钴原卟啉诱导血红素氧化酶-1表达上调对异种移植胰岛的保护作用

目的 研究不同浓度钴原卟啉(CoPP)与其诱导胰岛细胞血红素氧化酶-1(HO-1)表达的效应关系,探讨HO-1表达上调对异种移植胰岛的保护作用.方法 将分离和纯化的供者胰岛随机分为5组(A、B、C、D和E组),分别置于含有不同摩尔浓度CoPP的培养液中诱导,5组CoPP的浓度依次为0、5、25、50和75 mmol/L.检测各组胰岛细胞中HO-1mRNA和HO-1蛋白表达情况,并测定体外胰岛素释放水平,筛选出能诱导HO-1表达上调幅度最高的CoPP合适浓度.再将受者分为研究组和对照组(每组8只),对照组移植未经CoPP诱导的供者胰岛细胞;研究组移植经合适浓度CoPP诱导的供者胰岛细胞,术后每天监测受者血糖和排斥反应情况.结果 D组HO-1mRNA、蛋白表达上调幅度以及胰岛素分泌量均高于其他4组,差异均有统计学意义(P＜0.05),诱导胰岛细胞HO-1上调幅度最高的CoPP浓度为50 mmol/L.研究组受者移植D组供者胰岛后,维持正常血糖的时间为(14.63±1.19)d,对照组为(9.88±2.17)d,两组比较,差异有统计学意义(P＜0.01).结论 在异种胰岛移植中,经50 mmol/L的CoPP体外诱导供者胰岛HO-1表达上调幅度最高;HO-1的表达上调可以促进移植胰岛的功能恢复,抑制排斥反应,延长移植胰岛的存活时间.     

体内诱导胰岛血红素加氧酶-1过表达对异种胰岛移植物的保护作用

胞浸润数量明显少于其他两组(P<0.05).结论 CoPP诱导HO-1过表达可延长异种胰岛移植物存活,其机制可能与IL-10免疫调节有关.     

血红素加氧酶-1诱导性高表达保护异种移植胰岛功能的研究

目的 探讨通过钴原卟啉(cobalt protoporphyrin,CoPP)诱导供体胰岛血红素氧化酶-1(heme oxygenase-1,HO-1)高表达对异种胰岛移植物的保护作用.方法 采用大鼠对小鼠胰岛移植模型,分为对照组、体内诱导组、体外诱导组、体内阻断组及体外阻断组5组,改良Gotoh法提取胰岛,移植于糖尿病模型小鼠的肾包膜下.比较各组受体移植后血糖维持正常时间,糖刺激试验检测胰岛功能,PCR和ELISA法分别检测移植物内和血清IL-10、TNF-α、IL-1β及INF-γ及其mRNA表达.分别以PCR和Western印迹法检测胰岛组织内HO-1mRNA及蛋白表达.病理学检查移植物淋巴细胞浸润情况.结果 对照组小鼠血糖维持正常时间为(9.33±1.37)d,与体内诱导组的(16.33±1.51)d及体外诱导组的(14.33±1.51)d相比差异有统计学意义(P<0.05),体内诱导组优于体外诱导组(P<0.05).受体阻断的两组血糖控制时间则与对照组相近,分别为:体内阻断组(9.67±1.03)d,体外阻断组(9.17±1.47)d.体内、外诱导组及对照组胰岛于低糖刺激下胰岛素分泌量差异无统计学意义(P>0.05),而高糖刺激下,体内诱导、体外诱导及对照组分别为:(187.68±19.93)、(137.22±11.73)及(91.25±12.64)μIU·ml~(-1)·10islets~(-1)·45 min~(-1),差异有统计学意义(P<0.05).移植后,接受经诱导处理的胰岛的两组小鼠血清IL-10含量[体内诱导(72.97±9.74)pg/ml、体外诱导(70.84±3.56)pg/ml]明显高于未处理组[(30.57±3.93)pg/ml]及阻断两组[体内阻断:(39.78±3.00)pg/ml、体外阻断:(35.42±4.30)pg/ml].两诱导组胰岛移植物的IL-10 mRNA表达强于对照及阻断两组.IFN-γ、TNF-α及IL-1β在各组间差异无统计学意义.体内、体外诱导两组HO-1mRNA、蛋白表达高于对照组,其中体内诱导组HO-1mRNA高于体外组,但两组HO-1蛋白表达相当.体内、外阻断组HO-1mRNA、蛋白表达与对照组相当.病理学检查示两诱导组移植物内淋巴细胞浸润少于对照及阻断两组. 结论 CoPP体内、外诱导He-1高表达可促进胰岛功能,延长移植物生存时间,体内诱导对异种移植胰岛保护效应强于体外诱导.     

钴卟啉诱导胰岛血红素加氧酶-1过表达的初步研究

目的 探讨钴卟啉腹腔注射诱导大鼠胰岛血红素加氧酶-1(Ho-1)过表达的量/效关系及其对胰岛功能的影响.方法 大鼠分为5组,A、B、C、D 4组分别在提取胰岛前24 h予腹腔注射2.5、5、7.5及10 mg/ks的钴卟啉溶液,对照组给予1.5 Inl生理盐水.改良Goth法提取胰岛,计数胰岛得率、估定纯度,real time-PCR和Western印迹法检测胰岛组织内Ho-1mRNA及蛋白表达,糖刺激试验评价胰岛功能.结果 C、D组大鼠在注射CoPP溶液后出现机体损伤,D组部分大鼠死亡.A、B、C及对照组胰岛得率及纯度差异无统计学意义(P＞0.05),B组Ho-1mRNA及蛋白表达量为4组中最高(B组Ho-1mRNA表达量达对照组的84.18倍).A、B及对照组胰岛于低糖刺激时,胰岛素分泌量差异无统计学意义(P＞0.05),而高糖刺激下,胰岛素分泌量以B组最高,差异有统计学意义[A、B及对照组分别为:(172.37±16.4)、(187.68±19.93)及(91.25±12.64)μIU·ml-1·10islets-1·45min-1,P＜0.05].结论 腹腔注射5 mg/kg体重钴卟啉相对安全,可大幅提高大鼠胰岛组织中HO-1的表达量,并能增强胰岛功能.     

血红素氧合酶-1基因治疗减缓移植物血管病及机制

目的 观察血红素氧合酶-1(HO-1)基因治疗减缓同种移植物血管病的效果,探讨其机制.方法 以BN-Lewis大鼠血管移植为对象,依据基因治疗方案分为4组:同系对照组、空白对照组、载体对照组、腺病毒介导的HO-1( AdHO-1)组.移植后2个月,观察各组移植物纤维化和内膜增生,检测T细胞(CD3+)、B细胞(CD45RA)和巨噬细胞(CD68+)浸润数量,逆转录-聚合酶链反应(RT-PCR)和Western blot检测移植物HO-1基因和蛋白的表达,酶联免疫吸附试验(ELISA)法检测受体血清白细胞介素(IL)-10的浓度.结果 同系对照组无移植物血管病表现,空白对照组和载体对照组大量纤维沉积,AdHO-1组纤维沉积轻微.血管内膜/(内膜+中膜)百分比4组分别为7.6％、81.4％、85.9％、15.9％.每400倍视野浸润细胞数4组分别为T细胞(9.2±1.6、92.3±11.6、89.6±17.8、39.3±10.1)、B细胞(3.6±1.1、72.6±11.8、66.6±10.9、30.6±9.9)、巨噬细胞(7.5±1.2、78.5 ±21.7、72.5 ±19.8、34.5±18.7).血清IL-10浓度4组分别为(50.2±20.1)、(40.2±11.1)、(38.6±19.3)、(481.2 ±69.1)ng/L.AdHO-1组与空白对照组和载体对照组间差异有统计学意义(P＜0.05).AdHO-1基因治疗增高了移植血管HO-1基因和蛋白的表达.结论 AdHO-1基因治疗减缓同种移植物血管病,移植物纤维化和内膜增生明显减轻.AdHO-1基因治疗下调了T细胞、B细胞和巨噬细胞在移植物中的浸润,增加了HO-1和IL-10的表达,IL-10-HO-1通路的活化可能是移植血管得到保护的重要原因.     

血红素氧化酶-1在大鼠肝移植中保护作用的研究

目的 探讨改变血红素氧化酶-1(HO-1)的活性对大鼠同种异体原位肝移植的影响.方法 用二袖套法建立大鼠同种异体原位肝移植模型,实验分为4组:实验对照组(n=44):供体鼠术前24 h腹腔注射生理盐水5 ml/kg;氯化血红素组(n=44):供体鼠术前24 h腹腔注射氯化血红素100 mg/kg,在供肝4℃生理盐水保存期间,经门静脉注入氯化血红素100 mg/kg;锌原卟啉组(n=44):供体鼠术前24 h腹腔注射锌原卟啉5 mg/kg,在供肝4℃生理盐水保存期间,经门静脉注入锌原卟啉5 mg/kg;正常大鼠作为正常对照组(n=5).分别应用RT-PCR及免疫组化技术检测移植肝脏中HO-1 mRNA及蛋白表达,并用流式细胞仪分析移植肝脏肝细胞的凋亡率.结果 肝移植术后供体肝HO-1表达增强.肝移植术后氯化血红素组HO-I mRNA表达水平高于实验对照组,血清ALT和AST水平低于实验对照组(P<0.05);锌原卟啉组术后肝组织HO-1 mRNA表达水平低于实验对照组(P<0.05),血清ALT和AST水平高于实验对照组(P<0.05).肝移植术后48 h肝细胞凋亡率锌原卟啉组最高,氯化血红素组最低,实验对照组介于二者之间,组间差异有统计学意义(P<0.05).术后7 d实验对照组、氯化血红素组及锌原卟啉组3组的生存大鼠分别为7/12、9/12和4/12,组间差异均有统计学意义(P<0.05).结论 肝移植手术过程可促进大鼠肝脏H()-1表达增强;术前采用药物诱导HO-1可降低移植术后肝细胞凋亡率,减轻肝脏损伤程度,提高术后生存率.     

PPARγ激动剂15d-PGJ2在大鼠肝脏缺血-再灌注损伤中的保护作用及机理

目的探讨过氧化物酶体增殖物激活受体γ（PPARγ）激动剂15-脱氧前列腺素J2（15d—PGJ2）在大鼠肝脏缺血-再灌注损伤中的保护作用及机理。方法建立70％的大鼠肝脏缺血-再灌注损伤模型，40只SD大鼠随机均分为4组：假手术组、缺血-再灌注损伤组（缺血-再灌注组）、15d-PGJ2预处理组（15d-PGJ2组）及15d-PGJ2＋GW9662预处理组（15d—PCJ2＋GW9662组）。再灌注后，取静脉血检测肝血清酶（ALT、AST）水平，取肝脏组织检测髓过氧化物酶（MPO）活性、NF—κB活性、TNF—α含量和ICAM-1表达。结果与假手术组相比，其余3组血清ALT和AST水平、肝脏组织MPO和NF—JeB活性、TNF—α含量和ICAM-1表达均增加（P〈0.05）。与缺血-再灌注组相比，15d—PGJ2组血清ALT和AST水平、肝脏组织MPO和NF-κB活性、TNF-α含量和ICAM-1表达均明显降低（P〈0．05）；而15d-PGJ2＋GW9662组与缺血-再灌注组相比有差异但无统计学意义（P〉0．05）。与15d—PGJ2组相比，15小PGJ2＋GW9662组血清ALT和AST水平、肝脏组织MPO和NF—κB活性、TNF—α含量和ICAM-1表达明显增加（P〈0．05）。结论PPARγ激动剂15d—PGJ2对肝脏缺血-再灌注损伤有保护作用，其机理可能是通过PPARγ途径抑制NF—κB活性，减少TNF—α和ICAM-1炎症介质的释放实现的。     

微囊化大鼠胰岛异种移植的实验研究

目的　通过动物实验明确微囊化胰岛是否具有免疫隔离作用。方法　SD大鼠胰腺原位消化 ,Ficoll间断密度梯度离心法纯化、分离胰岛 ,气流吹喷制作海藻酸钠 /聚赖氨酸 /海藻酸钠(APA)微囊化大鼠胰岛 ,比较微囊化与未微囊化胰岛的胰岛素释放试验 ;将微囊化 (实验组 )与未微囊化 (对照组 )大鼠胰岛植入链脲佐菌素 (STZ)诱导的I型糖尿病小鼠中 ,作两组间血糖正常持续时间比较。结果　实验组与对照组的胰岛素释放试验差异无显著性 (P >0 .0 5 ) ;实验组血糖正常持续时间为 2 3～ 6 5d(平均 48d) ,对照组为 3～ 6d(平均 5d) ,两组差异有极显著性 (P <0 .0 1)。已排斥的实验组小鼠腹腔灌洗发现部分微囊化胰岛存活 ,部分已坏死 ,但微囊膜皆完整 ,囊壁无纤维化。结论　微囊具有良好的免疫隔离作用 ,可使胰岛移植物存活时间明显延长。同时推测微囊内移植物死亡与细胞因子、自由基作用或营养不足等有关。     

腺病毒介导的血红素氧合酶-1基因治疗对同种移植物慢性排斥反应损伤的保护作用

目的探讨腺病毒介导的血红素氧合酶-1（AdHO-1）基因治疗对同种移植物慢性排斥反应损伤的保护作用及其机制。方法选用血管移植和肾脏移植两种慢性排斥反应模型,对同种血管移植物和肾脏移植物进行体外AdHO-1基因转染,分析慢性排斥反应发生时移植物的结构和功能变化、目的基因和蛋白的表达以及免疫系统的相应反应。结果AdHO-1基因治疗缓解了慢性排斥反应对同种肾脏移植物的损伤,但弱于对血管移植物的保护效应;空载病毒加剧了同种肾脏移植物的损伤;AdHO-1基因治疗可减少慢性排斥反应发生时移植物内巨噬细胞和CD4^＋细胞的浸润。结论AdHO-1基因治疗可能通过保护移植物、下调免疫反应、诱导免疫偏移等作用减轻同种移植物的慢性排斥反应损伤。     

血红素加氧酶-1在异种移植适应现象中的研究进展

血红素加氧酶（heine oxygenase,HO）是血红素降解过程中的限速酶,HO-1在体内外实验已被证实具有抗炎症、抗凋亡、调节细胞分化作用,对器官移植的缺血再灌注损伤具有保护作用。适应（accommodation）是指受体体内存在抗供体的抗体,但移植物仍然可以被接受的现象,其发生机制目前还不清楚。新近报道在适应的异种移植动物模型中出现HO-1的高表达。HO-1高表达是否与异种移植免疫保护存在关联,是否参与适应机制的产生,目前还没有定论。HO-1是否通过其反应的下游产物产生保护作用,其机制尚不明了。目前,如何将异种移植物引入适应状态是异种移植研究的热点之一。理解HO-1与异种移植,尤其是与适应有关的异种移植之间的关系具有重要的临床应用前景。     

Lipotoxicity in human pancreatic islets and the protective effect of metformin

Human pancreatic islets from eight donors were incubated for 48 h in the presence of 2.0 mmol/l free fatty acid (FFA) (oleate to palmitate, 2 to 1). Insulin secretion was then assessed in response to glucose (16.7 mmol/l), arginine (20 mmol/l), and glyburide (200 micromol/l) during static incubation or by perifusion. Glucose oxidation and utilization and intra-islet triglyceride content were measured. The effect of metformin (2.4 microg/ml) was studied because it protects rat islets from lipotoxicity. Glucose-stimulated but not arginine- or glyburide-stimulated insulin release was significantly lower from FFA-exposed islets. Impairment of insulin secretion after exposure to FFAs was mainly accounted for by defective early-phase release. In control islets, increasing glucose concentration was associated with an increase in glucose utilization and oxidation. FFA incubation reduced both glucose utilization and oxidation at maximal glucose concentration. Islet triglyceride content increased significantly after FFA exposure. Addition of metformin to high-FFA media prevented impairment in glucose-mediated insulin release, decline of first-phase insulin secretion, and reduction of glucose utilization and oxidation without significantly affecting islet triglyceride accumulation. These results show that lipotoxicity in human islets is characterized by selective loss of glucose responsiveness and impaired glucose metabolism, with a clear defect in early-phase insulin release. Metformin prevents these deleterious effects, supporting a direct protective action on human beta-cells.     

A novel approach to xenotransplantation combining surface engineering and genetic modification of isolated adult porcine islets

BACKGROUND: Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly[ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2. METHODS: Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining. Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SCID mice reconstituted with human lymphocytes (5 x 10 8 /intraperitoneally/15 days before PIT). RESULTS: No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end"-capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P <.05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P <.001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100% of the animals given islets modified with DSPEG + AdBcl-2 during the observation time. CONCLUSIONS: Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.     

Effects of collagenase concentration on the purity and viability of isolated porcine pancreatic islets for use in xenotransplantation studies

Abstract: The use of lower concentrations of collagenase (0.8 mg/ml as compared to 1.25 and 1.5 mg/ml) resulted in isolation of porcine pancreatic islets retaining their original compact, wholesome appearance. In in vitro experiments, 100% of islet batches isolated with the low collagenase concentration responded to glucose stimulation, while only 63.1% of islet batches isolated with the high concentration of the enzyme did. The response to high glucose challenge of islets isolated with the low enzyme concentration was considerably higher compared to high collagenase concentration islets.
In in vivo experiments, intraperitoneal transplants of microencapsulated islets isolated using 0.8 mg/ml of collagenase reversed diabetes in diabetic BALB-c mice in a significantly larger proportion of recipients (87.5%) as compared to 33.3% of recipients grafted with 1.5 mg/ml collagenase islets.
The results of this study demonstrate that the use of a low collagenase concentration is conducive to isolation of islets of a superior morphology, purity, viability, and functioning ability.     

Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation

BACKGROUND: The long-term viability and function of transplanted encapsulated neonatal porcine islets was examined in a diabetic patient. METHODS AND RESULTS: A 41-yr-old Caucasian male with type 1 diabetes for 18 yr was given an intraperitoneal transplant of alginate-encapsulated porcine islets at the dose of 15,000 islet equivalents (IEQs)/kg bodyweight (total dose 1,305,000 IEQs) via laparoscopy. By 12 weeks following the transplant, his insulin dose was significantly reduced by 30% (P = 0.0001 by multiple regression tests) from 53 units daily prior to transplant. The insulin dose returned to the pre-transplant level at week 49. Improvement in glycaemic control continued as reflected by total glycated haemoglobin of 7.8% at 14 months from a pre-transplant level of 9.3%. Urinary porcine C-peptide peaked at 4 months (9.5 ng/ml) and remained detectable for 11 months (0.6 ng/ml). The patient was followed as part of a long-term microbiologic monitoring programme which subsequently showed no evidence of porcine viral or retroviral infection. At laparoscopy 9.5 yr after transplantation, abundant nodules were seen throughout the peritoneum. Biopsies of the nodules showed opacified capsules containing cell clusters that stained as live cells under fluorescence microscopy. Immunohistology noted sparse insulin and moderate glucagon staining cells. The retrieved capsules produced a small amount of insulin when placed in high glucose concentrations in vitro. An oral glucose tolerance test induced a small rise in serum of immuno-reactive insulin, identified as porcine by reversed phase high pressure liquid chromatography. CONCLUSION: This form of xenotransplantation treatment has the potential for sustained benefit in human type 1 diabetics.