部全层皮肤移植模型,受体小鼠随机分为对照组、CX-5461治疗组和FK506(阳性对照)治疗组,术后当天至术后14天,拍照记录急性排斥期移植物的生存面积;将观察窗延长到30天,监测CX-5461对移植物的长期保护作用;术后第9天和第14天取移植物和受体次级淋巴器官进行组织病理学检查:HE染色比较组织病理学变化,免疫组化检测免疫细胞浸润水平;药物的安全性评估:手术当天至14天记录小鼠的体重变化,术后第9天和第14天取每只小鼠的外周血作血常规检测。3.术后第9天和第14天,流式细胞术检测CX-5461治疗对于受体次级淋巴器官中CD3+、CD4+及CD8+T细胞增殖情况的影响;qRT-PCR检测移植物以及次级淋巴器官中相关炎症因子的表达水平。4.术后第9天和第14天,流式细胞术检测CX-5461治疗对受体次级淋巴器官中CD3+CD4+CD25+FOXP3+Treg细胞比例的影响;以及qRT-PCR检测Treg细胞的标志物TGF-β的表达水平;此外,在人原代T细胞中qRT-PCR检测CX-5461对Treg分泌的细胞因子IL-10和TGF-β表达水平的影响。研究结果:1.CX-5461在体外可以浓度依赖性地抑制人原代T细胞增殖和活化。2.在急性排斥期内,CX-5461治疗可以显著提高同种异体移植物的存活率。在长期实验中,CX-5461也可以对受体小鼠移植物发挥有效的保护作用。在急性排斥期内,CX-5461可以减轻移植物和脾脏的炎症水平,但不会造成非特异性的骨髓抑制现象,并且受体小鼠对CX-5461具有良好的耐受性。3.CX-5461在体内可以抑制小鼠脾脏、淋巴结中的CD3+、CD4+及CD8+T细胞增殖,下调移植物和次级淋巴器官中促炎因子的表达水平。4.术后 14 天,CX-5461 治疗促进小鼠脾脏和淋巴结中CD3+CD4+CD25+FOXP3+ Treg细胞分化,上调皮肤移植物和脾脏中TGF-β表达水平,此外,在人原代T细胞中给予CX-5461处理亦显著升高IL-10和TGF-β的表达水平。小结:体外实验中,CX-5461可以显著抑制T细胞增殖及活化,诱导其向Treg细胞分化。在小鼠同种异体皮肤移植模型中,CX-5461可以明显抑制急性排斥反应,抑制T细胞增殖及促炎细胞因子的表达,并促进Treg细胞分化。结论:CX-5461有可能作为一种新型免疫抑制剂用于预防器官移植后的急性排斥反应。更多还原

【Abstract】 Background:Organ transplantation is an effective treatment for end-stage organ failure.However,due to the individual differences of major histocompatibility antigens,innate and adaptive immune responses are initiated,resulting in acute rejection in the early stage.CD4+Th cells play a key role in mediating allogeneic tissue injury and subsequent inflammatory response.Therefore,it is very important to use immunosuppressants to inhibit the excessive proliferation and activation of recipient T cells after transplantation.CX-5461 is a specific RNA polymerase I(Pol I)inhibitor,which affects ribosomal biosynthesis by inhibiting the transcription of ribosomal DNA.It is now in phase II clinical trials as an anti-tumor drug and effectively inhibits a variety of proliferative vascular diseases.More importantly,we unexpectedly found that CX-5461 had significant anti-inflammatory activities.Based on the above results,in this study,we used mouse allogeneic skin transplantation model and human primary T cells cultured in vitro to explore the potential immunosuppressive effects of CX5461 and to clarify whether it had inhibitory effects on acute rejection after organ transplantation.Objective:To explore the pharmacological effects of CX-5461 on T cell function and acute allograft rejectionResearch contents:1.To detect the effects of CX-5461 on the function of human primary T cells in vitro2.To explore whether CX-5461 can effectively inhibit acute allograft rejection in vivoMethods:1.Human primary T cells were treated with different concentrations of CX-5461.The effects of CX-5461 on T cell proliferation was detected by EDU incorporation test and flow cytometry;The effects of CX-5461 on T cell activation was detected by qRT-PCR.2.The mice models of full-thickness back skin transplantation were established.The recipient mice were randomly divided into control group,CX-5461 treatment group and FK506(positive control)treatment group.From the day of operation to day 14 after operation,photos were taken to record the survival area of grafts in acute rejection stage;Extended the observation window to day 30 after operation to monitor the long-term protective effects of CX-5461;On the day 9 and 14 after operation,the grafts and recipient secondary lymphoid organs were taken for histopathological examinations:histopathological changes were detected by H&E staining,and the level of immune cell infiltration was detected by immunohistochemistry;Safety evaluation of drugs:the body weight changes of mice were recorded from the day of operation to day 14.The peripheral blood was taken for blood routine tests on the 9th and 14th day after operation.3.On the 9th and 14th day after operation,the effects of CX-5461 treatment on the proliferation of CD3+,CD4+and CD8+T cells in the recipient’s secondary lymphoid organs were detected by flow cytometry.qRT-PCR was used to detect the expression of related inflammatory cytokines in grafts and secondary lymphoid organs.4.On the 9th and 14th day after operation,the effect of CX-5461 treatment on the proportion of CD3+CD4+CD25+Foxp3+Treg cells in the recipient’s secondary lymphoid organs was detected by flow cytometry;TGF-β expression level-the marker of Treg cells was detected by qRT-PCR.In vitro,the effects of CX-5461 on expressions of cytokines IL-10 and TGF-β were detected by qRT-PCR in human primary T cells.Results:1.CX-5461 inhibited the proliferation and activation of human primary T cells in a concentration dependent manner in vitro.2.During the acute rejection period,CX-5461 treatment significantly improved the survival rate of grafts.In the long-term observation experiment,CX-5461 played protective effects on the recipient mouse graft.During the acute rejection period,CX-5461 reduced the inflammatory level of graft and spleen,but it did not cause non-specific bone marrow suppression,and the recipient mice had a good tolerance to CX-5461.3.CX-5461 inhibited the proliferation of T cells in spleens and lymph nodes and downregulated the mRNA expression of pro-inflammatory cytokines in skin grafts and secondary lymphoid organs in vivo.4.On day 14,the abundance of CD3+CD4+CD25+Foxp3+Treg cells in spleens and lymph nodes in CX-5461 treatment group was significantly increased.CX-5461 treatment upregulated the expression level of TGF-β in skin grafts and spleens.In addition,CX-5461 treatment in human primary T cells also significantly increased IL-10 and TGF-β expression levels.Summary:In vitro,CX-5461 can significantly inhibit the proliferation and activation of T cells,and can induces differentiation of Treg cells.In vivo,CX-5461 can significantly inhibit acute rejection of skin allografts,T cell proliferation,and the expression of proinflammatory cytokines.CX-5461 can also promote Treg differentiation in vivo.Conclusion:CX-5461 may be used as a novel immunosuppressant to prevent acute rejection after organ transplantation.更多还原