大量人胰岛分离技术的改进及相应胰岛功能的检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能。方法采用经典及改良技术分离、纯化28例人胰岛。胰岛收获量以胰岛当量(IEQ)表示。胰岛功能测定分别为体外测定胰岛素/DNA比率、静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内功能鉴定并行糖耐量试验,连续测血糖水平及其体内C肽浓度。结果 前13例采用经典方法,平均每个胰腺收获49123 IEQs,平均846 IEQs/g胰腺组织,平均纯度为87％。改进技术后15例的分离结果则分别为:501813 IEQs/胰腺,7003 IEQ/g胰腺组织及纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的基础。     

胰岛移植治疗糖尿病的研究进展

Islet transplantation is effective in treating diabetes, however, its clinical use is highly restricted by a series of influencing factors. This review elucidates the non-immune factors including islet purification, instant blood-mediated inflammatory response and revascularization deficit on the development of islet transplantation, and also highlights the application and perspective of islet xenotrasplantation in the treatment of diabetes.     

胰岛移植即刻经血液介导的炎症反应应对策略

胰岛移植作为治疗1型糖尿病和终末期2型糖尿病的有效手段,可以使患者获得较好的血糖控制能力。即刻经血液介导的炎症反应(IBMIR)是胰岛移植早期出现的非特异性炎症反应,发生后可迅速出现凝血级联和补体系统激活、炎症细胞聚集等,造成大量移植胰岛丢失,严重影响胰岛移植的疗效。如何减轻IBMIR对胰岛造成损伤是目前胰岛移植的研究热点,临床推荐的治疗胰岛移植IBMIR的药物有肝素和肿瘤坏死因子-α抑制剂依那西普。新近研究表明多种方法和药物可以减轻IBMIR对胰岛的损伤,本文就这些临床研究成果和临床前研究成果进行综述,以期为胰岛移植IBMIR的应对提供参考。     

Pancreatic islet transplantation

Type 1 diabetes mellitus is an autoimmune disease, which results in the permanent destruction of β-cells of the pancreatic islets of Langerhans. While exogenous insulin therapy has dramatically improved the quality of life, chronic diabetic complications develop in a substantial proportion of subjects and these complications generally progress and worsen over time. Although intensive insulin therapy has proven effective to delay and sometimes prevent the progression of complications such as nephropathy, neuropathy or retinopathy, it is difficult to achieve and maintain long term in most subjects. Reasons for this difficulty include compliance issues and the increased risk of severe hypoglycemic episodes, which are generally associated with intensification of exogenous insulin therapy. Clinical studies have shown that transplantation of pancreas or purified pancreatic islets can support glucose homeostasis in type 1 diabetic patients. Islet transplantation carries the special advantages of being less invasive and resulting in fewer complications compared with the traditional pancreas or pancreas-kidney transplantation. However, islet transplantation efforts have limitations including the short supply of donor pancreata, the paucity of experienced islet isolation teams, side effects of immunosuppressants and poor long-term results. The purpose of this article is to review recent progress in clinical islet transplantation for the treatment of diabetes.     

Pancreatic islet cell tumors in adolescents and young adults

BackgroundPancreatic islet cell tumors are rare in adolescents, and most studies published to date focus on older patients. We utilized a national database to describe the histology and clinical pattern of pancreatic islet cell tumors in adolescent and young adult (AYA) patients, and to compare AYAs to older adults. We hypothesized that AYAs with pancreatic islet cell tumors would have better overall survival.MethodsThe National Cancer Data Base (NCDB, 1998–2012) was queried for AYA patients (15–39 years) with a pancreatic islet cell tumor diagnosis. Demographics, tumor characteristics, treatment modalities, and outcomes were abstracted and compared to adults (≥ 40 years).Results383 patients (56.4% female, 65% non-Hispanic Whites) were identified, with a median age of 27 (IQR 16–34) years. Islet cell carcinoma was the most common histology. Of patients with known stage of disease, 49% presented with early stage (I or II). Seventy percent of patients underwent surgical resection, including local excision 44%, Whipple procedure 37.5%, or total pancreatectomy 19%. Chemotherapy was utilized in 27% and radiotherapy in 7%. All-cause mortality was 36%. AYA patients underwent more extensive resections (p = 0.001) and had lower mortality rates (p < 0.001), with no differences in tumor stage or use of adjuvant therapies, when compared to adults.ConclusionsAYA patients with pancreatic islet cell tumors had comparable utilization of adjuvant therapies but underwent more extensive resections and demonstrated a higher overall survival rate than adult counterparts. Further investigation into approaches to earlier diagnosis and tailoring of multimodality therapy of these neoplasms in the AYA population is needed.Levels of evidencePrognostic Study, Level II — retrospective study.     

胰十二指肠及肾一期联合移植5例报告

目的开展和进一步总结胰肾一期联合移植术的经验。方法回顾性总结４年来共实行胰十二指肠肾脏一期联合移植术５例的方法、疗效及并发症的预防和治疗。结果本组５例于术后移植胰腺和移植肾均发挥了正常功能,术后第１～１０天均停用胰岛素,空腹血糖在正常范围。术后并发症常见,计本组出现的并发症为胰周感染或脓肿３例,十二指肠残端瘘１例,化学性或细菌性膀胱炎４例,移植胰巨细胞病毒（ＣＭＶ）感染１例,代谢性酸中毒２例,肺部感染２例和急性排斥反应１例。并发症经处理后大多都能得到控制。本组中有２例已分别存活４年６个月和３年５个月,１例术后３周死于移植肾急性排斥反应多器官功能衰竭。结论胰十二指肠肾脏一期联合移植对治疗Ⅰ型糖尿病并发晚期尿毒症具有肯定的临床疗效,较其它移植有许多优点。术后并发症的预防和正确治疗是影响病人长期存活的重要因素     

胰岛细胞封装技术的研究进展

糖尿病发病率呈上升趋势,临床治疗极具挑战性。虽然药物降糖具有一定疗效,但存在血糖波动的危险,1型糖尿病的临床治愈仍难以实现。胰岛细胞移植是解决胰岛素注射引起的血糖波动问题的有效方法之一。然而,在胰岛细胞移植的临床实践中存在受者需要长期服用免疫抑制剂、移植后胰岛细胞大量丢失等问题,目前并未在临床广泛使用。胰岛细胞封装技术可减少胰岛细胞丢失,减少或消除排斥反应,是提高胰岛细胞存活率的关键环节。本文简要回顾了胰岛细胞封装技术的发展历程,分析了不同胰岛细胞封装技术面临的挑战,并对今后的研究加以展望,旨在为促进胰岛细胞移植的发展提供参考。     

两种酶对猪胰岛消化分离效果的对比

目的　研究单纯酶 (胶原酶Ⅳ )与复合酶 (由胶原酶Ⅰ、Ⅳ、弹性蛋白酶、纤维素酶组成 )对成年猪胰岛的分离效果。方法　采用经胰管灌注消化酶的方法分离胰岛 ,用双硫腙染色做胰岛计数 ,台盼蓝染色判定胰岛活化水平 ,胰岛培养第 2d、第 4d、第 6d测定胰岛素含量 ,培养第 6d做胰岛活性评价 ,电镜观察胰岛的形态结构。结果　经复合酶消化法制备的胰岛数量为 (4 915±l0 42 )个胰岛 /克胰腺 ;而经单纯酶消化的胰岛数量为 (30 12± 989)个胰岛 /克胰腺。两组比较差异显著 (P <0 .0 5 )。结论　复合酶消化法是一种优于单纯酶消化法的成年猪胰岛分离方法。     

采用改良的胰腺三管插管法分离成人胰岛的实验研究

目的 探讨胰腺插管方式对成人胰岛分离及纯化的影响.方法 共对17例成人胰腺进行了胰岛的分离和纯化.采用改进的腹部器官联合快速切取技术获取胰腺,分别采用标准法(3例)、单管法(11例)和三管法(3例)对胰腺进行灌注.标准法是将胰腺从胰颈处完全切断,沿主胰管分别向胰头和胰尾插管,主胰管人十二指肠处予以结扎.单管法为采用加长插管自主胰管插入,直至胰尾.三管法是在胰颈背侧切开胰腺至主胰管,经主胰管分别向胰头和胰尾方向插管,在主胰管进入十二指肠处插第3根插管.采用胶原酶LibarseHI消化,Ficoll连续密度梯度离心法纯化.双硫腙染色,鉴定胰岛的纯度,并计算胰岛当量(IEQ).丫啶橙/溴乙啶荧光染色,计数活细胞百分率.体外葡萄糖刺激试验鉴定胰岛功能.结果 标准法的灌注量平均为0.71 ml/g胰腺,单管法的灌注量平均为0.96 ml/g胰腺,三管法的灌注量平均为1.24 ml/g胰腺,明显多于前两种方法(P＜0.05).标准法的胰岛收获量平均为1914 IEQ/g胰腺,单管法为2270 IEQ/g胰腺,三管法为2514 IEQ/g胰腺,单管法和三管法明显高于标准法(P＜0.05);其胰岛纯度/活性分别为74 ％/79.3％、75.6 ％/79.4％和78.3 ％/84.0％,三者间的差异无统计学意义.标准法所获得的胰岛胰岛素释放指数平均为3.46,单管法为4.74,三管法为5.27,单管法和三管法明显高于标准法(P＜0.05).结论不同的插管灌注方式对成人的胰岛分离有一定影响,三管法有利于提高胰腺灌注量,增加胰岛的收获量.     

微囊化胰岛移植优化策略研究进展

胰岛移植是治疗糖尿病的有效方案之一,但目前仍存在供者短缺以及长期应用免疫抑制剂所致的不良反应等问题,导致移植后胰岛存活受限。微囊化胰岛移植技术可在一定程度上克服这些困难,但微囊内免疫隔离微环境破坏、氧气和营养物质供应不足等多种因素限制了微囊化胰岛移植的临床应用。近年来,关于提高微囊化胰岛移植效果的研究逐渐增多,干细胞在微囊化胰岛移植中的应用也逐步成为研究热点。因此,本文围绕微囊化胰岛移植的优化策略及干细胞在微囊化胰岛移植中的应用进行综述,探讨微囊化胰岛移植潜在的改进方法,旨在为微囊化技术在胰岛移植中的进一步临床应用研究提供参考。     

Current status of islet cell transplantation

Despite substantial advances in islet isolation methods and immunosuppressive protocol, pancreatic islet cell transplantation remains an experimental procedure currently limited to the most severe cases of type 1 diabetes mellitus. The objectives of this treatment are to prevent severe hypoglycemic episodes in patients with hypoglycemia unawareness and to achieve a more physiological metabolic control. Insulin independence and long term-graft function with improvement of quality of life have been obtained in several international islet transplant centers. However, experimental trials of islet transplantation clearly highlighted several obstacles that remain to be overcome before the procedure could be proposed to a much larger patient population. This review provides a brief historical perspective of islet transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, and outlines current challenges and future directions in clinical islet transplantation.     

与大鼠胰岛细胞联合移植的骨髓间充质干细胞的免疫调节作用

目的 研究同种异体或自体的大鼠骨髓间充质干细胞(MSC)与胰岛肝内联合移植对胰岛移植物的免疫调节作用及其机制.方法 以链佐星制备Lewis大鼠的糖尿病模型,作为胰岛移植受者,分为3组:单纯移植组大鼠经门静脉单独移植SD大鼠胰岛6000 IEQ/kg;同系MSC组大鼠经门静脉共同移植1×109/L的Lewis大鼠MSC 1 ml与SD大鼠胰岛6000 IEQ/kg;同种MSC组大鼠经门静脉共同移植1×109/L的SD大鼠MSC 1 ml与SD大鼠胰岛6000 IEQ/kg.检测受鼠的血糖变化,术后1、3 d大鼠外周血γ干扰素(IFN-γ)、白细胞介素(IL)-2、IL-4和IL-10的含量.结果 3组大鼠术后第1天血糖均下降到13.9 mmol/L以下.同系MSC组移植物存活时间为(11.38±4.03)d,同种MSC组为(5.50±2.07)d,单纯移植组为(2.88±1.25)d(P＜0.01).术后1、3 d,单纯移植组IFN-γ和IL-2的含量显著高于同系MSC组和同种MSC组(P＜0.01),同种MSC组IFN-γ和IL-2的含量高于同系MSC组(P＜0.05);单纯移植组IL-10的含量低于同系MSC组和同种MSC组(P＜0.01),同系MSC组IL-10的含量与司种MSC组相比较,差异无统计学意义(P＞0.05);各组IL-4含量的差异无统计学意义(P＞0.05).结论 MSC与同种胰岛共移植可以延长胰岛移植物存活时间,应用同系MSC的效果优于同种异体MSC.共移植的MSC主要通过减少TH1类细胞因子(IFN-y和IL-2)的表达使受者TH1/TH2平衡向TH2方向偏移.

Abstract：

Objective To compare the immune regulation of syngenic and allogenic mesenchymal stem cells (MSCs) in the transplantation combined with islets. Methods After induction of diabetes in 30 Lewis rats with streptozotocin (STZ), the recipient Lewis rats received islets from SD rats combined with syngenic (group B) or allogenic (group C) MSCs injection via the portal vein. The group of islets transplanted alone served as control (group A). The survival time of grafts in all groups was assessed by the level of blood glucose. ELISA was used to detect the levels of interferon-γ (IFN-γ), interleukin 2 (IL-2), IL-4 and IL-10 in the peripheral blood on the 1st and 3rd day after transplantation. Results The blood glucose levels in all three groups were decreased in a normal range (13. 9 mmol/L) and the survival time of grafts in group B (11.38 ± 4. 03 days) was significantly longer than in group C (5. 50± 2. 07 days) as well as group A (2. 88 ± 1.25 days). On the 1 st and 3rd day after transplantation, the levels of TH 1 cytokines IFN-γ and IL-2 in group A were significantly higher than in groups C and B (P＜0.05). Meanwhile the levels of TH 2 cytokine IL-10were increased in group B, but there was no significant difference between groups A and C (P＞0.05). The levels of IL-4 had no significant difference among these three groups (P ＞ 0.05).Conclusion Islet transplantation combined with MSCs could prolong the survival time of grafts.Syngenic MSCs, superior to allogenic ones, were more effective in changing the balance of TH1/TH2to TH2. Decreased expression of TH1 cytokine (IFN-γ, IL-2), which was closely related to the induction of immune tolerance, was beneficial to the long-term survival of grafts.     

Clinical islet transplantation in Japan

Introduction  The results of clinical islet transplantation in Japan are, here in, reported and discussed its efficacy and problems. Methods  Since the first islet transplantation was performed in 2004, 65 islet isolations and 34 islet transplantations to 18 type 1 diabetic patients have been performed in Japan. Results  Following islet transplantation, patients experienced decreased insulin requirements and lower hemoglobin A1C levels, and positive serum C-peptide levels. All patients achieved stabilized blood glucose levels and the disappearance of hypoglycemic unawareness. Although three patients achieved insulin independency for a limited period, persistent islet graft function was difficult to maintain. Overall islet graft survival was 86.5% at 6 months, 78.7% at 1 year, and 62.9% at 2 years after the first islet transplantation. In our institution, we carried out 23 islet isolations and six islet transplantations to four patients. Although insulin independency was not achieved, all patients showed a disappearance of hypoglycemic unawareness. Conclusions  Using data from the Japanese Trial of Islet Transplantation, the effectiveness of islet transplantation was shown even when using the pancreata from non-heart-beating donors. Although there are a number of problems to be solved and further improvement is needed, we can state that the introduction of clinical islet transplantation offers hope for type 1 diabetic patients.     

骨髓间充质干细胞对异种胰岛移植排斥反应的影响

目的 探讨骨髓间充质干细胞(mesenchymal stem cell,MSC)对异种胰岛移植排斥反应的影响.方法 建立人-Wistar大鼠异种胰岛移植模型,选取造模成功的30只糖尿病大鼠,随机分为对照组和MSC组,每组15只.用Lewis大鼠股骨与胫骨培养制成的MSC进行干预治疗,MSC组植入人胰岛细胞和MSC,对照...     

胰导管保护法提高大鼠胰岛获取量和功能的研究

目的 探讨胰导管保护法(即在胰腺获取时向胰导管内注射少量含胰酶抑制剂的UW液,TIUW液)能否提高冷保存后的大鼠胰腺的胰岛获取量和功能.方法 大鼠胰腺分为5组.组1:新鲜胰腺(n=10);组2:未应用胰导管保护法仅用TIUW液保存6 h的胰腺(n=10);组3:应用胰导管保护法且用TIUW液保存6 h的胰腺(n=10);组4:未应用胰导管保护法仅用TIUW液保存24 h的胰腺(n=10);组5:应用胰导管保护法且用TIUW液保存24 h的胰腺(n=10);双硫腙染色鉴定胰岛形态并计数,锥虫蓝染色评估胰岛死亡率,葡萄糖刺激试验评价胰岛离体功能,糖尿病裸鼠胰岛移植评价胰岛在体功能.结果 分离纯化后胰岛获取量分别为590±127、272±50、454±65、253±56、447±66(胰岛当量±标准差);胰岛死亡率分别为(5.7±4.2)%、(18.3±6.5)%、(11.7±4.2)%、(26.3±5.6)%、(15.0±5.3)%;刺激指数分别为7.32±2.32、4.81±1.17、7.56±2.44、2.88±1.00、5.71±1.90;糖尿病治愈率分别为100%、0%、100%、0%、70%.所有结果均显示差异有统计学意义(P＜0.05,组2与组3和组4与组5).结论 胰导管保护法提高了冷保存后大鼠胰腺的胰岛获取量和功能.     

脂肪间充质干细胞与胰岛联合移植对术后免疫状态及移植效果的影响

目的研究同系脂肪间充质干细胞(AMSC)与同种异体胰岛联合移植对胰岛移植术后免疫状态及移植效果的影响。 方法选择Lewis大鼠建立链脲佐菌素诱导的糖尿病大鼠模型作为胰岛移植受体,AMSC＋胰岛移植组经左肾包膜下联合移植Lewis大鼠AMSC及Wistar大鼠胰岛,单纯胰岛移植组经左肾包膜下单独移植Wistar大鼠胰岛,单纯AMSC移植组经左肾包膜下单独移植Lewis大鼠AMSC,阳性对照组为未进行移植的Lewis糖尿病大鼠,阴性对照组为正常Lewis大鼠。ELISA法检测血清细胞因子IFN-γ、IL-2、IL-4和IL-10浓度,流式细胞技术检测外周血CD4^＋、CD8^＋ T细胞比例,HE染色观察移植胰岛淋巴细胞浸润情况,观察血糖、胰岛素水平及胰岛移植物存活时间。采用单因素方差分析比较5组大鼠外周血淋巴细胞亚群比例和血清细胞因子水平,采用LSD法进行组间两两比较;血糖及血清胰岛素变化采用重复测量资料方差分析;采用独立样本t检验比较AMSC＋胰岛移植组与单纯胰岛移植组胰岛移植物淋巴细胞计数。采用Kaplan-Meier法绘制AMSC＋胰岛移植组与单纯胰岛移植组胰岛移植物生存曲线,并采用log-rank检验进行比较。P<0.05为差异有统计学意义。 结果AMSC＋胰岛移植组胰岛移植物平均生存时间为(26.8±3.0) d,长于单纯胰岛移植组的(19.0±1.3) d,两组大鼠胰岛移植物生存曲线差异有统计学意义(χ²＝4.494,P<0.05)。胰岛移植后各时间点,AMSC＋胰岛移植组大鼠移植后血糖和胰岛素水平均优于单纯胰岛移植组(P均<0.05)。胰岛移植前,5组大鼠外周血CD4^＋、CD8^＋ T细胞比例以及细胞因子IFN-γ、IL-2、IL-4和IL-10差异均无统计学意义(F＝0.425、0.476、0.256、0.418、0.281和0.313,P均>0.05)。胰岛移植后第7、14、28天,AMSC＋胰岛移植组大鼠外周血CD4^＋、CD8^＋T细胞比例均低于单纯胰岛移植组(P均<0.05),血清IFN-γ和IL-2浓度均低于单纯胰岛移植组(P均<0.05),IL-4和IL-10浓度均高于单纯胰岛移植组(P均<0.05)。胰岛移植第7天,AMSC＋胰岛移植组胰岛移植物平均淋巴细胞计数为(142±21)个/mm²,低于单纯胰岛移植组的(311±36)个/mm²(t＝8.245,P<0.05)。 结论与胰岛联合移植的AMSC能够提高胰岛移植的效果,可调节糖尿病大鼠体内IFN-γ、IL-2、IL-4和IL-10表达及抑制同种异体胰岛刺激的T细胞增殖,减轻胰岛移植物的免疫损伤。     

Current status and perspectives in clinical islet transplantation

Beta-cell replacement, by either pancreas or islet transplantation, is the only treatment to achieve an insulin-independent, constantly normoglycemic state avoiding hypoglycemic episodes in patients with type-1 diabetes mellitus. This article reviews the current state of the worldwide experience based on data provided by the International Islet Transplant Registry; it also illustrates the perspectives of clinical islet transplantation. Received: February 7, 2000 / Accepted: February 29, 2000     

New approaches for successful islet transplantation

The long-term control of glucose metabolism is expected to be achieved with insulin-secreting pancreatic islets transplanted as the whole pancreas or isolated islet tissue. The current clinical outcomes of such procedures are unsatisfactory compared with that of whole organ transplantation or experimental islet transplantation, although the safety of the transplantation procedure itself is assured. In this review, we first describe the immunological characteristics of purified islets in an attempt to explain the differences between the results for human and experimental islet transplantation. We then introduce new approaches currently undertaken in our laboratory that could, we hope, make islet transplantation more successful. Received: December 4, 1999 / Accepted: February 24, 2000