近系大鼠小肠移植模型排斥反应的研究

目的　动态观察近交系大鼠BN→F3 44组合模型小肠移植排斥反应的发生规律 ,探讨该模型对小肠移植排斥反应的研究价值。方法　选用近交系大鼠F3 44(RT1l)和BN (RT1n) ,根据改良Monchik法建立异位全小肠移植模型。实验分为同系移植组 (F3 44→F3 44,ITX 组 )和同种移植组 (BN→F3 44,ATX 组 ) ,每组供、受体大鼠各 8只。结果　 (1)ITX 组大鼠平均生存期 3 0d以上 ,ATX 组平均存活 12d。 (2 )ITX 组术后各时点的大体观察无明显差异 ,而移植小肠病理组织学术后 3d呈非特异性炎症反应表现 ,术后 0 ,5 ,7,9d与正常小肠的组织学特征基本相同。 (3 )ATX 组在术后 5 ,7,9d的大体表现与组织病理改变分别符合轻、中、重度排斥反应的诊断标准。结论　近交系大鼠BN→F3 44组合模型轻、中、重三个层次排斥反应特点鲜明 ,适用于小肠移植排斥反应免疫机制的研究。     

大鼠原位节段小肠移植模型的改进

目的 建立一种简便、稳定的大鼠原位节段小肠移植模型.方法 供、受体均为雄性SD大鼠,各40只,采用供体肠系膜上动脉-腹主动脉漏斗状袖片与受体肾下腹主动脉端侧吻合,供体门静脉与受体左肾静脉Cuff套管袖套吻合.切除受体大部分小肠,供体小肠近、远端分别与受体残留小肠近、远端行端端吻合.结果 供体手术时间(40 ±5)min,受体手术时间(50 ±8) min.热缺血时间(5±2)min,冷缺血时间(15±5) min.动脉吻合时间(5±2)min,静脉吻合时间(4 ±2)min.90.0％ (36/40)的大鼠术后存活时间＞10d.结论 该模型操作简便,手术时间短,模型成活率高,稳定性好.     

外源性腺苷三磷酸对移植胰腺再灌注损伤保护作用的实验研究

目的　探讨外源性三磷酸腺苷 (ATP)对大鼠移植胰腺再灌注损伤的作用及其机制。方法　应用同系大鼠异位全胰十二指肠移植模型 ,供体胰腺分别用EuroCollin液 (EC)或EC液添加ATP约 12～ 2 0ml(2～ 4℃ )进行低温灌注保存 6 0min ,光镜观察胰腺组织结构变化 ,放射自显影鉴定外源性ATP是否进入胰腺细胞内 ,高压液相色谱法 (HPLC)检测保存后移植物ATP和总腺苷核苷酸(TAN)。移植 2 4h后 ,检测血糖、血清中脂肪酶、淀粉酶 ,测定移植胰腺组织中髓过氧化酶 (MPO)活性 ,并进行组织学观察。结果　实验组保存的胰腺组织结构损伤明显轻于对照组。保存后实验组胰腺组织ATP和TAN水平 [(5 6 6± 0 37) μmol/ g ,(8 6 2± 0 88) μmol/g]明显高于对照组 [(2 82±0 2 4 ) μmol/ g ,(4 34± 0 4 1) μmol/ g],差异具有显著性 (P <0 0 5 )。放射自显影显示外源性 [α 3 2 P]ATP进入胰腺细胞内。移植胰腺早期功能指标检测 ,实验组血糖值 [(9 3± 2 2 )mmol/L]明显低于对照组 [(14 1± 2 9)mmol/L],实验组血清脂肪酶 [(139± 13)U/L]明显低于对照组 [(2 96± 2 5 )U/L],实验组胰腺组织MPO[(1 19± 0 16 )U/ g ]明显低于对照组 [(2 2 5± 0 2 8)U/ g],差异均具有显著意义 (P <0 0 5 )。结论　外源性ATP用于胰腺     

Protective effect of fructose-1,6-bisphosphate in the cold storage solution for liver preservation in rat hepatic transplantation

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods. FBP maintains ATP levels and thereby cellular energy metabolism, which is important to the liver during cold preservation. In the present study, we evaluated the effects of FBP on the composition of storage solutions for cold liver preservation. Adult male Wistar rats were randomly divided into three experimental groups. Hepatic perfusion and preservation were performed with UW, UW plus 10 mmol/L FBP (UWM), and FBP 10 mmol/L (FBPS) alone solutions. Biochemical measurements of AST, ALT, and TBARS were performed on samples of the cold storage solution at 0, 12, 18, and 24 hours preservation. FBPS and UW solutions showed similar preservation grades during 18 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade. FBP appears to protect the liver from injury caused by free radicals when the preservation time is less than 18 hours. Therefore, FBP may exert a protective effect for the preservation of livers during cold storage, and could represent an important component of new cold storage solutions.     

Metabolic aspects of small bowel transplantation in inbred rats

An inbred rat model of small bowel transplantation was used to study the metabolic consequences of systemic venous drainage of the graft. Lewis rats received either Lewis (isograft) or Lewis X Brown Norway F1 (allograft) small bowel grafts. Venous drainage of the isografts was to either the portal vein or the inferior vena cava. Allograft recipients underwent systemic venous drainage and were treated with a 4-week course of tapering cyclosporine. Ammonia levels in systemically drained isografts (108 +/- 5 microM/100 ml) were more than twice those in portally drained isografts (38 +/- 3, P less than 0.001), while amino acid analysis showed significant elevations in glycine, serine, asparagine, histidine, phenylalanine, and tyrosine. Ammonia levels decreased and amino acid alterations were generally corrected when animals were fed a modified protein diet low in aromatic and high in branched chain amino acids. Recipients of both systemically and portally drained isografts grew normally, while weight gain in allograft recipients was impaired. We conclude that systemic venous drainage of small bowel grafts results in altered ammonia and amino acid levels that resemble those found in models of hepatic encephalopathy; these changes can be significantly ameliorated by dietary modification; and the compromised growth seen in systemically drained allografted animals results from chronic rejection and/or cyclosporine rather than the partial porto-systemic shunt.     

大鼠小肠移植模型的改进

目的　通过改进技术 ,建立一种简便稳定存活率高的大鼠异位节段小肠移植模型。方法　“无损伤”游离 ,原位冷灌注 ,切取带有腹主动脉和肠系膜上静脉并门静脉的节段小肠 ,4℃乳酸林格氏液保存 1h。游离受体左肾静脉 ,切除左肾。采用显微外科技术行供体腹主动脉对受体腹主动脉的端侧吻合 ,门静脉与受体左肾静脉行袖式吻合。移植肠近端关闭 ,远端外置。结果　共进行 87次移植实验 ,其中 2 6次为正式实验。动脉、静脉吻合时间分别为 2 5～ 30min和 5min。 2 6只受体鼠中2 1只存活超过 3d ,平均存活 (8.93± 2 .5 9)d ,最长存活时间为 14d。结论　良好的血管吻合和充分补充液体是手术成功、移植肠具有良好活力的关键因素     

Protective effect of ulinastatin against ischemia-reperfusion injury in rat small bowel transplantation

INTRODUCTION: We aimed, to evaluate the protective effects of ulinastatin (UTI) against graft ischemia-reperfusion injury in rat small bowel transplantation (SBT). METHODS: Thirty-six recipients of rat SBT were randomly divided into three groups: 1, normal control, the graft was implanted immediately after harvest; 2, grafts preserved for 4 hours; and 3, grafts preserved for 4 hours and UTI administered to the recipients intravenously (50000 U/kg/d). Variables included pathological score and content of Na+-K+-ATPase, xanthine oxidase (XOD), malondialdehyde (MDA) and glutathione (GSH) in the transplanted small intestine. RESULTS: The cold preservation caused moderate injury to the graft which was manifested by pathological changes as well as elevated XOD and MDA and decreased Na+-K+-ATPase and GSH content. Application of UTI diminished these changes. CONCLUSIONS: UTI may exert protective effects against the ischemia-reperfusion injury of transplanted small intestine thereby promoting structural and functional recovery of the graft.     

缺血-再灌注不同时期应用1,6-二磷酸果糖对幼兔心脏的保护作用

目的　研究在缺血 再灌注的不同时期 1,6 二磷酸果糖 (FDP)对幼兔离体心脏的保护作用。方法　建立幼兔离体心脏做功模型 ,行缺血 再灌注试验 ,于缺血 再灌注的不同时期给予5mMFDP ,观察再灌注期的心功能、心肌组织含水量、心肌MDA、冠脉液CK含量以及心肌超微结构的变化。结果　缺血期给药组与缺血前给药组和再灌注期给药组比较 ,缺血期给药组的心功能恢复百分率、心肌超微结构优于后两组 ,缺血期给药组的冠脉液CK含量、心肌组织含水量和MDA含量显著低于后两组。结论　缺血期给 1,6 二磷酸果糖对幼兔心肌缺血 再灌注损伤的保护优于缺血前或者再灌注期给药     

大鼠异位节段小肠移植模型的建立

目的建立大鼠异位节段小肠移植模型。方法对100只雄性SD大鼠施行50次异位节段小肠移植，采用肠系膜上动脉-腹主动脉端侧吻合以及门静脉-左肾静脉套管吻合重建供肠血管，远端肠管腹壁造瘘。结果预实验阶段移植25只，存活3只，成功率12％；正式实验阶段25次手术，成功21次，成功率84％；供体手术时间（60±5）min，移植肠修整时间（15±5）min，受体手术时间（100±10）min。结论大鼠小肠移植中注重手术操作中的细节问题是建立稳定模型的关键。     

大鼠小肠移植排斥反应中细胞凋亡的动态研究

目的 研究小肠移植后排斥反应与细胞闻过则喜良心的关系以及雷帕霉素（RAPA）对移植后排斥及细胞凋亡的作用。方法 以SD大鼠为假移植组作对照（1组）,其他各组采用Wistar→SD大鼠异位小肠移植模型,移植后随机分为排斥反应组（2组）、雷帕霉素2mg.kg^-1组（3组）和雷帕霉素4mg.kg^-1.d^-1组（4组）,每组术后1、3、5、7d分别处死6只大鼠,获取移植肠组织行病理学检查和细胞凋亡检测。结果 各组在术后1、3d均未发现排斥反应,2组术后5、7d分别出现I度和Ⅱ度排斥,3组仅在术后7d出现早期排斥迹象,4组枯术后一直未见排斥证据,小肠细胞凋亡数量在排斥反应前期和排斥反应时显著增加,不同强度的排斥反应,细胞凋亡数量差异有显著性。结论 排斥反应中可出现大量的肠细胞凋亡,其始发时间早于排斥的组织学发现,凋亡细胞数量与排斥强度呈正相关,可作为小肠移植后排斥反应的另一种可信的标志物。雷帕霉素对排斥反应的抑制能力与剂量有关,并不能抑制肠细胞凋亡的发生。     

Metabolic effects of systemic venous drainage in small bowel transplantation

Small bowel transplantation has proved feasible in rats and in larger animals, but several important questions remain to be addressed before it becomes routine therapy in humans. One consideration is the site of venous outflow of the allograft. While portal drainage reestablishes the physiologic route of venous outflow, systemic drainage creates a partial mesocaval shunt, the metabolic consequences of which have not been studied in detail. Using a canine model of orthotopic small bowel autotransplantation, we compared the metabolic changes following transplantation with portal versus systemic venous drainage. Changes in blood ammonia levels, plasma amino acid composition, and hepatic blood flow were studied, since the Eck procedure produces metabolic changes of hyperammonemia and amino acid imbalances, while portocaval venous drainage of small bowel transplant produces a profile similar to that in controls. These data suggest that there is no metabolic disadvantage of systemic venous drainage as compared with controls. Because of its technical simplicity, systemic venous drainage may be preferable for small bowel transplantation.     

Effects of immunosuppressive therapy after experimental small bowel transplantation in rats

Significant improvements of graft and patient survival have been achieved over the past 20 years in the field of intestinal transplantation. Tacrolimus monotherapy with corticosteroids, or in combination with sirolimus is the most commonly used immunosuppressive regimen. Early (24h) after experimental allogenic small bowel transplantation in rats, sirolimus reduces the cellular and molecular inflammatory response with subsequent graft dysmotility more efficiently than tacrolimus, with contrary effects at 7 days after transplantation. This study evaluates three immunosuppressive strategies in the post-acute phase after intestinal transplantation - tacrolimus or sirolimus monotherapy and the combination therapy of tacrolimus with infliximab. After orthotopic intestinal transplantation between Brown Norway and Lewis rats, animals received 14 days of immunosuppressive treatment. Histology, infiltration of neutrophils, monocytes and macrophages, cytokine and mediator mRNA expression (real time RT-PCR) and smooth muscle function in a standard organ bath were assessed at 14 days after transplantation in all treatment groups and isogenic controls. Allogenic transplanted rats without immunosuppressive therapy and non-transplanted animals served as further control. Tacrolimus prevented acute rejection and graft dysmotility more effectively (p ≥ 0.05) than sirolimus. Reduced mRNA expression levels of CD4, IFN-γ, IL-6, IL-10, iNOS, NFκB, TNF-α and MCP-1 (p ≤ 0.05) were observed in tacrolimus treated animals compared to sirolimus. Additional infliximab application did not influence the cellular and molecular inflammatory response in the post-acute phase after transplantation. In conclusion, the severe cellular and molecular inflammatory response in allogenic transplanted grafts without immunosuppressive therapy is ameliorated by all three immunosuppressive regimens, but tacrolimus was found to be more efficient than sirolimus at 14 days after transplantation. Our findings do not rule out the usage of sirolimus as single immunosuppressive therapy, but indicate and confirm the potency and effectivity of tacrolimus as basis immunosuppressant in the field of intestinal transplantation.     

近交系大鼠小肠移植模型排斥反应的动态观察

目的:动态观察近交系大鼠小肠移植排斥反应发生规律,探讨该模型对小肠移植排斥反应研究的价值.方法:选用近交系大鼠F344(RT11)和BN(RT1n),根据改良Monchik法建立下腔静脉回流式异位全小肠移植模型.实验分为同系移植组(F344→F344,ITx组)和同种移植组(BN→F344,ATx组),每组各制作实验模型8只.结果:①ITx组大鼠平均生存期30d以上,ATx组大鼠平均存活12d.②ITx组大鼠术后各时点的大体观察无明显差异,而移植小肠病理组织学POD 3d呈非特异性炎症反应表现,POD 0、5、7、9d 与正常小肠的组织学特征基本相同.③IL-2Rα和γ-INF mRNA转录水平显著性改变早于排斥反应的病理诊断.④ATx组在POD 5、7、9d的大体表现与组织病理改变分别符合轻、中、重度排斥反应的诊断标准.结论:近交系大鼠BN→F344与近交系大鼠F344→Wistar适用于小肠移植排斥反应实验研究.     

脂肪酸、二磷酸果糖和谷氨酰胺对大鼠移植小肠粘膜细胞增殖和凋亡的作用

目的探讨ｎ３脂肪酸、１,６二磷酸果糖和谷氨酰胺对移植小肠粘膜细胞增殖和凋亡的作用。方法１９６只近交系Ｗｉｓｔａｒ大鼠分别作为供、受体行全小肠异位移植,术前和术后分别用ｎ３脂肪酸灌胃、１,６二磷酸果糖及谷氨酰胺静脉输注１０天,应用流式细胞术和细胞凋亡原位检测的方法分析小肠粘膜细胞增殖和凋亡的变化。结果移植小肠粘膜细胞增殖低下,凋亡增加。补充外源性ｎ３脂肪酸、１,６二磷酸果糖和谷氨酰胺后,移植小肠粘膜细胞的增殖加速,凋亡减少。结论ｎ３脂肪酸、１,６二磷酸果糖和谷氨酰胺特殊营养支持可显著地增加移植小肠粘膜细胞的增殖活性,同时也不同程度地抑制细胞凋亡发生,这种调控作用有助于改善移植小肠的结构和吸收功能。     

亲缘性活体部分小肠移植术

目的 介绍我国首例亲缘性活体部分小肠移植术的临床处理体会。方法 受体为男性,18岁,因短肠综合征而接受小肠移植。供体,男性,44岁,为受体之父。取供体回肠末段150cm,移植给患者,术后给予抗免疫排斥、抗感染、抗凝及营养支持等治疗。结果 目前,患者已健康生存19个月,移植肠功能恢复良好。结论 亲缘性活体部分小肠移植术是治疗短肠综合征的有效手段,良好的术后管理是确保活体小肠移植手术成功的关键。     

Host immune suppression after small bowel/liver transplantation in rats

Simultaneous liver grafting in the Lewis (RT1¹)-to-DA (RT1^a) rat strain combination protects small intestinal grafts from rejection. The present study examined host immune responses after combined small bowel/liver transplantation (SBL) in this model. Orthotopic liver transplantation and heterotopic small intestinal transplantation were performed simultaneously and compared with isolated small bowel allografts (SBA) and isolated small bowel isografts (SBI). All rats were sacrificed on postoperative day (POD) 7 or 14 for immunological and histological studies. The mean time to rejection of the SBA was 6.6±0.3 days. Incontrast, there was no clinical or histological evidence of intestinal rejection in SBL recipients during the 14 days of follow-up. The SBL recipients showed clinical and histological evidence of graft-versushost disease (GVHD). Lmphocyte proliferation and IL-2 production in response to donor antigens were suppressed after SBL transplantation compared with the SBA or the SBI controls (P<0.05). Cell-mediated cytotoxicity and lymphocytotoxic antibody production against donor cells were also significantly inhibited in the SBL recipients compared with the SBA control group (P<0.05). We conclude that SBL transplantation in the Lewis-toDA rat strain combination: (1) suppresses host alloimmune responses, (2) prevents early intestinal rejection, and (3) favors the development of GVHD.     

Laparoscopic harvesting of small bowel graft for small bowel transplantation

Background: Small bowel transplantation represents a valid therapeutic option for patients with intestinal failure, obviating the need for long-term total parenteral nutrition. Recently, reports have shown the feasibility of performing living related intestinal transplantation using segmental small bowel grafts. The limitations of this technique include inadequate harvested small bowel lengths, as compared with the lengths obtained in cadaveric small bowel harvests, and large incisions for the donor. In this pilot study, we evaluated the feasibility of laparoscopically harvesting long segments of proximal jejunum for small bowel transplantation using a porcine model. The results can be used to evaluate the potential for applying this technique in human cases. Methods: For this study 10 yorkshire pigs were used. Under general anesthesia, each pig underwent laparoscopic segmental resection of 200 cm of proximal jejunum on a vascular pedicle. The harvested graft then was autoreimplanted using an open technique by anastomosing the vascular pedicle to the superior mesenteric vessels. Success was determined 2 hours after anastomosis by visually identifying a pink graft with viable-appearing mucosa, an artery with a strong thrill, and palpable venous flow. The animals were then sacrificed. Results: The mean operation time required to laparoscopically harvest the small bowel graft was 80 min (range, 35–120 min), and the mean length of harvested graft was 220 cm (range, 200–260 cm). The mean length of the graft's vascular pedicle was 4.5 cm (range, 4–5 cm). All 10 grafts were successfully harvested laparoscopically and then reimplanted using an open technique. All the grafts maintained good vascular flow, and showed no evidence of mucosal necrosis at necropsy. Obviously, further studies would be required to examine the long-term results of reimplanting a laparoscopically harvested small bowel graft, but proposals for such studies is beyond the scope of this report. Conclusion: Minimally invasive techniques can be used to harvest proximal small bowel grafts for living related small bowel transplantation.     

1,6-二磷酸果糖对大鼠脑缺血再灌注损伤的保护作用

目的 探讨1，6-二磷酸果糖对脑缺血再灌注损伤大鼠MAPK信号转导通路的影响。方法雄性SD大鼠180只，随机分为3组：假手术组（C组）、缺血再灌注组（I组）、1，6-二磷酸果糖组（F组），每组60只，每组随机分为6个亚组（再灌注2、6、12、24、48、72h）（n=10）。I、F组制作大鼠脑缺血再灌注损伤模型，凝断双侧椎动脉24h时夹闭双侧颈总动脉5min重新开放，C组只暴露椎动脉和颈总动脉，F组再灌注开始时静脉注射1，6-二磷酸果糖1．5ml／kg，I、C组均给予等量生理盐水。再灌注2、6、12、24、48、72h时，每组随机处死5只大鼠，取新鲜脑组织，测定超氧化物歧化酶（SOD）、丙二醛（MDA）水平；余下5只采用免疫组织化学染色方法测定P38、Ref-1的表达，并用TUNEL细胞原位凋亡检测法计数神经细胞凋亡数目。结果与C组比较，I组再灌注各时点脑组织SOD活性降低，MDA含量增高，P38、Ref-1表达增强，凋亡细胞数增多（P〈0．05）；1，6-二磷酸果糖减弱了缺血再灌注引起的上述指标的改变。结论MAPK细胞信号转导通路参与了1，6-二磷酸果糖对大鼠脑缺血再灌注损伤的保护作用。