大鼠移植小肠急性排斥反应期组织病理学研究

目的　探讨大鼠异位小肠移植急性排斥反应期移植小肠病理学特点及意义。方法　实验分 4组 ,每组 6只。A组为非手术对照组 ;B组为异系移植组 ;C组为同系移植组 ;D组为异系移植加环孢霉素A治疗组。术后 3 ,5 ,7,10d取移植小肠进行病理学检查 ,测量黏膜厚度 ,绒毛高度和隐窝深度 ;并检测 3 ,5 ,7d移植小肠黏膜细胞凋亡。结果　A组小肠黏膜正常 ;B组移植小肠炎性细胞浸润、黏膜结构破坏程度和黏膜细胞凋亡数目明显高于C ,D组 ,随移植时间的延长黏膜细胞凋亡数目增加 ,黏膜结构破坏程度加重 ;C组移植小肠黏膜结构损伤程度较B组轻 ;D组仅有少量炎性细胞浸润 ,间质轻度水肿 ,未见黏膜结构破坏。结论　炎性细胞浸润、黏膜细胞凋亡和黏膜结构破坏是移植小肠急性排斥反应损伤病理学变化的主要特点。动态观察移植小肠病理学变化和细胞凋亡 ,对诊断小肠移植急性排斥反应和估计损伤程度有一定的价值。     

移植物浸润T淋巴细胞肿瘤坏死因子相关凋亡诱导配体及受体DR4和DR5表达与大鼠小肠移植急性排斥反应的相关性

目的 探讨浸润T淋巴细胞七的DR4、DR5表达同小肠移植急性排斥反应的关系.方法 将2种近交系大鼠(SD、Wistar)54只按随机配对法分为A、B、C3组.A组大鼠为对照组(18只)行虚拟手术;B组(18只)大鼠行同系小肠移植;C组(18只)大鼠行不同品系小肠移植.各组大鼠于术后5 d取移植肠样本分别做HE染色和免疫荧光双标记染色.采用免疫荧光染色、激光共聚焦技术测定各组标本浸润T淋巴细胞肿瘤坏死因相关凋亡诱导配体及DR4、DR5的表达情况.结果 A组大鼠小肠黏膜正常,B组大鼠小肠表现为免疫耐受,C组大鼠表现为急性排斥反应.C组大鼠高T淋巴细胞表达肿瘤坏死因子相关凋亡诱导配体与A、B组大鼠比较差异有统计学意义(P < 0.01);A、B组大鼠浸润T淋巴细胞DR4、DR5均呈高表达,C组大鼠呈低表达,C组大鼠与A、B组比较差异有统计学意义(P < 0.01);A、B组大鼠比较差异无统计学意义(P > 0.05).结论 急性排斥反应的发生可能与浸润T淋巴细胞DR的低表达有关.减少浸润淋巴细胞DR表达的下调,或者上调DR将有助于控制急性排斥反应,诱导免疫耐受.     

移植物浸润T淋巴细胞肿瘤坏死因子相关凋亡诱导配体及受体DR4和DR5表达与大鼠小肠移植急性排斥反应的相关性

目的 探讨浸润T淋巴细胞七的DR4、DR5表达同小肠移植急性排斥反应的关系.方法 将2种近交系大鼠(SD、Wistar)54只按随机配对法分为A、B、C3组.A组大鼠为对照组(18只)行虚拟手术;B组(18只)大鼠行同系小肠移植;C组(18只)大鼠行不同品系小肠移植.各组大鼠于术后5 d取移植肠样本分别做HE染色和免疫荧光双标记染色.采用免疫荧光染色、激光共聚焦技术测定各组标本浸润T淋巴细胞肿瘤坏死因相关凋亡诱导配体及DR4、DR5的表达情况.结果 A组大鼠小肠黏膜正常,B组大鼠小肠表现为免疫耐受,C组大鼠表现为急性排斥反应.C组大鼠高T淋巴细胞表达肿瘤坏死因子相关凋亡诱导配体与A、B组大鼠比较差异有统计学意义(P < 0.01);A、B组大鼠浸润T淋巴细胞DR4、DR5均呈高表达,C组大鼠呈低表达,C组大鼠与A、B组比较差异有统计学意义(P < 0.01);A、B组大鼠比较差异无统计学意义(P > 0.05).结论 急性排斥反应的发生可能与浸润T淋巴细胞DR的低表达有关.减少浸润淋巴细胞DR表达的下调,或者上调DR将有助于控制急性排斥反应,诱导免疫耐受.     

骨髓细胞胸腺内注射对移植肠CD4、 CD8和CD25的影响

目的 探讨异基因骨髓注射在大鼠小肠移植中的免疫耐受作用和意义.方法 选用近交系大鼠F344/N和Wistar/A进行全小肠异位移植,实验组在异基因移植前7d取供体BMC行受体胸腺内注入,对照单纯同基因及异基因大鼠移植模型了解异基因供体骨髓在受体胸腺内注射能否减少移植后急性排斥反应的发生,观察其生存时间、病理结果及CD4、CD8、CD25的变化.结果 (1)A组移植大鼠平均存活时间为(7.33±1.03) d;B组为(43.76 ±4.57) d;C组为(55.28±7.48)d.(2)异基因大鼠异位全小肠移植术后3、7、15 d可出现典型的轻、中、重度排斥反应,而同基因组和实验组中未出现排斥反应.(3)异基因移植组术后CD4、CD25+,高于其他组(P＜0.05).而CD8的变化差异无统计学意义(P＞0.05).结论 移植术前7d异基因供体骨髓在受体胸腺内注射能显著减少小肠移植后急性排斥反应的发生,并可以抑制移植肠CD4、CD25细胞的表达.     

移植物浸润T淋巴细胞肿瘤坏死因子相关凋亡诱导配体及受体DR4和DR5表达与大鼠小肠移植急性排斥反应的相关性

目的 探讨浸润T淋巴细胞七的DR4、DR5表达同小肠移植急性排斥反应的关系.方法 将2种近交系大鼠(SD、Wistar)54只按随机配对法分为A、B、C3组.A组大鼠为对照组(18只)行虚拟手术;B组(18只)大鼠行同系小肠移植;C组(18只)大鼠行不同品系小肠移植.各组大鼠于术后5 d取移植肠样本分别做HE染色和免疫荧光双标记染色.采用免疫荧光染色、激光共聚焦技术测定各组标本浸润T淋巴细胞肿瘤坏死因相关凋亡诱导配体及DR4、DR5的表达情况.结果 A组大鼠小肠黏膜正常,B组大鼠小肠表现为免疫耐受,C组大鼠表现为急性排斥反应.C组大鼠高T淋巴细胞表达肿瘤坏死因子相关凋亡诱导配体与A、B组大鼠比较差异有统计学意义(P < 0.01);A、B组大鼠浸润T淋巴细胞DR4、DR5均呈高表达,C组大鼠呈低表达,C组大鼠与A、B组比较差异有统计学意义(P < 0.01);A、B组大鼠比较差异无统计学意义(P > 0.05).结论 急性排斥反应的发生可能与浸润T淋巴细胞DR的低表达有关.减少浸润淋巴细胞DR表达的下调,或者上调DR将有助于控制急性排斥反应,诱导免疫耐受.     

肠康复治疗用于小肠移植的免疫安全性研究

目的 :观察肠康复治疗对大鼠小肠移植急性排斥反应的影响 ,评价其应用的安全性。方法 :48只异基因异位小肠移植受体大鼠 (SD→Wistar)根据肠康复治疗及环孢素A应用与否随机分为 4组 ,观察至术后 14天各时相点移植小肠病理改变、固有层淋巴细胞IL 2受体表达、血浆IL 2水平以及受体大鼠脾淋巴细胞转化试验和IL 2分泌能力。结果 :肠康复治疗能够增强小肠移植受体大鼠的细胞免疫功能 ,加重急性排斥反应。应用环孢素A(10mg·kg-1·d-1)能阻断这种免疫增强作用 ,在这种情况下 ,肠康复治疗不能诱发或加重急性排斥反应。结论 :肠康复治疗能够加重小肠移植的排斥反应 ,但在应用环孢素A的情况下 ,免疫增强作用受抑制 ,不会诱发或加重急性排斥反应。     

脾细胞移植诱导大鼠小肠移植微嵌合体形成的实验研究

目的研究脾细胞输注移植促进微嵌合体形成与大鼠小肠移植免疫耐受的关系。方法制备供体大鼠的脾细胞悬液,于移植前四周将脾细胞悬液经舌静脉注入受体大鼠体内,行同种异体节段性小肠移植,PCR技术检测雌性受体外周血及皮肤Y染色体特异性DNA片段证实微嵌合体形成,通过移植肠段病理组织学变化观察急性排斥反应与微嵌合体形成的关系。结果大鼠接受小肠移植后的存活时间显示,对照组移植后平均存活时间为7.1±1.2d,显著少于实验组的18.4±3.6d(P<0.05),差异具有统计学意义。预先经脾细胞输注的实验组小肠移植受体的微嵌合体检出率高于对照组;嵌合体阳性的受体急性排斥反应较嵌合体阴性者轻。结论脾细胞输注移植可促进小肠移植嵌合体形成,微嵌合体形成可诱导大鼠小肠移植的免疫耐受。     

输血诱导免疫耐受对大鼠小肠移植急性排斥反应的影响

目的 观察供体特异性输血(DST)诱导免疫耐受对大鼠小肠移植后急性排斥反应的影响.方法 采用SD至Wistar大鼠异位小肠移植模型,实验组分别予以DST,环孢素(CsA)及DST联合CsA干预,Wistar大鼠同系间移植作为对照,于3、5、7 d观察移植肠管病理变化及受体血清中肿瘤坏死因子(TNF)-α和干扰素(IFN)-γ表达程度.结果 病理显示CsA干预组在7 d出现轻度移植排斥反应;DST联合CsA干预组与对照组结果相似,在3、5、7 d均无明显的移植排斥反应发生.并且DST联合CsA干预组TNF-α表达程度低于单独CsA干预组,在第7天时与对照组比较差异无统计学意义(P＞0.05);IFN-γ表达程度低于单独CsA干预组,在第5、7天时与对照组比较差异无统计学意义(P＞0.05).结论 异基因大鼠间小肠移植在CsA配合应用的情况下,进行DST可诱导其产生一定的免疫耐受,预防及减轻大鼠小肠移植急性排斥反应的发生及反应程度.     

肝肠联合移植对小肠移植物免疫保护作用的实验研究

目的研究肝肠联合移植中肝脏对小肠移植物的免疫保护作用。方法建立大鼠肝肠联合移植、单独小肠移植及单独肝移植模型 ,每组取 12只受体大鼠观察生存期 ,另取 6只大鼠分别于移植术后 5、7、14d收集小肠及肝脏移植物 ,进行组织病理学研究并利用半定量RT PCR方法检测移植物中IL 2、IL 4、穿孔素以及颗粒酶B的mRNA表达。结果联合移植组受体生存时间 (2 7 83±4 4 7)d较小肠移植组 (11 5 8± 3 2 6 )d明显延长 (t=7 19,P <0 0 1) ,与肝移植组 (2 8 92± 2 39)d相比 ,差异无显著性 (t=0 75 ,P >0 0 5 )。联合移植组小肠移植物排斥反应较小肠移植组明显减轻 ,穿孔素及颗粒酶B表达水平降低。结论肝 /肠联合移植可以为小肠移植物提供免疫保护。     

猪肝肠联合移植免疫耐受的实验研究

目的 研究猪肝肠联合移植中肝移植物对同源小肠移植物免疫耐受的作用.方法 70头杂交长白猪分为4组,A、B、C组为辅助性同种异体肝肠联合移植(每组20头);D组为节段性间种异体小肠移植(10头).移植后A、D组未用免疫抑制剂治疗,B、C组分别采用常规剂量和小剂量的环孢素和甲基强的松龙治疗.结果 A组术后小肠移植物较D组排斥反应时间延迟,程度明显减轻(P＜0.05).常规剂量的B组与小剂量的C组在术后存活时间、排斥反应开始时间以及排斥反应程度方面差异无统计学意义(P＞0.05).结论 猪同种异体肝肠联合移植中肝移植物可以诱导同源小肠移植物免疫耐受.     

骨髓来源半成熟树突状细胞诱导大鼠小肠移植免疫耐受

目的 观察髓样分化因子88(MyD88)沉默的半成熟树突状细胞(DC)诱导大鼠小肠移植模型免疫耐受.方法 供体乃44大鼠,受体Wistar大鼠.随机分为3组(n=6).A组(半成熟DC组)移植前7d经阴茎背静脉注射半成熟MyD88沉默DC(2×106细胞数),B组(未成熟DC组)移植前7 d经阴茎背静脉注射未成熟DC(2×106细胞数)和C组(阴性对照组)移植前7 d经阴茎背静脉注射1 ml生理盐水.3组均于注射7 d后行大鼠异位节段性小肠移植术,观察统计各组受体存活情况,检测受体血清中的促炎症因子白细胞介素(IL)-2、干扰素(IFN)-γ等的变化.结果 半成熟MyD88沉默DC组[(13.7±1.2)d,n=6]较未成熟DC组[(8.0±1.0)d,n=6,P<0.05]和阴性对照组[(6.0±0.8)d,n=6,P<0.05)存活时间显著延长,同时受体血清中IL-2和IFN-γ等促炎症因子的表达明显降低(P<0.05),病理改变亦较轻微.结论 MyD88沉默的半成熟DC具有独特的表形和功能,能够诱导受体产生特异性免疫耐受,显著延长受体术后的存活时间.     

猪节段性小肠移植免疫耐受的研究

目的 探讨免疫耐受在移植领域的应用前景,建立猪节段性小肠移植免疫耐受模型。方法 １２只幼猪为受者,随机分为实验组及对照组,另选体重相近的异性猪为供者,实验组用供者骨髓细胞预处理,２周后行节段性小肠移植,术后不使用免疫抑制剂;对照组不作预处理。结果 实验组部分移植小肠获长期存活,Ｔ细胞亚群明显下降,外周血白细胞介素（ＩＬ－６均无明显变化,外周血白细胞发现嵌合现象,病理表现为轻度急性排异和慢性排异或无     

Rat‐to‐mouse small bowel xenotransplantation: A novel model for studying acute vascular and hyperacute xenograft rejection and xenogenic cell migration

Kiyochi H, Kellersmann R, Blömer A, Garcia BM, Zhang Z, Zhong R and Grant DR. Rat-to-mouse small bowel xenotransplantation: A novel model for studying acute vascular and hyperacute xenograft rejection and xenogenic cell migration. Xenotransplantation 1999; 6: 00-00. ©Munksgaard, Copenhagen Abstract: The present study was undertaken to establish a rat-to-mouse vascularized small bowel xenotransplantation model to study acute vascular and hyperacute xenograft rejection, and xenogenic cell migration. Lewis rat small bowel grafts were transplanted heterotopically to group 1, Balb/c mice, and group 2, Balb/c mice pre-sensitized with a donor spleen cell injection. The grafts were examined by serial pathology and flow cytometry. In group 1, acute vascular rejection was present by the 5th post-operative day (POD). Immunohistology showed a strong endothelial deposition of IgG, IgM and C3, associated with a minimal lymphocytic infiltrate. There was a vigorous cell migration from the recipient to the graft, in which recipient origin cells comprised 80.1± 6.9% of the graft mesenteric lymph node by POD 3. However, there was almost no cell migration from the graft to the recipient. The intestinal xenografts in the group 2 showed massive hemorrhage, fibrin deposition, vascular congestion and thrombosis 60 min after transplantation. IgG and C3 were present on the endothelium as early as 1 min after reperfusion. The vigorous humorally-mediated vascular damage and rapid elimination of donor cells seen with intestinal xenograft rejection are distinct from the usual picture of allograft rejection. Hyperacute rejection can be induced by recipient pre-sensitization with donor spleen cells. The potential advantages of studying xenotransplantation in this model include: (1) the wide range of immunologic reagents available for mice; (2) the opportunity to study the progression of vascular damage easily by performing serial biopsies in the same animal; and (3) the opportunity to study, in vivo, two-way cellular response by examining cell trafficking in the mesenteric lymph nodes.     

大鼠肝/小肠联合整块移植模型建立

目的建立大鼠肝/小肠联合整块移植模型。方法分别以Wistar和SD大鼠为供、受体进行肝/小肠联合移植,并进行单独肝移植或小肠移植作为对照,不同移植组各取12只受体进行生存期观察。结果肝/肠联合移植手术52次,手术成功43例,成功率82.7%。联合移植组受体有9只(75%)存活时间超过60 d,且小肠移植物存活。单独小肠移植组受体均于20 d内死亡,单独肝移植组10只受体(83.3%)存活60 d以上。结论肝/小肠联合整块移植模型是研究肝/肠联合移植免疫学特性的有力模型。     

受体大鼠肠道缺血预处理对肠道黏膜屏障及移植肝脏的影响

目的探讨肠道缺血预处理(IPC)对肠道黏膜屏障的作用及其对大鼠移植肝脏形态和功能的影响。方法清洁级SD大鼠,随机分为3组:S组为假手术组(n=8);A组为肝脏移植组(n=20);B组为肠道缺血预处理肝脏移植组(n=20)。A,B组在移植后1h各取8只,测定其肠道MPO与MDA含量,血清SOD,NO含量及肝功能各项指标;光镜下观察小肠组织病理改变;测定黏膜湿重、微绒毛高度和宽度及观察移植肝脏肝小叶形态结构的变化,余动物观察存活率。结果 B组1 d生存率与A组比较无统计学差异(P0.05),但1周生存率有显著提高(P0.05)。A组小肠MPO活性与MDA含量明显高于S组和B组,血清SOD活性和NO含量则显著减低,空肠黏膜湿重、绒毛高度和宽度显著低于S组与B组(均P0.01)。光镜观察见A组受鼠空肠与移植肝脏的形态学损伤性改变较B组与S组为重。结论受体大鼠肠道缺血预处理对大鼠移植肝脏具有保护作用。     

Long-lasting donor passenger leukocytes after hepatic and intestinal transplantation in rats

BACKGROUND: Donor passenger leukocytes (DPLs) that migrate after organ transplantation stimulate the recipient immune system and normally cause rejection and graft vs. host reaction. However, DPLs also contribute to the unresponsiveness to the donor organ. The quantity and quality of these migrating cells are considered dependent on individual transplanted organs. We compared the DPLs of the liver, which might contain somatic stem cells, with those of intestinal grafts that have highly immunogenetic cells. To study DPLs over a long period, we used green fluorescent protein (GFP) transgenic (Tg) rats developed by us as donors. METHODS: We performed orthotopic liver transplantation (OLT) and small bowel transplantation (SBT) from GFP Tg rats to wild recipients. A short course of tacrolimus (0.64 mg/kg, intramuscularly) was used to prevent antigenicity of the GFP. The fate of the DPLs in the peripheral blood and the recipient bone marrow was monitored by flow cytometry. Using long-surviving recipients, the GFP(+) cells in the graft and various host immunologic organs were measured and characterized by immunohistochemical staining. RESULTS: In both groups, the numbers of the GFP(+) cells in the peripheral blood increased transiently and then gradually decreased to undetectable levels. While no GFP(+) cells were identified in the long-surviving-recipient bone marrow, there were a few GFP(+) cells in the graft liver, graft mesenteric lymph nodes and the recipient spleen. These cells showed major histocompatibility complex (MHC) class II antigen. There was no significant difference in the migration patterns of the GFP(+) cells in the OLT and SBT rats. CONCLUSIONS: In both the OLT and SBT groups, the DPLs migrated transiently in the recipient peripheral blood. A small numbers of MHC class II-positive DPLs were present at the graft site and in the host spleen, but not in the bone marrow. There were no significant differences in the migration patterns of the DPLs between the OLT and SBT rats over the long term.     

Donor-derived alloantigen-presenting cells persist in the liver allograft during tolerance induction

The predictive value of chimerism was evaluated in three different transplantation models in the rat without immunosuppression: small bowel- (SBTx), liver- (LTx), and liver/small bowel transplantation (LSBTx) were performed in the Brown Norway (BN)-to-Lewis- (LEW) strain combination. Immunohistochemistry and flow cytometry were used to identify donor cells in the recipient's spleen. Their number did not change significantly during transient rejection or tolerance after LTx and LSBTx. However, the amount of donor-derived nonparenchymal cells within the liver allograft including antigen-presenting cells (APCs), such as dendritic and Kupffer cells, clearly mirrored the recipient's immune status: as expected, their number decreased during rejection, but recovered considerably during and after tolerance induction. We conclude that donor cells in the periphery of the recipient correlate with the presence of the allograft, but do not seem to influence graft acceptance actively. However, the kinetics of the detected donor APC population in the liver suggests their important role in modifying the recipient's immune response towards tolerance. Received: 29 March 1999/Revised: 19 August 1999/Accepted: 3 November 1999     

Impact of graft length on surgical damage after intestinal transplantation in rats

BACKGROUND: Intestinal grafts greatly affect nutrition and immunology in the host. The growth of the recipient and incidence of graft-versus-host disease depend on graft length. A larger graft may affect the host immune system, but little is known about how the length of the intestinal graft severely affects surgical intervention. We developed a cervical small bowel transplantation (SBT) rat model that minimized technical variations using a cuff method and studied the effects of graft length on surgical damage in SBT. MATERIALS AND METHODS: We transplanted a whole (70 cm) or partial (15 cm) intestine into a syngeneic rat combination of LEW (MHC haplotype: RT1(l)) to LEW and evaluated changes in perioperative hemodynamics and the endogenous endotoxin level. Natural killer (NK) cell activity in the peripheral blood and the immunologic response of the recipient spleen were also studied. RESULTS: In the whole SBT model, body weight loss was more severe than in the segmental SBT model; the rats in the former model often died, while all in the latter survived indefinitely. The systemic blood pressure markedly decreased in the whole SBT group immediately after reperfusion. The proliferative activity of splenic lymphocytes stimulated by concanavalin A was also more severely inhibited in the former model than in the latter postoperatively. NK cell activity in the whole SBT rats declined more severely than the segmental SBT rats 3 days postoperatively. CONCLUSION: The longer graft severely induced surgical intervention; and influenced host immunosuppression, resulting in the higher mortality in rats undergoing whole SBT.