本文選題：肝移植　切入點(diǎn)：近交系大鼠　出處：《廣西醫(yī)科大學(xué)》2008年博士論文

【摘要】： 第一部分近交系大鼠肝移植自發(fā)性免疫耐受模型的構(gòu)建 目的:選擇近交系BN大鼠和LEW(LEW)大鼠組合,擬建立大鼠肝移植自發(fā)性免疫耐受模型,為開展肝移植免疫耐受的實(shí)驗(yàn)研究提供模型及實(shí)驗(yàn)依據(jù)。 方法:采用Kamada二袖套法,利用封閉群Wistar大鼠進(jìn)行建模技能訓(xùn)練,在此基礎(chǔ)上建立近交系大鼠BN和LEW組合之間的肝移植模型。實(shí)驗(yàn)動(dòng)物分三組,即A組:同系移植LEW→LEW(n=15);B組:同種移植LEW→BN(n=10)和C組同種移植BN→LEW(n=16)。根據(jù)臨床表現(xiàn)和Banff標(biāo)準(zhǔn)判斷是否發(fā)生排斥反應(yīng)以及排斥反應(yīng)發(fā)生的強(qiáng)度。以BN大鼠為供體,術(shù)后60天以上的C組受體(n=2)和A組受體(n=2)為受體,分別行頸部異位心臟移植,了解移植心是否被排斥。 結(jié)果:共完成171例次ROLT操作,總的手術(shù)成功率60.8%(104/171),其中模型訓(xùn)練的前期70例24h成活率僅17.1%(12/70),以后隨著手術(shù)技巧成熟,手術(shù)成功率逐漸達(dá)到90%以上。大鼠肝移植手術(shù)死亡的主要原因?yàn)槁樽硪馔�、出血性休克、肺水腫、腔血栓形成及狹窄和原發(fā)性移植物無功能等。A組同系移植,術(shù)后一周內(nèi)出現(xiàn)肝功能受損,組織學(xué)檢查發(fā)現(xiàn)肝臟間質(zhì)水腫,但無排斥反應(yīng)表現(xiàn)。B組同種移植自術(shù)后3d開始,出現(xiàn)Ⅰ級(jí)排斥反應(yīng),9d以后逐漸達(dá)到高峰,14d內(nèi)全部死于Ⅲ級(jí)排斥反應(yīng)。C組同種移植自術(shù)后5d開始,出現(xiàn)Ⅰ～Ⅱ級(jí)排斥反應(yīng),但無進(jìn)行性加重,至術(shù)后28天左右,移植肝組織結(jié)構(gòu)恢復(fù)正常。以BN大鼠為供體,術(shù)后60天以上的C組受體和A組受體為受體,進(jìn)行心臟移植,結(jié)果發(fā)現(xiàn)移植后60d的C組受體并不排斥植入的心臟,但兩例A組受體則分別于第7d和第9d排斥了供體的心臟。 結(jié)論:(1)良好的手術(shù)技巧以及對(duì)細(xì)節(jié)加以注意是大鼠肝移植模型構(gòu)建成功的關(guān)鍵(2)BN→LEW肝移植組合能夠產(chǎn)生穩(wěn)定的自發(fā)性免疫耐受,其產(chǎn)生免疫耐受的時(shí)間估計(jì)在術(shù)后60天以上,是研究肝移植免疫耐受的較好動(dòng)物模型。(3)近交系大鼠BN和LEW組合在組織結(jié)構(gòu)上有其自身特點(diǎn),手術(shù)耐受性相對(duì)較差,建模難度相對(duì)高于常用的遠(yuǎn)交系大鼠。 第二部分T淋巴細(xì)胞凋亡誘導(dǎo)大鼠肝移植自發(fā)性免疫耐受的實(shí)驗(yàn)研究 目的:探討T淋巴細(xì)胞凋亡及其誘導(dǎo)的免疫調(diào)節(jié)在大鼠肝移植自發(fā)性免疫耐受中的作用,旨在為臨床肝移植免疫耐受的誘導(dǎo)及其免疫抑制劑的合理應(yīng)用提供理論依據(jù)。 方法:將健康雄性清潔的近交系BN和LEW大鼠隨機(jī)分為4組,A、B和C組每組15對(duì),D組12對(duì)。A組:BN→LEW同種肝移植(免疫耐受組);B組:LEW→BN同種肝移植(急性排斥組);C組:BN→LEW同種肝移植(甲基強(qiáng)的松龍干預(yù)組);D組:LEW→LEW同系肝移植(對(duì)照組)。C組甲基強(qiáng)的松龍干預(yù)劑量:術(shù)日12mg/kg(i.v.),術(shù)后第一天8mg/kg(i.m.)。采用Kamada二袖套法,建立上述BN和LEW組合之間的肝移植模型。A、B和C組受體分別于術(shù)后3d、5d及7d,三個(gè)時(shí)間點(diǎn)殺死三只大鼠,D組于這三個(gè)時(shí)間點(diǎn)殺死2只大鼠,收集肝組織和血清分別做相應(yīng)檢測(cè),另留取部分新鮮肝組織立即置入液氮保存,供Fas、IL-10、TGF-β和Foxp3基因熒光定量PCR檢測(cè)。剩余的大鼠用于觀察肝移植術(shù)后長(zhǎng)期生存率。 結(jié)果:(1)施行大鼠原位肝移植術(shù)57例,手術(shù)成功率91.2%。各組肝移植術(shù)后長(zhǎng)期存活率分析,提示B組生存期最短,C組次之,而A與D組生存期明顯較長(zhǎng)(P＜0.01)。(2)A組大鼠術(shù)后3天移植肝內(nèi)出現(xiàn)較多淋巴細(xì)胞凋亡,術(shù)后5天達(dá)高峰,然后開始下降。而B,C和D組大鼠術(shù)后移植肝內(nèi)淋巴細(xì)胞較少凋亡。A組術(shù)后第3,5天移植肝內(nèi)淋巴細(xì)胞凋亡率與B,C和D組相比,有顯著性差異(P＜0.05)(3)A組術(shù)后第3d血清IL-2和IFN-γ水平升高十分明顯,與B、C和D組相比較差異十分顯著(P＜0.01),隨后A組血清IL-2和IFN-γ水平逐漸下降。B組血清IL-2和IFN-γ水平于術(shù)后緩慢上升,于第7d達(dá)高峰。(4)術(shù)后3d和5d內(nèi),各組肝內(nèi)CD4+CD25+Treg細(xì)胞的比例差異不明顯,但術(shù)后7d A組肝內(nèi)CD4+CD25+Treg細(xì)胞占CD4+的比例明顯高于B,C和D組(p＜0.05)。(5)A組術(shù)后高表達(dá)Fas基因,于術(shù)后第5天達(dá)高峰,隨后開始下降。經(jīng)統(tǒng)計(jì)學(xué)分析,A組第3,5天Fas基因的相對(duì)表達(dá)量與B,C和D組比較,差異具有顯著性(P＜0.05)。(6)A組術(shù)后3d和5d IL-10和TGF-β基因的相對(duì)表達(dá)量與B,C和D組比較,無顯著性差異(P＞0.05),但術(shù)后第7d A組IL-10和TGF-β基因的相對(duì)表達(dá)量明顯高于B,C和D組(P＜0.05)。(7)各組術(shù)后3d和5d Foxp3 mRNA的表達(dá)無明顯差異,但術(shù)后7d A組Foxp3基因的相對(duì)表達(dá)量與B,C和D組比較,差異具有顯著性(P＜0.05),表明術(shù)后7d開始A組大鼠Foxp3基因的表達(dá)明顯增強(qiáng)。 結(jié)論:(1)T淋巴細(xì)胞凋亡在大鼠肝移植自發(fā)性免疫耐受中具有重要作用,這種T細(xì)胞凋亡的機(jī)制可能是通過Fas/FasL死亡受體途徑實(shí)現(xiàn)的;(2)CD4+CD25+Treg細(xì)胞可能是誘導(dǎo)大鼠肝臟移植自發(fā)免疫耐受的因素之一,CD4+CD25+Treg細(xì)胞的免疫調(diào)節(jié)功能可能與Foxp3、IL-10和TGF-β等基因的表達(dá)量有關(guān);(3)凋亡所誘導(dǎo)的免疫調(diào)節(jié)可能與肝移植自發(fā)性免疫耐受的長(zhǎng)期維持有密切關(guān)系。(4)術(shù)中或術(shù)后早期大劑量類固醇激素的應(yīng)用可能因?yàn)橐种埔浦哺蝺?nèi)浸潤(rùn)淋巴細(xì)胞凋亡,而不利于長(zhǎng)期免疫耐受的形成。
[Abstract]:Construction of spontaneous immune tolerance model of rat liver transplantation in the first part of inbred line
Objective: to establish a rat model of liver transplantation spontaneous immune tolerance by selecting inbred BN rats and LEW (LEW) rats, and provide a model and experimental basis for the experimental study of immune tolerance in liver transplantation.
Methods: using Kamada two cuff technique using outbred Wistar rats model of skill training, on the basis of the establishment of liver transplantation model of inbred line between BN and LEW in rat. The experimental animal were divided into three groups: group A: LEW, LEW isotransplantation (n=15); group B, allograft, LEW BN (n=10) and C group, LEW BN transplantation (n=16). According to the clinical manifestations and Banff criteria to determine whether the occurrence of rejection and rejection strength. Using BN rats as donors and more than 60 days after the operation of group C receptor (n=2) and group A (n=2) receptor receptor, respectively. For cervical heterotopic heart transplantation, heart transplantation is understanding rejection.
Results: a total of 171 cases of ROLT operation, the overall success rate was 60.8% (104/171), the model in the early stage of training in 70 cases of 24h survival rate is only 17.1% (12/70) after surgery, with mature techniques, the surgical success rate has reached more than 90%. The main causes of death in rat liver transplantation for anesthesia accident, bleeding shock, pulmonary edema, cavity thrombosis and stenosis and primary graft non function.A group isotransplantation, appear to damage liver function within a week after operation, histological examination showed that liver interstitial edema, but no rejection in allograft group.B began 3D after operation, appear grade rejection 9D, then gradually reached the peak, 14d all died of grade rejection in group.C after 5D transplantation began to appear, I ~ II rejection, but no progressive increase, to 28 days after operation, liver transplantation structure return to normal. Using BN rats as donors, The recipients of C group and A group received cardiac transplantation for more than 60 days after operation. After heart transplantation, it was found that the C group receptor of 60d did not exclude the implanted heart, but two cases of A group rejected the donor heart at 7d and 9D respectively.
Conclusion: (1) excellent operation skills and attention to detail is the key to the success of the construction of rat liver transplantation model (2), BN LEW liver transplantation combined to produce stable spontaneous immune tolerance, the tolerance time is estimated at more than 60 days after the operation, is a good animal model for the study of liver transplantation immune tolerance. (3) inbred rat BN and LEW combination has its own characteristics in the organizational structure, the relatively poor surgical tolerance, the difficulty of modeling is relatively higher than the commonly used outbred rats.
Experimental study on spontaneous immune tolerance induced by second part of T lymphocyte apoptosis in rat liver transplantation
Objective: To explore the role of T lymphocyte apoptosis and its immunomodulation in the spontaneous immune tolerance of rat liver transplantation, in order to provide a theoretical basis for the induction of immune tolerance and the reasonable application of immunosuppressive agents in clinical liver transplantation.
鏂規(guī)硶:灝嗗仴搴烽泟鎬ф竻媧佺殑榪戜氦緋籅N鍜孡EW澶ч紶闅忔満鍒嗕負(fù)4緇，

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