摘要
肝移植已成为治疗终末期肝病最有效的手段,然而,感染仍是肝移植术后严重影响受体生存率和移植物存活率的主要并发症之一。由于受体术前基础疾病的严重性以及肝移植手术的特殊性,术后感染表现为病原菌种类复杂、病情凶险、死亡率极高。并且,感染与移植后排斥反应关系密切,病毒感染已被证实可促进移植后排斥反应的发生,而细菌感染与术后排斥反应的关系目前尚未完全清楚。重症感染时,机体免疫系统功能受到抑制,T细胞亚群比例失调、功能受损,呈现一种“免疫麻痹”现象,这一概念已得到越来越多证据的支持。在众多因素中,DCs的表达变化引起了广泛观注,它可通过异质性改变,影响效应性T细胞的活化,发挥免疫抑制效应。相对于此,免疫调理对脓毒症的治疗作用也越来越明确。但是,由于担心诱发排斥反应的发生,器官移植术后感染免疫调理治疗仍有争议。本研究采用大肠杆菌诱导近交系大鼠肝移植术后腹腔感染,在此基础上,给予胸腺肽α1免疫调理治疗,通过检测受体细胞免疫功能的变化,从而为临床治疗提供一定的基础理论依据。
目的
建立近郊系大鼠原位肝移植术后腹腔细菌感染模型,研究细菌感染对移植肝急性排斥反应的影响,进而研究其对受体T细胞免疫功能的影响及脾脏DCs的增殖情况和其生物学效应,并探讨胸腺肽α1对其的免疫调理作用。
方法
1.建立封闭群大鼠原位全肝移植模型:以Kamada经典二袖套法为基础,采用快速切取供肝,一针法连续缝合肝上下腔静脉,进行120例大鼠原位肝移植,观察各手术步骤操作时间、术后并发症及术后生存期。
2.建立大鼠原位肝移植急性排斥反应模型:分3种移植组合:SD→SD组(同基因对照组),SD→Wistar组及DA→Lewis组,每组32例。分别于术后3、5、7、10天各随机处死4只,观察外周血肝功能变化(ALT和TBIL)与肝脏病理改变,将余大鼠留作生存分析。
3.建立大鼠原位肝移植急性排斥反应模型并留置腹腔导管,于术后3天、5天两个时间点灌注ATCC25922大肠杆菌活菌,每个时间点分为5个感染组:2×108cfu/ml、2×107cfu/ml、5×106cfu/ml、1×106cfu/ml、5×105cfu/ml,1个对照组:腹腔灌注灭菌生理盐水2ml。于灌注前1d、灌注后1d、3d、5d、7d共5个时间点,每个时间点4只大鼠,另设8只大鼠观察感染后一般情况及生存时间。观察受体一般情况、腹水细菌培养及白细胞计数、外周血白细胞计数、TCO2、HCO3-、PH、ALT、TB以及肝肾肺HE染色,评价感染的状态及严重程度。
4.以DA或LEW大鼠为供体,LEW大鼠为受体,建立大鼠肝移植模型,根据有无诱导感染分为4组:G1组为LEW→LEW,无感染组;G2组为LEW→LEW,感染组;G3组为DA→LEW,无感染组;G4组为DA→LEW,感染组。注菌时间为术后第三天,浓度1×106cfu/mL。每组于感染前1d、感染后1、3、5d,即术后第2、4、6、8d,共4个时间点,每个时间点4只大鼠。通过观察肝脏病理改变、免疫组化法及荧光定量PCR检测移植肝内CXCR3、CXCL10变化及TUNEL法检测肝脏淋巴细胞凋亡情况,评价细菌感染对移植肝免疫排斥反应的影响。
5.实验动物分组、处置及取材时间同步骤4,应用流式细胞术检测外周血T淋巴细胞亚群比例,单相混合淋巴细胞培养鉴定其功能,ELISA法检测血清细胞因子改变,观察细菌感染对受体T淋巴细胞亚群、功能和分化的影响。
6.实验动物分组、处置及取材时间同步骤4,通过免疫磁珠分选术分离受体脾脏DCs,流式细胞术检测其表型、单相混合淋巴细胞培养鉴定其功能的改变,观察细菌感染对受体脾脏DCs的影响。
7.以DA大鼠为供体,LEW大鼠为受体,建立大鼠肝移植模型,根据有无诱导感染和/或药物干预分为4组:G1组为移植对照组;G2组在G1组基础上药物干预;G3组为感染组;G4组感染后药物干预组。感染诱导方法同步骤4,胸腺肽α1一次性2mg于术后6天或感染后72h注入腹腔。每组于感染前1d、胸腺肽α1处理后2d,即术后第8d,共2个时间点,每个时间点4只大鼠,每组另设6只大鼠观察生存期。观察大鼠一般情况、生存期、移植肝病理变化、外周血T细胞亚群比例及功能变化,探讨免疫调理对大鼠肝移植术后腹腔感染的治疗作用。
8.统计学处理:应用SPSS16.0软件进行单因素方差分析、t检验、秩和检验、生存分析等,P<0.05判定为有统计学意义。
结果
1.大鼠原位肝移植至操作稳定阶段,各主要步骤的时间为:供体手术30.2±2.5min,修肝5.7±1.6 min,肝上下腔静脉吻合9.1±0.8 min,门静脉重建1.6±0.5 min,肝下下腔静脉重建1.5±0.4 min,无肝期15.4±1.1 min,胆道重建1.1±0.3 min,受体手术39.5±1.4 min。手术成功率100%,1周存活率97.7%,晚期并发症如胆道梗阻发生率为3.3%。
2.SD→SD组均生存超过120d;SD→Wistar组中位生存时间为100d,95%可信区间为(83.369～116.631)d;两组差异无统计学意义(P=0.317);DA→Lewis组中位生存时间为12d,95%可信区间为(10.125～13.848)d,生存时间明显较SD→Wistar组缩短,差异有统计学意义(P=0.000)。SD→SD组血清ALT、TBIL浓度术后随时间的延长,逐渐下降,肝功能好转;SD→Wistar组血清ALT、TBIL浓度随时间延长,于术后5d下降相对较慢,随后逐步下降;DA→Lewis组血清ALT、TBIL浓度术后随时间的延长,呈进行性升高。至术后10d,ALT:SD-SD< SD-Wistar 3.生存期的比较,术后5d腹腔注菌组感染后存活时间最短(大部分在1~3d)。术后3d注菌,G1、G2、G3组大多数在感染后2~3d死亡;G4组存活时间在感染后5~9d;G5组存活时间在感染后9~10d;Log-Rank:G4组与G5组比较,χ2=13.322,ν=1,P = 0.000,差异有显著统计学意义;G4组与G0组比较,χ2=11.829,ν=1,P = 0.001,差异有显著统计学意义;G5组与G0组比较,χ2=0.013,ν=1,P = 0.909,差异无统计学意义。腹水白细胞计数比较,G0组术后早期轻度升高,后期降至诊断标准以下;G4组持续显著升高;G5组先升高后下降,感染后7d与G4组比较差异有显著统计学意义(P<0.01)。G4组腹水细菌培养持续阳性,G5组后期腹水细菌培养阴性。随着时间推移,G4外周血WBC、ALT、TBIL均持续升高,而PH值、HCO3-、TCO2持续下降,表现为较重的酸碱平衡紊乱、肝功能异常,至观察终点与G0组比较均有统计学差异(P<0.05);G5感染后早期亦有不同程度异常,但后期均好转,至观察终点,与G4组比较均有统计学差异(P<0.05)。HE染色结果显示,各组至观察终点肾肺均未见明显异常病理改变。
4.HE结果显示,感染后第5d(术后第8d),G3组4只大鼠病理表现均呈重度急性排斥反应,G4组仅有1只受体大鼠移植肝呈重度急性排斥反应表现,而肝实质损害加重,RAI评为为(8.0±0.8 vs 6.5±1.3,P<0.05)。CXCL-10及其配体CXCR-3免疫组化结果及mRNA水平的相对表达量,G3组呈持续上升趋势;而G4组观察时间内早中期升高,感染后5d下降,此时与G3组的比较差异有统计学意义(P<0.05)。各组移植肝内凋亡细胞均持续增多,但G3组凋亡细胞主要分布在肝实质,随时间推移,汇管区凋亡细胞增多;G4组凋亡细胞主要分布在肝实质,随感染加重,汇管区凋亡细胞明显增多,术后8d与G3组的比较差异有统计学意义(P<0.05)。
5.混合淋巴细胞培养结果显示:G1组T淋巴细胞功能术后轻度升高,G2组淋巴细胞功能呈上升趋势,G3组淋巴细胞功能明显升高,G4组淋巴细胞功能持续升高,G2、G4组淋巴细胞功能上升幅度均不及G3组,且在感染后上升趋势明显变缓。G1、G2、G3组术后第8d淋巴细胞功能比较差别均有统计学意义(0.3727±0.05479 vs 0.7795±0.00995 vs 0.9550±0.03594,P<0.05);G4组感染后第5d(术后第8d),淋巴细胞功能低于G3组(0.7560±0.00787 vs 0.9550±0.03594,P<0.05)。流式细胞术结果显示:G1组T淋巴细胞亚群及其比值均变化不大;G2组感染后5d,T细胞亚群比值降低;G3组CD4+和CD8+T细胞在术后前三个时间点有所下降,至术后8d方回升,CD8+/CD4+T细胞改变升高,与术后2d比较差异有统计学意义(P<0.05);G4组随着感染的加重,CD4+和CD8+T细胞均较快下降,两者比值亦持续下降,至感染后5d,其比值在1.43~1.46之间,较之感染前差异有统计学意义(P<0.05)。感染后5d(术后8d),三者差异G4组与G1、G3组差异均有统计学意义。外周血IL-10水平的变化:G1组术后比较稳定,变化不大;G2组感染后呈低水平升高;G3组术后持续降低;G4组感染后持续显著升高;术后第8d,G3 6.经免疫磁珠新鲜分离的大鼠脾脏DCs具有典型的树突状特征,每个脾脏可得到约1.5～3×106个OX62+细胞。代表DCs成熟状态的表面分子MHC-Ⅱ、CD80、CD86三者的变化趋势在各组术后各时间点基本保持一致。G1组术后高表达;G2组在感染后表达降低;G3组术后表达上升;G4组随着感染加重,表达水平明显降低,DCs趋于幼稚化。G2组三者表达率术后2d与术后8d差异均有统计学意义(P<0.05);G3组MHC-Ⅱ表达率术后2d与术后8d差异有统计学意义(P=0.017),CD80表达率虽不具统计学差异,但还是有所升高,CD86表达率有统计学差异;G4组三者表达率,自感染后第一天起,各时间点比较差异均有统计学意义(P<0.01);术后8d,三者表达率在各组之间均有明显差异(P<0.01)。DCs刺激同种异体T细胞增殖的能力,G4组术后8d明显下降,G3组上升,(0.27±0.05 vs 0.93±0.02,P<0.01)。
7.G1组与G2组中位生存时间均为12d,95%CI分别为(10.400～13.600)和(10.868～13.132),两者比较差异无统计学意义(P = 0.416);G3组与G4中位生存时间分别为9d和10d,95%CI分别为(7.400～10.600)和(8.868～11.132),两者比较差异无统计学意义(P = 0.671)。至观察终点,G1、G2组RAI评分均为8～9分,属于重度急性排斥反应;G3、G4组术后第8d移植肝RAI评分6～8分,介于中到重度排斥反应之间;G1与G2、G3与G4组比较差异均无统计学意义(P>0.05)。处理后,G2组、G4组淋巴细胞亚群数量及比值均升高,T淋巴细胞功能升高,G2组与G1组及G4组与G3组比较,差异均有统计学意义(P<0.05)。
结论
1.本实验建立了稳定的封闭群大鼠原位肝移植模型,手术时间短、成功率高、稳定可靠。在此基础上,成功建立了稳定的近交系大鼠原位肝移植急性排斥模型,结果稳定、可靠、重复性好,可用于相关领域的基础研究。
2.本实验采用腹腔内大肠杆菌灌注建立肝移植术后腹腔细菌感染的模型可行、可靠、可重复:1)可比较确切的证实腹腔内感染的存在,2)无需二次手术诱导感染,减少创伤对受体大鼠的打击,可满足实验需要。
3.随着感染加重,一定程度上加重了肝实质损害,但部分缓解了肝移植排斥反应病理表现程度。趋化因子CXCL-10及其受体CXCR-3在感染后期表达减少以及移植肝汇管区及小叶中央静脉周围浸润淋巴细胞凋亡增多是移植肝排斥反应减轻的原因之一。
4.排斥反应和感染早期T淋巴细胞功能均增加,移植术后感染后期淋巴细胞功能严重降低;排斥反应使机体CD4+/CD8+T细胞比值升高;感染使CD4+/CD8+比值早期增加,晚期降低;二者的协同作用则使CD4+/CD8+T细胞比值显著下降。排斥反应发生时,血清IFN-γ水平升高;感染作用下,IL-10水平升高;两者共同作用下,促进Th1细胞向Th2细胞转变。机体抗炎反应占优势,表现为一种局部和全身免疫抑制状态。
5.细菌感染早期或急性免疫排斥反应均可促进脾脏DCs快速成熟,促进其刺激同种异体T细胞增殖的功能;至感染后期,尤其是排斥反应和感染同时作用时,DCs表现为未成熟状态,刺激同种异体T细胞增殖的功能明显下降。
6.大鼠肝移植术后合并腹腔细菌感染,短时间内,给予免疫调理治疗未明显延长受体生存时间,但亦未发现加重移植肝急性排斥反应的程度,却能提高肝移植术后受体T淋巴细胞亚群、比值及淋巴细胞功能。
Liver transplantation(LT) has become the most effective treatment for patients with end-staged liver diseases, however, infections is still common after LT and seriously decreases the recepients and graft survival. As the severity of illness of recepients pretransplant, as well as the particularity of LT, posttransplantive infections charactered various of pathogen species, severity, and high morbidity and mortality. Moreover, infection related with rejection posttransplant. Viral infection has been shown to promote chronic rejection posttransplant, whilethe relationship between bacterial infection and rejection are not entirely definited. Severe infections, accompanied with imbalance and functional impairment of T cell subsets, showed "immune paralysis" of the immune system. DCs play an immunosuppressive effect role during this course via their heterogeneity. There has been growing evidences to support this. The effection of immune regulation treatments to sepsis has been more explicit. However, it is controversial in organ transplant due to fear of rejection. In this study, the intraabdominal infection was induced by E. coli after orthotopic liver transplantation in rats(ROLT), and thymosin-alpha1 was injected intraperitoneally then, the changes of cellular immune response was monitored.
Objective
To momitor the immume function changes of T cells and the proliferation and biological effect of dendritic cells derived from spleen and to explore the role of thymosinα1 in the immune conditioning of recepients by established a model of intraabdominal bacterial infection following orthotopic liver transplantation in rat.
Methods
1. Established the orthotopic liver transplantation model of closed colony rats: based on the classical two-cuff technique by Kamada, 120 cases of rat orthotopic liver transplantation were performed with fast resecting the donor liver, continuous sutureing suprahepatic inferior vena cavum(SHVC) to observe the time of surgical procedures, postoperative complications and recepients survival rate.
2. Establishment of rats orthotopic liver transplantation with acute rejection models: according to the different donor-recipient combinations,the research animals were divided into three groups: SD→SD group (Isograft group), SD→Wistar group and DA→Lewis groups, each group with 32 cases. Six rats killed randomly to observe the changes in blood liver function (ALT and TBIL) and liver pathological changes in 3, 5, 7, 10 days respectively after transplantation. The residual rats were retained for survival analysis.
3. Peritoneal catheter was retented after rats orthotopic liver transplantation with acute rejection model. E. coli ATCC25922 was perfused through the tube 3 days and 5 days posttransplant. The research groups were randomly divided into five infection groups, including 2×108cfu/ml, 2×107cfu/ml, 5×106cfu/ml, 1×106cfu/ml, 5×105cfu/ml, and one control group (perfused sterile saline 2ml intraperitoneally) at each time point. Rats were sacrificed in 1 day before perfusion, 1 day, 3 days, 5 days, and 7 days after perfusion resceptively, each time point with four rats, another eight rats involved in each group was retained for survival analysin. To observed recepients’general condition, ascites bacterial culture and leukocyte count, peripheral blood white blood cell count, TCO2, HCO3-, PH, ALT, TB, as well as liver and kidney lung HE staining to evaluate the status and severity of infection.
4. Used DA or LEW rats as donors and LEW rats as recepients, established rat liver transplantation model. Based on whether induction of infection or no, the experimental animals were divided into four groups: G1 group (LEW→LEW, non-infection group), G2 group (LEW→LEW, infection group), G3 group (DA→LEW, non-infection group), G4 group (DA→LEW, infection group). The bacteria were perfused through peritoneal catheter with concentration of 1×106cfu/mL three day after transplantation. Rats were sacrificed in 1 day before perfusion, 1 day, 3 days, and 5 days after perfusion resceptively, each time point with four rats. Through three means: liver pathology, CXCR3 and its ligand CXCL10 changes detected by immunohistochemistry and fluorescence quantitative PCR, and apoptosis of liver cells assayed by TUNEL to evaluate the change of the graft immune rejection responses.
5. Experimental animal groups, disposition, and time of specimen collection were similar with step 4. The proportion of peripheral blood T lymphocyte subsets and its function were identified by flow cytometry (FCM) and one-way mixed lymphocyte culture respectively. Serum cytokine changes were detected by ELISA to evaluated the T lymphocyte subsets, function and differentiation after bacterial infection.
6. Experimental animal groups, disposition, and time of specimen collection were similar with step 4. The spleen dendritic cells(DCs) of recepients were isolated freshly by magnetic cell sorting technique. Their phenotype and function were detected by flow cytometry and one-way mixed lymphocyte culture to evaluate the effects on spleen DCs by bacterial infection on recepients.
7. Used DA rats as donors and LEW rats as recepients to established rat liver transplantation model. According to whether induction of infection and/or drug intervention or no, rats were divided into four groups: G1 group was the control group, G2 group was drug intervention based on G1, G3 group was infected group, G4 group was drug intervention based on G3. Infection-induced method was similar to the step4. Thymosinα1 with a dose of 2mg was injected into the abdominal cavity in a one-time six days after transplantataion (72h after infection). Rats were sacrificed 1 day pre-infection and 2 days after drug intervention (eight days posttransplantation) respectively. Each time point and each group included four rats. Six rats were retained to survival analysis in each group. The general conditions and survival rate were observed. The pathological changes in liver grafts, the proportion of peripheral blood T lymphocyte subsets and their functional changes were inditified to evaluate the effection of immune regulation on rat liver transplantation with intra-abdominal bacteria infection.
8. The data were analyzed through SPSS16.0 software. One-way ANOVA, t test, rank sum test methods and Kaplan-Meier survival analysis were applied. P<0.05 was considered statistically significant.
Results
1. In the stabile stage of rat orthotopic liver transplantation, the time of the main steps of operation were as follow: donor operation 30.2±2.5min, donator liver trimming 5.7±1.6 min, suprahepatic inferior vena cava(SHVC) anastomosis 9.1±0.8 min, portal vein (PV) reconstruction 1.6±0.5 min, infrahepatic vena cava(IHVC) reconstruction of 1.5±0.4 min, anhepatic period 15.4±1.1min, biliary duct reconstruction 1.1±0.3min, recepients operation 39.5±1.4 min. Surgical success rate was 100%, one-week survival rate was 97.7%, late complications such as biliary obstruction incidence was 3.3%.
2. The survival time of SD→SD group was more than 120d; the median survival time of SD→Wistar group was 100d, its 95%CI was (83.369~116.631)d, no statistical difference was found between the two groups(P=0.317). The median survival time of DA→Lewis Group was 12d, its 95%CI was (10.125 ~13.848)d, the difference had remarkable statistical significance (vs SD→Wistar group, P=0.000). In regard to serum ALT and TBIL levels, downward trend was found in SD→SD group gradually, the same was seen in SD→Wistar group but slower during the first five days posttransplant, while in DA→Lewis Group they increased progressively. At 10 days after transplantation, ALT and TBIL presented SD-SD < SD-Wistar < DA-Lewis simultaneously, (58.38±12.44 vs 100.96±16.50 vs 1162.25±84.34) and (15.99±5.92 vs 76.65±9.45 vs 175.06±16.67) respectively, the difference all had statistical significance (DA→Lewis Group vs SD→SD group and SD→Wistar group, P<0.05). There was no acute rejection episodes in SD→SD group with rejection activity index (RAI) score 1 to 2 score. The same representations was found in SD→Wistar group except that 1 rat showed severe acute rejection with RAI 8 score at 10 days postoperation. In DA→Lewis group, acute rejection episodes appeared in some rats at three days after operation and the pathological grading of damages all intensified quickly with RAI score 8 to 9 at 10 days posttransplant. And the result of Kruskal-Wallis H test in day 10 showed:χ2=20.107,ν=2, P=0.000, obviously statistical significance was found between three groups.
3. The survival time were extremely short in all intra-abdominal injection groups induced in 5d postoperative (mostly of the 1~3 days after infection). Among the intra-abdominal injection groups induced in 3d posttransplant, the survival time after infection were most of 2~3 days in G1, G2 and G3 group, 5~9 days in G4, 9~10 days in G5. The result of Log-Rank analysis were follow: (χ2=13.322,ν=1,P = 0.000, G4 vs G5), (χ2=11.829,ν=1,P = 0.001,G4 vs G0) and (χ2=0.013,ν=1,P = 0.909,G5 vs G0). In regard to ascites white blood cell count, a continuously increased trend was found in G4, while G5 showed increased firstly and then decreased. Remarkly statistical difference was found with P<0.01 in 7 days after infection (G4 vs G5). Ascites bacterial culture was negative in G5 group during the late phase of infection, it might be that the body resisted bacterial spontaneously. While in G4, it was continuously positvie. As time goes by, peripheral WBC, ALT and TBIL increased continuously, PH, HCO3- and TCO2 value decreased, showing serious acid-base balance disturbances and abnormal liver function in G4 group. Statistical difference was found with P<0.05 in end point of study (G4 vs G0). There were abnormalities during earlier phase of infection in G5, but normalities during later phase, statistical difference was also found with P<0.05 in end point of study (G4 vs G5). HE staining showed that kidneys and lung were normal in pathology in each group at the end of the observation.
4. HE staining showed that all the four rats presented severe acute rejection in G3 group. While only one recepient showed severe acute rejection pathologically in G4 group, but parenchyma of liver graft was damaged more seriously than G3. The RAI score of this two groups were (8.0±0.8 vs 6.5±1.3,P<0.05). In regard to CXCL-10 and its ligand CXCR-3 either in immunohistochemistry results or the relative expression of mRNA levels, a continuously increased trend was found in G3 group, while G4 showed increased firstly and then decreased. statistical difference was found with P<0.05 in end point of study (G4 vs G3). The amount of apoptotic cells in liver graft increased continuously in all groups, they distributed mainly in the hepatic parenchyma in G3 but mainly in portal area in G4. Statistical difference was found with P<0.05 in terminal point of study (G4 vs G3).
5. The result of one way MLR showed that T lympholeukocyte function elevated slightly in G1, increased in G2, elevated remarkably in G3, increased constituously in G4. The increased magnitude of G2 and G4 were lower than G3. Statistical differences were found between G1, G2 and G3 (0.3727±0.05479 vs 0.7795±0.00995 vs 0.9550±0.03594, P<0.05) at day 5 after infection. The difference was seen between G3 and G4 (0.7560±0.00787 vs 0.9550±0.03594, P<0.05). Correspondingly, the trends of T lympholeukocyte subpopulation and CD4+/CD8+ ratio in various group was different. No obvious changes were found in G1 group. Rising trend was found in G2 group except day 5 postinfection. The subpopulations of T lympholeukocyte decreased in the first three time point in G3 except day 8, the CD4+/CD8+ ratio elevated continuously ( day 8 vs day 2, P<0.05). Obvious decreased trend of CD4+ and CD8+ T cells and CD4+/CD8+ were seen in G4 with a ratio of 1.43~1.46 at day 5 after infection (P <0.05, vs G1 and G3). Regarding to serum cytokines, the trends of IL-10 level were unchanged in G1, mildly elevated in G2, significantly reduced in G3, and remarkably elevated in G4. Pairwise comparisons between groups differences were obviously statistical significant (P <0.01) in day 5 after infection. Contrastively, IFN-γlevel was significantly increased in G3 group (and acute rejection occurred synchronously), but reduced notedly in G2 and G4 at day 5 after infection (vs day 3, P<0.01). Pairwise comparisons between groups differences were remarkably statistical significant (P<0.01) in day 5 after infection. Another serum cytokines we examined was IL-12, the trends of it in all experimental groups were similar to IFN-γlevel, but the level decreased more significantly in day 5 post-infection in G4 than in G2 (P<0.05).
6. Rats spleen DCs were with typical dendritic features isolated using immunomagnetic beads freshly. About 1.5~3×106 cells were available from each spleen. The expression trends of MHC-Ⅱ, CD80 and CD86 remained almost coherence at all time points in all group. G1 was high expression postoperative, G2 group decreased after infection, G3 increased continuously during the whole process, the expression levels in G4 were significantly lower, DCs oriented toward immature. Pairwise comparisons between groups differences were significantly statistical significant (P<0.01) in day 8 after surgery. Differences were all statistical significant on three surface molecules in G2 and G4 groups at day 8 vs day 2 posttransplant (P<0.05). The same results were seen in G3 except CD80. In regard to the function (to stimulate allogeneic T cell proliferation capacity) of DCs at day 8 after operation, decreased in G4 group but increased in G3 group (0.27±0.05 vs 0.93±0.02, P<0.01).
7. The median survival time were 12 days in both G1 and G2 group, 95% CI was (10.400~13.600) and (10.868 ~13.132), respectively, the difference was not statistical significant (P>0.05). The median survival time were 9 days (95% CI, 7.400~10.600) in G3 and 10 days (95% CI, 8.868～11.132) in G4, the difference was not statistical significant (P>0.05). At the terminal of research, HE staining result showed severe acute rejection with RAI score of 8~9 in G1 and G2, and moderate to severe acute rejection with RAI score of 6~8 in G3 and G4. Difference was not statistical significant (P>0.05) between G3 and G4. The subsets of T cells and CD4+/CD8+ ratio both increased in treatment groups G2 (vs G1) and G4 (vs G3) (P<0.05).
Conclusions
1. A high survival rate, stable and reliable closed colony rat orthotopic liver transplantation was established in this study. Futhermore, a inbred rat orthotopic liver transplantation with acute rejection model was successful established too.The results were stable, reliable, reproducible, can be used for basic research in related fields.
2. The model of intraabdominal infection induced by E. coli after liver transplantation in the inbred rat was feasible and reliable and repeatable with following advantages e.g. more precise confirmation of the existence of intraabdominal infection and reducement of trauma to the rats. The model could meet the needs for experimental study.
3. Intraperitoneal bacterial infection after liver transplantation in the rats worsened the hepatic parenchymal damage in some extent, but released the pathological manifestations of acute rejection of allograft partly. The decreased expression of chemokine CXCL-10 and its receptor CXCR-3 in the late stage of infection, as well as the augmentation of apoptosis of infiltrating lymphocytes around the centrilobular vein and periportal space of allograft may be one of the causes contributed to the improvement of liver acute rejection.
4. The T lymphocyte function increased during the early stage of acute rejection and/or infection. It reduced significantly in the late phase of acute rejection accompanied with infection. The ratio of CD4+/CD8+ T cells increased in the acute rejection model, and increased during the early stage of infection model but decreased in the later period, and it dropped dramatically in the infection folling acute rejection model. The levels of serum IFN-γelevated when acute rejection occurred; the serum levels of IL-10 increased in the infection model. The synergistic action of these two factors promoted the change from Th1 cells to Th2 cells. Serum IL-12 secretion reduction may have contributed to this change. Anti-inflammatory response and systemic immunosuppression took the place during this time.
5. The spleen DCs presented rapid mature and full function to stimulate allogeneic T cell proliferation during early stage of acute rejection and/or infection. But it was immaturing and lost the function during the late phase of infection, in particular under the comorbid state of infection and rejection, which may be involved in local and systemic immune suppression of the recepients.
6. Immune regulating treatment did not significantly prolong the survival time of recepients and not augmented acute rejection during the short-term of rats liver transplantation complicated with abdominal bacterial infection. But it improved the subproportions, ratio and function of T lymphocyte according to our small size samples. A large scale of sample size was needed to enhance the reliability of this results.
引文
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