靶向树突状细胞CCR7反义肽核酸抑制大鼠肝移植急性排斥的实验研究

发布时间：2019-06-21 02:01

【摘要】： 移植排斥反应实质上是受体免疫系统对供体移植物抗原的免疫应答,是T淋巴细胞依赖的细胞性反应过程。但器官移植后受体的免疫系统并不能直接识别供体抗原,供体抗原信息必须经过抗原提呈细胞传递给受体T淋巴细胞使之活化才能产生后续的免疫应答。树突状细胞(Dendritic cells, DCs)是功能最强的抗原递呈细胞,唯有DCs才能有效激活初始T细胞,其在体内递呈抗原的过程是一个动态的迁移过程,携带抗原信息的DCs从外周迁移到淋巴组织的T细胞区完成抗原递呈并激活初始T细胞。目前认为外周DCs向淋巴组织的迁移动力主要通过淋巴组织趋化因子作用于DCs趋化因子受体CCR7这条途径。因此,CCR7成为一个理想的目标。肽核酸(peptide nucleic acid ,PNA)是一种新型的核酸类似物,由多肽骨架替代核酸中的磷酸核糖骨架并与4种碱基相连而成,能够按照碱基互补的原则识别相应的碱基序列。PNA较寡核苷酸链具有更高的特异性和亲和力,不易被蛋白酶及核酸酶降解,而且对机体无毒,从而成为更有前途的反义反基因制剂。目的(1)通过建立大鼠原位肝移植急性排斥和免疫耐受动物模型,观察两组大鼠肝移植后体内DCs的迁移动态变化并对其在移植排斥中的作用进行探讨。(2)设计靶向趋化因子受体CCR7的反义PNA,分离、培养大鼠骨髓来源DCs,在体外观察反义PNA对CCR7基因表达的下调作用及其对DCs趋化活性和凋亡的影响。(3)在上述动物模型和体外实验的基础上,进一步研究体内应用反义PNA对大鼠肝移植急性排斥反应的影响,并对其可能机制进行探讨。通过以上三部分实验,来阐明大鼠肝移植后DCs的迁移特征及其在移植排斥反应中的作用,应用反义PNA下调CCR7基因表达,阻断DCs的定向迁移和抗原递呈,切断抗原信息与免疫系统的联系,从而为抑制移植排斥反应和诱导免疫耐受提供一种新的策略。方法(1)采用改良Kamada两袖套法建立大鼠原位肝移植模型,分为两组:急性排斥组,供体为Wistar大鼠,受体为SD大鼠;免疫耐受组,供受体同为Wistar大鼠。于术后第3、5、7天切取移植肝组织及腹腔淋巴结(n=4),依据肝脏病理学变化诊断肝移植术后急性排斥反应,免疫组织化学染色法检测DCs在肝组织、淋巴结的动态变化以及T细胞在淋巴结的活化增殖。(2)体外培养大鼠骨髓来源DCs,设计靶向CCR7 mRNA翻译起始区的反义PNA,以随机PNA、空白组为对照处理DCs,分别于24h,48h, 72h后收集细胞,利用免疫细胞化学法、Western blotting和逆转录聚合酶链反应(RT-PCR)技术检测反义PNA对CCR7分子表达的影响,通过趋化实验和流式细胞术检测DCs的趋化活性和凋亡率。(3)同上方法建立大鼠肝移植排斥模型,在供肝冷保存时和肝移植术后应用反义PNA,以随机PNA、空白组为对照,观察大鼠术后生存时间。于术后第7、10天取标本(n=4)观察肝功能变化和肝脏组织病理学改变,免疫组化染色法检测淋巴结DCs数量变化以及T细胞的活化增殖。结果(1)急性排斥组大鼠术后第5天出现典型的急性排斥反应表现和肝组织病理学改变,免疫耐受组大鼠术后无明显排斥反应表现,肝组织仅发生轻微的形态学变化。急性排斥组大鼠肝组织DCs数量在术后第3天明显增多,于第5天达到高峰,第7天则出现下降,淋巴结DCs数量从第3天开始明显增多,第5天、7天持续上升,与免疫耐受组相比差异有显著性。急性排斥组术后第3天即可观察到明显的T淋巴细胞活化增值反应,先于肝组织病理学变化发生,而免疫耐受组未见明显的T淋巴细胞活化增殖。(2)体外培养的大鼠骨髓来源DCs经PNA处理后,在24h,各组DCs其CCR7表达无明显差别。在48h,反义PNA组CCR7蛋白表达水平明显低于对照组,而在mRNA水平却无明显差别。在72h,反义PNA组CCR7的表达在不同水平均明显低于对照组。趋化实验和流式细胞术结果显示,在48h以后反义PNA组DCs趋化活性受到明显抑制,凋亡率明显增加。(3)大鼠肝移植术后,反义PNA组与随机PNA、空白组相比淋巴结DCs数量明显减少,T细胞活化增殖受到显著抑制,术后移植排斥反应延迟,受体生存时间明显延长。结论(1)Wistar→SD大鼠的肝移植组合是高排斥组合,而Wistar→Wistar的组合则无排斥,表现为免疫耐受。排斥组合大鼠肝移植术后,DCs的迁移动力学发生显著变化,主要表现在抗原摄取和递呈加速,形成大量DCs向淋巴结抗原递呈区的迁移和积聚,从而对T细胞产生强烈而持久的刺激,最终激活T细胞导致移植排斥反应发生。这使我们有理由相信,通过阻断DCs迁移,使抗原递呈过程不能发生,从而切断抗原信息与免疫系统的联系,可能达到抑制排斥反应的目的;或者将迁移DCs的数量和速度控制在一定的低量状态,有可能诱导免疫耐受。(2)在体外试验,靶向趋化因子受体CCR7的反义PNA显著下调DCs CCR7基因表达,不仅明显抑制DCs的趋化活性而且还可以诱导其发生凋亡。(3)在体内试验,通过反义PNA下调CCR7基因表达,阻断DCs迁移和抗原递呈,能有效抑制急性排斥反应,延长受体存活时间,尽管并没有使移植物达到长期存活和诱导免疫耐受,但是利用反义制剂阻断DCs的迁移和抗原递呈以达到抑制移植排斥反应,可能较目前所使用的免疫抑制方法更具选择性和安全性。
[Abstract]:The graft rejection is essentially the immune response of the recipient's immune system to the donor-graft antigen, a cellular response to T-lymphocyte-dependent. However, the immune system of the recipient after organ transplantation cannot directly identify the donor antigen, and the donor antigen information must be passed through the antigen-presenting cell to the receptor T-lymphocytes to activate to produce a subsequent immune response. Dendritic cells (DCs) are the most potent antigen-presenting cells. Only DCs can effectively activate the initial T-cells. The process of presenting the antigen in vivo is a dynamic migration process. The DCs carrying the antigen information migrate from the periphery to the T cell region of the lymphoid tissue to complete the antigen presentation and activate the initial T cells. It is believed that the migration of peripheral DCs to the lymphoid tissue is mainly through the lymphatic tissue chemokines on the CCR7 pathway of the DCs chemokine receptor. CCR7 is therefore an ideal target. Peptide nucleic acid (PNA) is a novel nucleic acid analog, which is formed by replacing the ribose nucleic acid in the nucleic acid by the polypeptide skeleton and is connected with the four bases, and the corresponding base sequence can be identified according to the base complementary principle. The PNA is more specific and affinity with the oligonucleotide chain, is not easy to be degraded by the protease and the nuclease, and is non-toxic to the body, thus being a more promising antisense anti-gene preparation. Objective (1) To observe the dynamic changes of the migration of DCs in the two groups of rats after liver transplantation by establishing an animal model of acute rejection and immune tolerance in orthotopic liver transplantation in rats. Objective: To study the effect of antisense PNA on the expression of CCR7 gene and its chemotaxis and apoptosis in vitro. The effect of antisense PNA on acute rejection of liver transplantation in rats was further studied on the basis of the above animal model and in vitro experiment. In this paper, the migration characteristics of DCs after liver transplantation in rats and their role in the rejection of transplantation were explained by the above three-part experiments. The expression of CCR7 gene was down-regulated by antisense PNA, the directional migration and antigen presentation of DCs were blocked, and the antigen information and the immune system were cut off. Contacting to provide a new way to inhibit graft rejection and to induce immune tolerance The model of rat orthotopic liver transplantation was established by using the modified Kamada two-cuff method. The model was divided into two groups: the acute rejection group, the donor was Wistar rats, the receptor was SD rats, and the immune tolerance group, for the receptor, was Wis. In the third, fifth and 7th day of the operation, the transplanted liver tissue and the celiac lymph node (n = 4) were cut, and the acute rejection after the liver transplantation was diagnosed according to the pathological changes of the liver. The dynamic changes of the DCs in the liver and the lymph nodes and the T cells in the lymph nodes were detected by the immunohistochemical staining. (2) In-vitro culture of rat bone marrow-derived DCs, antisense PNA was designed to target CCR7 mRNA translation initiation region, and the cells were collected at 24 h,48 h and 72 h, respectively, after 24 h,48 h and 72 h, respectively. The effect of antisense PNA on the expression of CCR7 was detected by cytochemical method, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). (3) The rat liver transplantation rejection model was established by the same method, and the antisense PNA was used after the liver transplantation and after the liver transplantation, and the rats were observed with the random PNA and the blank group. The changes of liver function and the pathological changes of the liver were observed on the 7th and 10th day after the operation. The changes of the number of DCs in the lymph nodes and T cells were detected by the immunohistochemical staining. Results (1) The expression of acute rejection and the pathological changes of the liver in the fifth day after the operation of the acute rejection group showed no obvious rejection in the immune tolerance group. The number of DCs in the acute rejection group increased significantly in the third day after the operation, and reached the peak on the fifth day. The number of the DCs in the lymph node decreased from day 3 to day 5, and the number of lymph nodes continued to rise on the 7th day, and it was associated with the immune tolerance group. No significant T-lymphocyte activation value-added reaction was observed on the third day after the acute rejection group, and no significant T-lymphocyte activation value was found in the immune tolerance group. Lymphocyte activation and proliferation. (2) In vitro cultured rat bone marrow derived DCs were treated with PNA, and at 24 h, each group of DCs was CCR. The expression of CCR7 protein in the antisense PNA group was significantly lower than that of the control group at 48 h, while the expression of CCR7 protein in the antisense PNA group was significantly lower than that of the control group at 48 h. The expression of CCR7 in the antisense PNA group was at different levels at 72 h. The results of chemotaxis and flow cytometry showed that the chemotactic activity of the antisense PNA group was significantly inhibited after 48 h. The rate of apoptosis was significantly increased. (3) The number of DCs in the antisense PNA group and the random PNA and blank group was significantly reduced after liver transplantation in rats, and the activation and proliferation of T cells were significantly inhibited. Conclusion (1) The combination of liver transplantation in Wistar rats with SD rats is a combination of high rejection, while the combination of Wistar rats and Wistar rats There was no rejection and immune tolerance. The migration kinetics of DCs after liver transplantation in combination rats showed significant changes, which were mainly manifested in the uptake and delivery of antigen, the migration and accumulation of large amount of DCs to the lymph node antigen presenting region, and thus the T-fine. Strong and lasting stimulation of the cell, and the final activation of T-fine It is a reason for us to believe that by blocking the migration of the DCs, the antigen presenting process cannot occur, so that the contact of the antigen information with the immune system can be cut off, and the purpose of inhibiting the rejection reaction may be achieved; or the number and speed of the migration DCs will be The degree of control is in a certain low amount. It is possible to induce immune tolerance in a state. (2) In vitro, the antisense PNA targeting the chemokine receptor CCR7 significantly downregulates the expression of the DCs CCR7 gene, not only significantly inhibiting the chemotactic activity of the DCs, and can also induce the apoptosis. (3) in vivo tests, the expression of the CCR7 gene is down-regulated by the antisense PNA, the migration of the DCs and the antigen presentation are blocked, the acute rejection reaction can be effectively inhibited, the survival time of the receptor is prolonged, Long-term survival and induction of immune tolerance, but the use of antisense preparations to block the migration of DCs and the presence of antigens to achieve the inhibition of graft rejection, may be more immunosuppressive than currently used
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392;R657.3

【参考文献】