胰腺移植动物模型新选择——门静脉回流胰腺腹腔移植小鼠模型

目的胰腺移植是1型糖尿病和2型糖尿病的推荐治疗方法。在胰腺移植术式中,移植胰腺静脉血经门静脉回流对患者预后有益存在争议。因此,需要一种符合门静脉回流的胰腺动物模型来进行相关的基础研究。小鼠在实验室中被广泛应用,在胰腺免疫损伤和内分泌研究方面具有优势,但至今没有经门静脉回流的小鼠胰腺移植模型。建立经门静脉回流的胰腺移植小鼠模型。方法供体胰腺保留含有肠系膜上动脉与腹腔动脉的一段腹主动脉和门静脉。受体游离脾静脉和腹主动脉,采用插管技术连接移植胰腺和受体的血管。结果我们利用显微外科技术,成功建立了一种新的经门静脉回流胰腺移植小鼠模型。通过30组小鼠验证,移植后生存率为93.3%(28/30),胰腺有功能率为92.9%(26/28),模型的其他相关指标(生存时间、手术时间、冷/热缺血时间、移植胰腺功能)也令人满意。结论该模型具有可重复性和可靠性,可用于对门静脉回流胰腺移植手术探讨、胰腺灌注保存、胰腺缺血再灌注损伤和胰腺免疫损伤的研究。     

造血干细胞移植治疗1型糖尿病

1型糖尿病(type 1 diabetes mellitus,T1DM)是一种T细胞介导的胰腺β细胞破坏的自身免疫疾病,具有较高的发病率和死亡率.胰岛素的使用只是控制患者血糖水平,延缓糖尿病进展并不能治愈糖尿病.供体的短缺及预防排斥的长期免疫抑制限制了胰腺及胰岛细胞的移植.随着国内外对造血干细胞的深入研究,造血干细胞移植治疗1型糖尿病有着广泛的应用前景.     

中国胰肾联合移植现状

由于胰腺外分泌处理和移植胰腺排斥反应难以诊断的特殊性．胰腺移植在大器官移植总数和移植效果上曾远远落后于肾、心脏和肝等器官移植。直至近10余年,随着新型强效免疫抑制剂的临床应用、器官保存技术的改进和移植手术方式的日趋成熟,胰腺移植受者和移植胰腺的存活率均显著提高。胰、肾联合移植已成为治疗l型糖尿病、部分2型糖尿病合并尿毒症的有效方法。     

胰岛微包囊

目前，1型糖尿病病人越来越多，胰腺供体缺乏和胰腺移植的长期免疫抑制，严重阻碍胰岛移植治疗糖尿病的发展。异种胰岛的免疫隔离可以解决将来大量的移植。用海藻酸钠一聚赖氨酸一海藻酸钠包裹胰岛进行移植可在不使用免疫抑制剂的情况下延长胰岛移植物的存活时间，逆转高血糖。     

临床胰腺和胰、肾联合移植的现状

由于胰腺外分泌处理和移植胰腺排斥反应难以诊断的特殊性，胰腺移植在移植总数和移植效果上曾远远落后于肾、心和肝等器官移植。直至近10余年，随着新型强效免疫抑制剂的临床应用、器官保存技术的改进和移植手术方式的日趋成熟，胰腺移植受者和移植胰腺的存活率均显著提高。胰、肾联合移植已成为治疗1型糖尿病、部分2型糖尿病合并尿毒症的有效方法。本文概述临床胰腺与胰、肾联合移植的现状与进展。     

胰岛细胞移植治疗糖尿病的历史、现状与未来

1胰腺和胰岛细胞移植治疗糖尿病的简史根据糖尿病发病机制的不同分为1型糖尿病和2型糖尿病。1型糖尿病是一种自身免疫性疾病,占糖尿病人群的5%～10%[1]。尝试采用外科手段来治疗糖尿病的历史可追溯到20世纪中叶,德国     

胰腺与胰肾联合移植免疫抑制剂的应用

目前已公认胰腺与胰肾联合移植是治疗Ⅰ型糖尿病(IDDM)及其并发症的安全、有效方法。移植的最终目的是彻底根治IDDM，完全停用外源性胰岛素，消除或改善糖尿病并发症，提高生活质量。由于糖尿病病变的特殊性、移植胰排斥反应发生率和移植物丢失率高以及术后免疫抑制剂引起的副作用，如：高血压、高脂血症和移植后糖尿病(PTDM)等因素，胰腺与胰肾联合移植术后免疫抑制剂的选择与应用比单纯肾移植更复杂，涉及问题更多。本文就胰腺及胰肾联合移植术后免疫抑制的临床应用及进展作一概述。     

异种胰岛移植研究进展

对于1型糖尿病(胰岛素依赖型糖尿病),使用胰岛素治疗虽能有效控制血糖,但不能防止其并发症的发生与发展。胰腺移植存在着创伤大、并发症多和供者不足等缺点,而胰岛移植较胰腺移植具有简单、安全、不良反应轻等优点,仅需占胰腺2%的B细胞即可控制血糖。然而,临床同种胰岛移植的开展受到供者严重短缺和移植物排斥反应的限制。异种胰岛来源广泛,特别是猪胰岛,可以解决供者来源短缺的问题。近年来,随着免疫隔离技术和免疫耐受技术的进展,使异种胰岛移植成为可能。     

胰岛移植治疗糖尿病的研究进展

胰岛移植是一种极具潜力治愈1型糖尿病的方法.其临床应用主要面临两大挑战:移植疗效欠稳定和胰腺供体匮乏.影响临床胰岛移植疗效的因素包括免疫因素及非免疫因素导致的胰岛损伤、丢失.     

第一届胰腺移植国际共识大会胰腺移植临床实践指南解读

2019年, 第一届胰腺移植国际共识大会制定了第一个胰腺移植临床实践指南, 并于2021年正式发表。指南涉及49条关于胰腺移植对糖尿病患者治疗效果的专家组评审意见, 认为所有类型胰腺移植均可显著改善受者术后远期生存、生活质量以及减缓糖尿病慢性并发症的发病进程, 优先将供者肾脏分配给胰肾联合移植(simultaneous pancreas-kidney transplantation, SPK)受者是合理的, 同时提出了涉及胰腺移植供受者选择、免疫方案、手术技术以及术后预防方案等临床实践的110条专家推荐意见。本文通过对该指南进行解读, 对推荐意见进行重新归类整理, 以增进理解, 提高临床实用性。     

Growing new endocrine pancreas in situ

Type 1 diabetes mellitus is a major cause of endstage renal disease in young adults. Maintenance of normoglycemia in type 1 diabetics using exogenous insulin is difficult under the best of circumstances. Transplantation therapies are limited by the scarcity of human donor organs, rendering a priority the identification of an alternative source for replacing insulin-secreting cells. Embryonic pancreatic primordia transplanted into diabetic animal hosts undergo selective endocrine differentiation in situ and normalize glucose tolerance. Pancreatic primordia can be transplanted across isogeneic, allogeneic, and both concordant (rat-to-mouse) and highly disparate (pig-to-rodent) xenogeneic barriers. Successful transplantation of pancreatic primordia depends on obtaining them at defined windows during embryonic development within which the risk of teratogenicity is eliminated, growth potential is maximized, and immunogenicity is reduced. Here we review studies exploring the potential for pancreatic organogenesis post-transplantation of embryonic primordia as a therapy for type 1 diabetes.     

同种异体胰岛及胰腺干细胞来源的胰岛样结构序贯移植治疗糖尿病

目的 观察同种异体大鼠胰岛及胰腺干细胞来源的胰岛样结构序贯移植在糖尿病治疗中的作用.方法 分离胰腺组织获得胰岛及胰腺导管上皮细胞,将具有干细胞潜能的胰腺导管上皮细胞在体外培养27d.将新鲜分离的胰岛(200±50)个及诱导分化2周的胰腺干细胞来源的胰岛样结构(2×106)个序贯移植到糖尿病大鼠的肾被膜下观察大鼠的血糖及生存情况.结果 将胰岛及胰腺干细胞来源的胰岛样结构序贯移植到同一糖尿病大鼠3周后血糖仍在5 mmol/L水平,对照组血糖无明显下降.结论 胰腺干细胞可诱导分化为分泌胰岛素的胰岛样结构,胰岛及胰腺干细胞来源的胰岛样结构序贯移植对大鼠糖尿病有治疗作用.     

Engineering of Pseudoislets: Effect on Insulin Secretion Activity by Cell Number,Cell Population,and Microchannel Networks

Engineered pseudoislets reconstituted from a suspension of pancreatic α and β cells have the potential to relieve the shortage of donor islets for transplantation in the treatment of type 1 diabetes. However, the methods to fabricate pseudoislets are not well developed. In this study, we attempted to generate pseudoislets, which show a higher potential for glucose-induced insulin secretion, by altering total cell number, adjusting the cell ratio of pancreatic α and β cells, and fabricating microchannel networks with the use of alginate hydrogel beads. To effectively aggregate α and β cells and hydrogel beads, we used a previously established rapid aggregation method. When pseudoislets were reconstituted with 8,000 cells in a 1:8 α/β-cell ratio, we observed that the glucose-induced insulin secretion was enhanced by 3.1 times compared with the pseudoislets formed with β cells only. In addition, embedding of microchannel networks increased the insulin secretion rate by 4.4 times compared with the pseudoislets without the microstructures. These findings demonstrated that active modification was effective in reconstituting higher functional pseudoislets, which may be useful for islet transplantation.     

Long-term engraftment following transplantation of pig pancreatic primordia into non-immunosuppressed diabetic rhesus macaques

BACKGROUND: Transplantation therapy for human diabetes is limited by a shortage of donor organs, and transplant function diminished over time by cell death and limited potential for expansion of beta cells in pancreas or islets. Outcomes are complicated by immunosuppression. A way to overcome supply and expansion problems is to xenotransplant embryonic tissue. Previously, we have shown that beta cells originating from embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of streptozotocin (STZ)-diabetic (type 1) Lewis rats or Zucker Diabetic Fatty (ZDF) diabetic (type 2) rats engraft and normalize glucose tolerance without the need for host immune-suppression. METHODS: In this study, we transplant E28 pig pancreatic primordia in the mesentery of STZ-diabetic rhesus macaques. RESULTS: Long-term engraftment of pig beta cells within liver, pancreas and mesenteric lymph nodes post-transplantation of E28 pig pancreatic primordia into STZ-diabetic rhesus macaques is demonstrated by electron microscopy, positive immune-histochemistry for insulin, and positive RT-PCR and in situ hybridization for porcine proinsulin mRNA. Insulin requirements were reduced in one macaque followed over 22 months post-transplantation and porcine insulin detected in plasma using sequential affinity chromatography, HPLC and mass spectrometry. Of potential importance for application of this transplantation technology to treatment of diabetes in humans and confirmatory of our previous findings in Lewis and ZDF rats, no host immunosuppression is required. CONCLUSIONS: Under selected circumstances, pancreatic primordia elicit a muted immune response relative to more differentiated tissue, such that engraftment occurs in non-immunosuppressed hosts. Our findings that pig pancreatic primordia engraft long-term in non-immunosuppressed STZ-diabetic rhesus macaques establishes the potential for their use in human diabetics.     

Applications of organ precursor cell therapy: can lessons from embryonic kidney transplantation be applied to the endocrine pancreas?

PURPOSE OF REVIEW: The purpose of this review is to provide an update relating to a novel approach to endocrine pancreas replacement therapy based in part on a technology developed for the transplantation of developing kidneys. The approach is to use organ primordia, and in this way transplant kidneys or pancreas in cellular form. RECENT FINDINGS: Cellular allotransplantation and xenotransplantation of both kidney and pancreatic anlagen has been successfully performed such that functioning organs develop in situ. SUMMARY: The number of human organs available for transplantation is limited. We and others have shown that it is possible to 'grow' new kidneys or endocrine pancreas from organ-specific precursor cells in situ. For the kidney, this technology takes advantage of the fact that a developing renal anlagen can attract its blood supply from an appropriate vascular bed post-transplantation, enabling the transplantation of kidneys in 'cellular' form. Techniques developed for the transplantation of embryonic kidneys can be applied to the transplantation of embryonic pancreas. Pancreatic anlagen implanted into a host peritoneum develop into a novel organ consisting of functional islets of Langerhans surrounded by stroma. The transplantation of developing pancreas could represent a novel 'cellular' treatment for diabetes mellitus, a major cause of end-stage renal disease.     

Glucose tolerance normalization following transplantation of pig pancreatic primordia into non-immunosuppressed diabetic ZDF rats

Pancreas or pancreatic islet transplantation in humans is limited by organ availability, and success of the latter is negatively impacted upon by tissue loss post-transplantation and limited potential for expansion of beta cells. A way to overcome the supply and expansion problems is to xenotransplant embryonic tissue. Previously, we have shown that beta cells originating from embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of streptozotocin-diabetic (type 1) Lewis rats engraft without the need for host immune-suppression and normalize glucose tolerance. Here we show long-term engraftment of pig beta cells within liver, pancreas and mesenteric lymph nodes post-transplantation of E28 pig pancreatic primordia into diabetic ZDF rats, a model for type 2 diabetes. Porcine insulin is present in circulation after an oral glucose load. Glucose tolerance is normalized in transplanted ZDF hosts and insulin sensitivity restored in formerly diabetic ZDF males. Release of porcine insulin in vitro from tissue originating in transplanted rats occurs within 1 min of glucose stimulation characteristic of first-phase secretion from beta cells. Of potential importance for application of this transplantation technology to treatment of type 2 diabetes in humans and confirmatory of our previous findings in Lewis rats, no host immunosuppression is required for engraftment of E28 pig pancreatic primordia.     

The importance of pancreatic embryonic epithelium for mesenchyme-to-epithelial transition during islet development

Stem or progenitor cells are a promising potential alternative source of pancreatic islets for transplantation in the treatment of juvenile-onset diabetes. However, to derive islets from such cells, it is important to elucidate the mechanisms of normal pancreatic development. Previous work in our laboratory has shown that, contrary to previous thinking, pancreatic mesenchyme when combined with pancreatic epithelium can contribute cells to islets. However, the signals and role of individual tissues involved in this mesenchyme-to-epithelial transition (MET) have yet to be elucidated. The aim of this study was to investigate whether MET can occur in the absence of pancreatic epithelium. Chick and quail eggs were incubated for 4 days and the dorsal pancreatic buds and stomach rudiments were microdissected. Mesenchyme and epithelium of the organ rudiments were separated after collagenase treatment. Separated pancreatic mesenchyme were cultured alone and in combination with stomach (nonpancreatic). After 7 days of culture, the specimens were analysed using immunohistochemistry for quail-specific nucleolar antigen (QCPN), insulin, and islet precursor cell marker (ISL-1). Pancreatic mesenchyme when cultured in the absence of epithelium did not differentiate into islets, but differentiated into fibroblast-like cells. When pancreatic mesenchyme were cultured in combination with stomach epithelium, there was no evidence of mesenchymally derived islets. We have demonstrated that pancreatic mesenchyme require pancreatic epithelium to differentiate into islet cells. These findings further increase our understanding of normal pancreatic islet development and may help to elucidate the molecular mechanisms of MET in islet development.     

Islet Transplantation in Genetically Determined Diabetes

Hyperglycemia induced in animals by beta cell toxins or by pancreatectomy can be reversed by pancreatic islet transplantation. Abnormal carbohydrate metabolism in juvenile onset human diabetics has also been corrected, albeit temporarily because of graft rejection, by pancreatic transplantation. It does not necessarily follow that naturally occurring diabetes in animals or adult onset diabetes in man would respond to similar treatment. Islet transplantation was studied in mice with chemically induced or genetically determined diabetes. Streptozotocin-induced diabetic mice were permanently cured by syngeneic islets and, when immunosuppressed, were rendered normoglycemic for six weeks after receiving xenogeneic rat islets. In contrast, histocompatible islets from normoglycemic coisogenic donors were ineffective in hyperglycemic db/db recipients as were xenogeneic rat islets in immunosuppressed db/db hosts. However, when islets were isolated from db/db donors and transplated to genetically normal coisogenic mice, which had been rendered hyperglycemic with streptozotocin, they became normoglycemic. Apparently the metabolic defect in the db/db mice, which is similar in some ways to human maturity onset diabetes, does not reside in their islets as these cells can function normally if transplanted to genetically nondiabetic hosts. In two other types of genetic diabetes (ob/ob and NZO) islet transplantation was more effective. Pancreatic transplantation is unlikely to be the proper treatment for all types of diabetes even if technical and immunological problems are overcome.     

Regulation of cell proliferation using tissue engineering in MIN6 cells

Pancreatic islet transplantation for patients with diabetes mellitus has been hindered by the problem of donor shortage, as is the case for transplantation of other organs. Among several measures to overcome this problem, cell transplantation using xenogenic cell lines has been considered. For the treatment of diabetic patients, a murine pancreatic beta-cell line MIN6 is a potential source of cell transplant. In order to restrict otherwise unlimited proliferation of transplanted MIN6 cells, cells are rendered to form spheroidal aggregates (SMIN6) on nonadherent culture dishes. SMIN6 stopped its growth around day 7 with a diameter of 220 +/- 40 microm and kept its size almost constant at least until day 28. SMIN6 cells, however, had reduced responsiveness of insulin secretion to glucose concentration compared with MIN6 cells cultured in a monolayer. On the other hand, spheroid MIN6 cells formed in the presence of extracellular matrix gel (SMIN6E) possessed the capacity for glucose-dependent insulin secretion comparable with conventional MIN6 cells. SMIN6E encapsulated in agarose beads (SMIN6E-B) was also viable for at least 1 month in vitro with a constant diameter and favorable glucose responsiveness. The development of spheroid-type MIN6 may contribute to the future clinical application of MIN6 or other beta-cell lines for treatment of diabetes mellitus.