【作者】 姚宇锋；

【导师】 蔡端；

【作者基本信息】 复旦大学 ， 外科学， 2007， 博士

【摘要】 目的：1．建立体外培养、扩增受体C3H小鼠骨髓源性树突状细胞(BM-DC)的方法，并对其进行鉴定；观察其对供体C57BL／6小鼠可溶性抗原提呈的有效性。同时探讨B7反义肽(B7 antisense peptide，B7AP)抑制单向混合淋巴细胞反应的作用，并明确B7AP显著抑制单向混合淋巴细胞反应的最适工作浓度。2．通过体外供体抗原间接识别反应体系，观察B7AP预处理的负载供体抗原的受体树突状细胞(dendritic cell，DC)诱导针对供体抗原的特异性免疫低反应；同时通过体内实验进一步观察其可行性，并对其机制进行初步分析。3．建立同种异基因小鼠颈总动脉原位移植模型(C57BL／6→C3H)，观察阻断间接识别途径对移植动脉慢性排异的影响，并初步探讨此种效应的局部机制。方法：1．体外分离受体C3H小鼠骨髓细胞，在重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和肿瘤坏死因子-α(rmTNF-α)的刺激下，手法筛选、培养扩增受体小鼠骨髓源性树突状细胞；通过细胞形态、功能、表面分子表达对其进行鉴定。在培养过程中加入制备的供体C57BL／6小鼠可溶性抗原，通过与受体C3H小鼠脾脏T细胞进行单向混合淋巴细胞反应，观察受体DC负载供体抗原的有效性。进一步利用不同浓度的B7AP与负载供体抗原的受体DC进行充分孵育，并与受体脾脏T细胞进行单向混合淋巴细胞反应(MLR)，建立B7AP浓度对数值与单向混合淋巴细胞反应cpm值(每分钟液闪数)的曲线，应用统计学方法寻找B7AP显著抑制单向混合淋巴细胞反应的最适工作浓度。2．建立体外供体抗原间接识别反应体系，利用B7AP预处理的负载供体抗原的受体DC与受体T细胞行初次单向混合淋巴细胞反应，收集反应后受体T细胞，与负载供体抗原、第三供体抗原的受体DC和供体DC行再次单向混合淋巴细胞反应，观察反应后受体T细胞针对间接途径提呈的供体抗原、第三供体抗原以及直接途径提呈的供体抗原的免疫活性。同时通过B7AP封闭的负载供体抗原的受体DC对受体小鼠进行预处理后，分离受体脾脏T细胞与负载供体抗原、第三供体抗原的受体DC和供体DC行再次单向混合淋巴细胞反应，了解B7AP封闭的负载供体抗原的受体DC体内有效性，并分别在早期和2月时对受体小鼠进行供体抗原刺激后取脾脏进行细胞因子和淋巴细胞凋亡(apoptosis)分析，初步探讨免疫耐受的其他可能机制。3．建立小鼠同种异基因颈总动脉原位移植模型(C57BL／6→C3H)，通过术前3天对受体小鼠进行股静脉注射B7AP预处理的负载供体抗原的受体DC，2月后取移植动脉进行病理分析，观察移植动脉内膜增生情况，作计算机图像分析，并通过对移植动脉的免疫组化分析，初步了解此效应的局部机制。结果：1．在rmGM-CSF和TNF-α刺激下，可从受体C3H小鼠骨髓前体细胞培养扩增出大量的成熟DC。形态学符合成熟DC，表面高表达CD80、CD86、MHC-Ⅱ类抗原，功能上具有强烈的刺激不同品系小鼠T细胞增殖的能力。2．在培养过程中加入供体抗原的受体DC具有显著刺激受体T细胞增殖的能力。3．B7AP预处理后的负载供体抗原的受体DC能够有效减弱对受体T细胞的刺激增殖作用，并且在反义肽终浓度为10mg／L，或者与DC孵育时浓度为20mg／L时，能够保证DC表面B7分子被完全封闭。4．再次单向混合淋巴细胞反应结果显示，B7AP预处理的负载供体抗原的受体DC能够诱导产生针对供体抗原的免疫低反应，但仍然保持对第三抗原的免疫反应。5．体内实验也显示B7AP封闭的负载供体抗原的受体DC对受体小鼠进行预处理后，受体脾脏分离T细胞也能产生针对供体抗原的免疫低反应，同时受体在受到供体抗原刺激后脾脏细胞因子mRNA半定量RT-PCR结果显示Th2分泌细胞因子IL-10明显升高，而Th1分泌细胞因子IL-2、IFN-γ减弱，此种效应在一定时间内可以维持；但是受体脾脏淋巴细胞凋亡无改变。6．体内外实验均发现阻断间接识别途径形成的预处理受体T细胞仍能产生对直接途径提呈的供体抗原的免疫反应。7．成功建立同种异基因小鼠颈总动脉原位移植模型(C57BL／6→C3H)。8．2月时取移植动脉，制作病理切片HE染色，观察见预处理小鼠组移植动脉内膜增生不明显，计算机图像分析系统分析显示预处理组小鼠移植动脉内膜相对厚度为0.071±0.034，而对照组的移植动脉相对内膜厚度为0.179±0.056，两者差别具有统计学意义；同时移植动脉壁TGF-β1免疫组化分析显示预处理组TGF-β1的表达相对于对照组明显减弱，与动脉内膜增生程度一致。结论1．C3H小鼠骨髓前体细胞在rmGM-CSF和TNF-α的刺激下，可以扩增培养大量成熟DC。2．受体C3H小鼠的成熟DC能够有效的提呈供体C57BL／6小鼠可溶性抗原。3．负载供体抗原的受体C3H小鼠DC经B7AP预处理后能够抑制其和受体小鼠T细胞的单向混合淋巴细胞反应。4．B7AP预处理的负载供体抗原的受体DC能够诱导针对供体抗原的特异性免疫低反应，同时可能通过诱导Th1→Th2免疫偏移来加强这种效应。5．阻断间接途径产生的受体T细胞的特异性免疫低反应不影响其对直接途径提呈的供体抗原的免疫反应。6．B7AP预处理的负载供体抗原的受体DC能够有效抑制小鼠同种异基因颈总动脉原位移植(C57BL／6→C3H)后移植动脉的内膜增生，TGF-β1在局部可能发挥一定的作用。7．B7AP阻断间接识别途径能够有效抑制同种异体移植慢性排异反应。更多还原

【Abstract】 Objectives: 1. To establish the method of culturing and propagation of the recipient mouse bone marrow-derived dendritic cells(BM-DC) in vitro and verify its validity of donor soluble antigen presentation. Also to investigate the effect of the inhibition of the one-way mixed lymphocyte reaction (MLR) by B7 antisense peptide(B7AP) and find the most suitable B7AP concentration of marked inhibition to one-way MLR. 2. To investigate donor-specific immune hyporesponsiveness induced by the B7AP-pretreated donor antigen-loaded recipient derived DC using the the response system of the recipient-donor indirect pathway in vitro, then to observe the validity in vivo and analyze the mechanism initially. 3. To investigate the effect and its regional mechanism of chronic rejection of the transplanted artery via the blockade of indirect recognition after establishing the allogeneic murine model of carotid artery orthotopic transplantation.Methods: 1. The recipient C3H BM-DC were generated by culturing bone marrow precursors with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and tumor necrosis factor-alpha (TNF-α). BM-DC were identified by the cell morphous、function and the expression of the molecules on the cell surface. In the culturing process, donor C57BL/6 soluble antigens were added, then the validity of the recipient DC loading the donor antigen was investigated by the one-way MLR using the spleen T cells of the recipient C3H and the cultured recipient DC. Also the MLRs of the cultured recipient DC and the T cells of the recipient spleen were performed after the cultured recipient DC were incubated with B7APs of different concentrations, to display the curve of the logarithm of the B7AP concentrations and the CPMs of the MLRs and to find the most suitable concentration of the B7APs associated with marked inhibition on the one-way MLR. 2. The response system in vitro of the donor antigen indirect presentation to the recipient was established. The primary one-way MLR of the B7AP-pretreated donor antigen-loaded recipient DC and the recipient spleen T cells were taken , then the secondary MLR of the collected recipient T cells after the primary MLR and the donor-antigen or the third antigen loaded recipient DC or donor DC to investigate its immunological competence. Also the recipient spleen T cells were isolated after the recipient was pretreated by the B7AP-pretreated donor antigen-loaded recipient DC and the MLR was taken with the donor antigen or the third party antigen loaded recipient DC or donor DC to identify the vadility of the B7AP-pretreated donor antigen-loaded recipient DC in vivo. Meanwhile the cytokine genes(IL-2, IL-10, and INF-γmRNA) and the lymphocyte apoptosis of the recipient spleen were analyzed to find the other possible mechanism of the immune tolerance initially after stimulated by the donor antigen in the earlier period or 2 months later. 3. The reciepient was pretreated by B7AP-blocked donor antigen-loaded recipient DCs, then the allogeneic murine model of carotid artery orthotopic transplantation was established (C57BL/6→C3H) . The transplanted artery was harvested to be analyzed pathologically after 2 months. Also the TGF-β1 immunohistochemistry analysis of the transplanted artery were performed to find the regional mechanism of the immune tolerance.Results: 1. Under the stimulation of the rmGM-CSF and TNF-α, large number of the mature BM-DC were propagated from the mouse bone marrow. They match with the DC morphologically, highly express the CD80、CD86n MHC- II on their surface and stimulate the proliferation of the other strain’s T cells intensively. 2. The recipient DC which were cultured by adding the donor antigen could stimulate the proliferation of the recipient T cells significantly. 3. B7AP-pretreated donor antigen-loaded recipient DC could effectively inhibit the proliferation of the recipient T cells and it was sure that the B7 molecular could be fully blocked when the concentration of B7AP was 10mg/L or 20mg/L incubated with DC. 4. The result of the secondary MLR showed B7AP-pretreated donor antigen-loaded recipient DC could induce the immune hyporeponsiveness to the donor antigen, but immune responsiveness to the third party antigen. 5. The experiment in vivo also showed the recipient spleen T cells would hyporesponse to the donor antigen after the recipient was pretreated by the B7AP-blocked donor antigen-loaded recipient DC. Meanwhile after stimulated by donor antigen, the Th2 cytokines (IL-10) mRNA of the recipient spleen were up-regulated and the Th1 cytokine (IL-2、IFN-γ) mRNA were down-regulated, which was maintained in some time, but the apoptosis of the recipient spleen lymphocytes were not changed. 6. The recipient T cells pretreated by the blockade of indirect recognition were hyperresponsive to the donor antigen presented via direct recognition. 7. We successfully established the allogeneic murine model of carotid artery orthotopic transplantation. 8. The transplanted artery was harvested in 2 month and the intimal proliferative lesion was observed pathologically. The result showed the relative thickness of the intima pretreated was 0.071±0.034, but 0.179±0.056 of the control group. The difference was statistically significant. The TGF-β1 in the artery wall pretreated was low-expressed by the immunohistochemical analysis, which was matched with the levels of intimal proliferative lesion.Conclusions: 1. A large quantity of mature DC was propagated from mouse bone marrow precursors under rmGM-CSF and TNF-αstimulation. 2. The recipient DC could present the donor antigen effectively. 3. The donor-loaded recipient DC pretreated by the B7AP could inhibit the one-way MLR with the recipient T cells. 4. The B7AP-pretreated donor antigen-loaded recipient DC could induce the specific hyporesponsiveness to the donor antigen, which may be enhanced by the immune deviation (Th1→Th2). 5. The specific hyporesponsiveness of the recipient T cells to the donor antigen via the blockade of the indirect recognition would not take effect on its response to the donor antigen via direct recognition. 6. The B7AP-pretreated donor antigen-loaded recipient DC could effectively inhibit the intimal proliferative lesion of the transplanted artery in the allogeneic murine model of carotid artery orthotopic transplantation, and the TGF-β1 may play a regionnal role. 7. Blockade of indirect recognition by B7AP can effectively inhibit chronic allograft rejection.更多还原