【摘要】： 移植排斥反應(yīng)實(shí)質(zhì)上是受體免疫系統(tǒng)對(duì)供體移植物抗原的免疫應(yīng)答,是T淋巴細(xì)胞依賴的細(xì)胞性反應(yīng)過(guò)程。但器官移植后受體的免疫系統(tǒng)并不能直接識(shí)別供體抗原,供體抗原信息必須經(jīng)過(guò)抗原提呈細(xì)胞傳遞給受體T淋巴細(xì)胞使之活化才能產(chǎn)生后續(xù)的免疫應(yīng)答。樹突狀細(xì)胞(Dendritic cells, DCs)是功能最強(qiáng)的抗原遞呈細(xì)胞,唯有DCs才能有效激活初始T細(xì)胞,其在體內(nèi)遞呈抗原的過(guò)程是一個(gè)動(dòng)態(tài)的遷移過(guò)程,攜帶抗原信息的DCs從外周遷移到淋巴組織的T細(xì)胞區(qū)完成抗原遞呈并激活初始T細(xì)胞。目前認(rèn)為外周DCs向淋巴組織的遷移動(dòng)力主要通過(guò)淋巴組織趨化因子作用于DCs趨化因子受體CCR7這條途徑。因此,CCR7成為一個(gè)理想的目標(biāo)。肽核酸(peptide nucleic acid ,PNA)是一種新型的核酸類似物,由多肽骨架替代核酸中的磷酸核糖骨架并與4種堿基相連而成,能夠按照堿基互補(bǔ)的原則識(shí)別相應(yīng)的堿基序列。PNA較寡核苷酸鏈具有更高的特異性和親和力,不易被蛋白酶及核酸酶降解,而且對(duì)機(jī)體無(wú)毒,從而成為更有前途的反義反基因制劑。 目的(1)通過(guò)建立大鼠原位肝移植急性排斥和免疫耐受動(dòng)物模型,觀察兩組大鼠肝移植后體內(nèi)DCs的遷移動(dòng)態(tài)變化并對(duì)其在移植排斥中的作用進(jìn)行探討。(2)設(shè)計(jì)靶向趨化因子受體CCR7的反義PNA,分離、培養(yǎng)大鼠骨髓來(lái)源DCs,在體外觀察反義PNA對(duì)CCR7基因表達(dá)的下調(diào)作用及其對(duì)DCs趨化活性和凋亡的影響。(3)在上述動(dòng)物模型和體外實(shí)驗(yàn)的基礎(chǔ)上,進(jìn)一步研究體內(nèi)應(yīng)用反義PNA對(duì)大鼠肝移植急性排斥反應(yīng)的影響,并對(duì)其可能機(jī)制進(jìn)行探討。通過(guò)以上三部分實(shí)驗(yàn),來(lái)闡明大鼠肝移植后DCs的遷移特征及其在移植排斥反應(yīng)中的作用,應(yīng)用反義PNA下調(diào)CCR7基因表達(dá),阻斷DCs的定向遷移和抗原遞呈,切斷抗原信息與免疫系統(tǒng)的聯(lián)系,從而為抑制移植排斥反應(yīng)和誘導(dǎo)免疫耐受提供一種新的策略。 方法(1)采用改良Kamada兩袖套法建立大鼠原位肝移植模型,分為兩組:急性排斥組,供體為Wistar大鼠,受體為SD大鼠;免疫耐受組,供受體同為Wistar大鼠。于術(shù)后第3、5、7天切取移植肝組織及腹腔淋巴結(jié)(n=4),依據(jù)肝臟病理學(xué)變化診斷肝移植術(shù)后急性排斥反應(yīng),免疫組織化學(xué)染色法檢測(cè)DCs在肝組織、淋巴結(jié)的動(dòng)態(tài)變化以及T細(xì)胞在淋巴結(jié)的活化增殖。(2)體外培養(yǎng)大鼠骨髓來(lái)源DCs,設(shè)計(jì)靶向CCR7 mRNA翻譯起始區(qū)的反義PNA,以隨機(jī)PNA、空白組為對(duì)照處理DCs,分別于24h,48h, 72h后收集細(xì)胞,利用免疫細(xì)胞化學(xué)法、Western blotting和逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)技術(shù)檢測(cè)反義PNA對(duì)CCR7分子表達(dá)的影響,通過(guò)趨化實(shí)驗(yàn)和流式細(xì)胞術(shù)檢測(cè)DCs的趨化活性和凋亡率。(3)同上方法建立大鼠肝移植排斥模型,在供肝冷保存時(shí)和肝移植術(shù)后應(yīng)用反義PNA,以隨機(jī)PNA、空白組為對(duì)照,觀察大鼠術(shù)后生存時(shí)間。于術(shù)后第7、10天取標(biāo)本(n=4)觀察肝功能變化和肝臟組織病理學(xué)改變,免疫組化染色法檢測(cè)淋巴結(jié)DCs數(shù)量變化以及T細(xì)胞的活化增殖。 結(jié)果(1)急性排斥組大鼠術(shù)后第5天出現(xiàn)典型的急性排斥反應(yīng)表現(xiàn)和肝組織病理學(xué)改變,免疫耐受組大鼠術(shù)后無(wú)明顯排斥反應(yīng)表現(xiàn),肝組織僅發(fā)生輕微的形態(tài)學(xué)變化。急性排斥組大鼠肝組織DCs數(shù)量在術(shù)后第3天明顯增多,于第5天達(dá)到高峰,第7天則出現(xiàn)下降,淋巴結(jié)DCs數(shù)量從第3天開始明顯增多,第5天、7天持續(xù)上升,與免疫耐受組相比差異有顯著性。急性排斥組術(shù)后第3天即可觀察到明顯的T淋巴細(xì)胞活化增值反應(yīng),先于肝組織病理學(xué)變化發(fā)生,而免疫耐受組未見(jiàn)明顯的T淋巴細(xì)胞活化增殖。(2)體外培養(yǎng)的大鼠骨髓來(lái)源DCs經(jīng)PNA處理后,在24h,各組DCs其CCR7表達(dá)無(wú)明顯差別。在48h,反義PNA組CCR7蛋白表達(dá)水平明顯低于對(duì)照組,而在mRNA水平卻無(wú)明顯差別。在72h,反義PNA組CCR7的表達(dá)在不同水平均明顯低于對(duì)照組。趨化實(shí)驗(yàn)和流式細(xì)胞術(shù)結(jié)果顯示,在48h以后反義PNA組DCs趨化活性受到明顯抑制,凋亡率明顯增加。(3)大鼠肝移植術(shù)后,反義PNA組與隨機(jī)PNA、空白組相比淋巴結(jié)DCs數(shù)量明顯減少,T細(xì)胞活化增殖受到顯著抑制,術(shù)后移植排斥反應(yīng)延遲,受體生存時(shí)間明顯延長(zhǎng)。 結(jié)論(1)Wistar→SD大鼠的肝移植組合是高排斥組合,而Wistar→Wistar的組合則無(wú)排斥,表現(xiàn)為免疫耐受。排斥組合大鼠肝移植術(shù)后,DCs的遷移動(dòng)力學(xué)發(fā)生顯著變化,主要表現(xiàn)在抗原攝取和遞呈加速,形成大量DCs向淋巴結(jié)抗原遞呈區(qū)的遷移和積聚,從而對(duì)T細(xì)胞產(chǎn)生強(qiáng)烈而持久的刺激,最終激活T細(xì)胞導(dǎo)致移植排斥反應(yīng)發(fā)生。這使我們有理由相信,通過(guò)阻斷DCs遷移,使抗原遞呈過(guò)程不能發(fā)生,從而切斷抗原信息與免疫系統(tǒng)的聯(lián)系,可能達(dá)到抑制排斥反應(yīng)的目的;或者將遷移DCs的數(shù)量和速度控制在一定的低量狀態(tài),有可能誘導(dǎo)免疫耐受。(2)在體外試驗(yàn),靶向趨化因子受體CCR7的反義PNA顯著下調(diào)DCs CCR7基因表達(dá),不僅明顯抑制DCs的趨化活性而且還可以誘導(dǎo)其發(fā)生凋亡。(3)在體內(nèi)試驗(yàn),通過(guò)反義PNA下調(diào)CCR7基因表達(dá),阻斷DCs遷移和抗原遞呈,能有效抑制急性排斥反應(yīng),延長(zhǎng)受體存活時(shí)間,盡管并沒(méi)有使移植物達(dá)到長(zhǎng)期存活和誘導(dǎo)免疫耐受,但是利用反義制劑阻斷DCs的遷移和抗原遞呈以達(dá)到抑制移植排斥反應(yīng),可能較目前所使用的免疫抑制方法更具選擇性和安全性。
[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
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392;R657.3

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