胰岛移植研究新进展

本文叙述了胰岛移植中的最近进展，诸如供胰的选择，获取及保存，胰岛的分离及纯化、胰岛的培养和冻存，移植的部位和数量，以及有关问题作了介绍。     

成人胰岛大容量分离技术的改进及胰岛功能检测

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,为利用同种异体胰岛移植治疗1型和部分2型糖尿病提供理论依据和技术基础。方法 采用改良的自动分离技术连续分离28例人胰岛,然后用连续性密度样度离心法纯化胰岛。胰岛收获量以国际标准的胰岛当量(islet equivalent,IEQ)表示。胰岛功能的测定分别为体外测定胰岛的胰岛素／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胰岛功能鉴定并随后进行腹腔糖耐量试验,连续测定移植鼠血糖水平及其体内C肽浓度。结果 28例成人胰腺分离的胰岛收获量为5000～1030000IEQ／胰腺,平均为291635IEQ／胰腺,前13例平均每个胰腺收获49123IEQ,平均每克组织收获846IEQ。平均纯度为87％,随着技术的改进后15例的分离结果则分别为：平均每个胰腺501813IEQ,平均每克组织7003IEQ,平均纯度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肽水平亦接近正常鼠。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实验基础。     

胰岛移植进展

近年来 ,随着胰岛分离、纯化技术的不断改进以及新型免疫抑制剂的问世和应用 ,临床同种胰岛移植已取得令人瞩目的进展[1 2 ] ,有可能在今后数年内得以广泛开展。本文对这些方面的进展作一综述。一、国际胰岛移植登记处的报告据国际胰岛移植登记处 (ITR)最近公布的结果 ,截止到2 0 0 0年 12月 31日 ,全球共有 5 1个研究中心开展了 4 93例成人胰岛移植 ,术后不依赖胰岛素的持续时间超过 12、2 4、36和 4 8个月的例数分别为 4 0、2 2、11和 6例。自体胰岛移植后停用胰岛素最长者已达 13年 ;Ⅰ型糖尿病 (IDDM )患者同种异体胰岛移植后停用胰…     

胰岛移植研究新进展(文献综述)

本文叙述了胰岛移植中的最近进展,诸如供胰的选择、获取及保存,胰岛的分离及纯化、胰岛的培养和冻存,移植的部位和数量,以及有关问题作了介绍.     

实验大鼠胰岛分离移植技术方法的比较分析

目的 探索高效的大鼠胰岛分离移植技术方法.方法 应用Wistai-Furth大鼠,于体内或体外胶原酶经胰管灌注膨化胰腺,联合不同密度Ficoll液或Histopaque液纯化胰岛细胞,评估胰岛的数量、纯度、胰岛当量以及肾被膜下胰岛移植的有效性.结果 体外经胰管灌注膨化胰腺结合Histopaque液纯化提取胰岛的数量、纯度和胰岛当量值均显著高于体内灌注组各数值(P＜0.01),其提取时间无显著差别.1000个胰岛细胞移植进入左肾被膜下,有效的逆转了糖尿病大鼠高血糖,其远期糖耐受结果优于500和800个胰岛细胞移植组.结论 体外灌注膨化消化胰腺结合Histopaque液纯化胰岛的分离方法是一种满意的分离技术.1000个胰岛细胞是保证肾被膜下胰岛移植成功的最低有效数量.     

人工生物胰的体外分泌功能研究

目的 探讨海藻酸钠-氯化钡微囊对成人胰岛细胞体外分泌功能的影响.方法 对6例成人胰腺组织称重后采用V型胶原酶消化法分离,采用Ficoll间断密度梯度离心纯化法纯化,采用DTZ染色显微镜下评价胰岛细胞的数量、纯度.采用海藻酸钠氯化钡为材料包裹成人胰岛,体外培养及放射免疫法测定培养液中基础胰岛素浓度.结果 成人胰腺经胶原酶消化分离后,胰岛平均收获量(3600±447)个胰岛/g胰腺；Ficoll间断密度梯度离心纯化后,每克胰腺组织平均收获量(2140±207)个胰岛,纯度大于70％；成人胰岛细胞培养2、4、6d后,微囊化组第2、4、6天的基础胰岛素平均浓度(每100个胰岛单位mU/L)分别为3.302±1.63、3.504±1.10、2.921±1.13,未微囊化组的基础胰岛素平均浓度(每100个胰岛单位mU/L)分别为3.814±1.49、4.175±1.60、3.617±1.34,两组基础胰岛素浓度差异无统计学意义(P＞0.05).结论 人工生物胰具有良好的体外分泌功能,包裹成人胰岛的海藻酸钠-氯化钡微囊对胰岛体外分泌胰岛素的功能无影响.     

胰岛移植临床应用进展

近年来 ,随着胰岛分离、纯化技术的不断改进以及新型免疫抑制剂的问世和应用 ,胰岛移植已取得突破性进展 ,胰岛移植既能纠正糖代谢异常 ,又能防止微血管病变的发生、发展 ,是治疗胰岛素依赖性糖尿病 (insulindependentdiabetesmellitus,IDDM)即 1型糖尿病较理想的方法 ,在今后数年内可以广泛开展。1　胰岛移植的适应证　　目前临床实施的胰岛移植分为自体胰岛移植和同种异体胰岛移植。自体胰岛移植主要适应证为慢性胰腺炎全胰切除后所致的外科源性糖尿病 ,同种异体胰岛移植主要适应证为IDDM和上腹部恶性肿瘤联合脏器切除后所致的外科源…     

胰岛分离、纯化制备的改进

目的 探讨大型哺乳动物胰岛机械化大量分离、纯化的方法,为人类胰岛移植物的大量制备摸索创造条件.方法 应用改进的机械化胰岛分离、纯化系统,用HCA和UW液顺序原位灌洗犬胰腺,主副胰管插管,4℃胶原酶-V(1.5 g/L)+胰酶抑制剂pefabloc(0.4 mmol/L)灌注后,Ricordi-Chamber消化罐消化,4℃COBE2991连续密度梯度离心纯化,测定胰岛当量(IEQ)、胰岛纯度及存活率、胰岛素及C-肽的释放量、培养24 h后光镜及电镜观察.结果 胰腺消化时间为(25.0±6.0)min,胰岛外分泌腺包裹率为(9.4±2.4)%,消化后胰岛收获量为(17.2±3.6)×104IEQ/每个胰腺,纯化后胰岛收获量为(8.3±2.0)×104IEQ/每个胰腺,胰岛纯度为(89.7±3.5)%.纯化胰岛体外低糖与高糖刺激下胰岛索分泌量及C-肽的释放量良好,培养24 h后形态结构及功能正常.结论 本实验室改进的胰岛机械分离方法及各设备运行可靠,获得的胰岛形态功能良好,可望用于临床人类胰岛的大量制备.     

微重力培养的新鲜和冻存胰岛联合移植治疗1型糖尿病

目的经微重力培养的新鲜和冻存胰岛联合移植提高1型糖尿病的治疗效果。方法将分离纯化的大鼠胰岛分为（1）体外实验组：实验组1．1：冻存的胰岛经微重力培养；实验组2．1：冻存的胰岛在普通培养基中培养；对照组1：新鲜大鼠胰岛经微重力培养。观察胰岛收获率和体外胰岛素分泌情况。（2）胰岛移植组：即将新鲜和冻存的胰岛经微重力或普通培养7d后分别移植入受体鼠体内，观察移植效果。结果经微重力培养的各组胰岛收获率、DNA含量和胰岛素含量高于普通组。普通培养组在培养后期胰岛素分泌明显下降，并且胰岛素刺激指数明显低于经微重力培养组（P〈0．05）。移植经过微重力培养的500 IEQ新鲜胰岛和1500 IEQ冻存胰岛在移植后1周内可达100％纠正糖尿病，全部受体维持正常血糖耐受曲线一直到观察结束。结论采用细胞内低温保存液（HTS）结合细胞冻存液（DMSO）对大鼠胰岛进行冻存前后经微重力培养可以明显提高胰岛冻存质量，是目前胰岛冻存的最佳选择。使经微重力培养的新鲜和冻存胰岛联合移植一次治愈糖尿病成为可能，并有效的节约了胰岛资源，提高了移植效果。     

成人胰岛细胞的分离

目的　探讨成人胰岛细胞的分离及胰腺冷缺血时间与胰岛细胞活率的关系。方法　采取胰、肾联合切取 ,经腹主动脉插管 ,用自制的灌注器以高渗枸橼酸盐嘌呤溶液 (HC A液 ) 15 0 0～2 0 0 0ml进行原位灌洗。将肾与胰腺、十二肠、脾分离 ,采用胶原酶P消化胰腺 ,分离并纯化胰岛细胞 ,以双硫腙及丫腚橙染色 ,测定所得到的胰岛细胞的纯度及活率。结果　胰腺的冷缺血时间为 2 .5～ 8h ,温缺血时间为 0～ 3min。胰岛收获量为 (2 .38± 0 .6 7)× 10 3 IEQ/g ,纯化后胰岛细胞的活率为 19%～ 83% ,冷保存指数 (或输注指数 )为 9.8～ 6 6 .4。胰岛细胞的活率与胰腺的冷缺血时间呈负相关 ,冷保存指数和胰岛细胞活率呈负相关。结论　以高渗枸橼酸盐嘌呤溶液灌洗、胶原酶P消化胰腺所得到的胰岛细胞活率高 ,但胰腺的冷缺血时间不宜超过 5h。     

同供体肝细胞预先输入对异种胰岛移植物存活影响的实验研究

应用胶原酶消化分离大鼠胰岛细胞，以Ｆｉｃｏｌｌ密度梯度离心纯化，同时分离大鼠肝细胞。以链脲霉素制备小鼠糖尿病模型２４只。随机分为３组。实验组小鼠预先输入供体大鼠肝细胞，６在后再经门静脉植入同供体经培养的胰２岛细胞。对照１组为空白对照组，不予任何处置，对照２组单纯植入胰岛细胞。     

猪胰岛在糖尿病大鼠不同部位异种移植的实验研究

研究了猪胰岛在糖尿病大鼠脑室，胸腺，肾包膜下，门静脉等具有“免疫学特穗”的部位异种移植的效果。结果显示，在不使用免疫抑制剂的情况下，猪胰岛在这四个部位均能纠正大鼠的高血糖状态，并能维持正常血糖一段时间，其中脑组的移植物平均功能存活时间较胸腺组，肾包膜下组，门静脉组明显延长。     

胰岛移植物质量鉴定方法的研究

胰岛移植物的质量直接影响胰岛移植的效果，用经胶原酶消化，Ｆｉｃｏｌｌ纯化的成年大鼠胰岛，比较乙醇和二甲亚砜配制的双硫腙，作Ｂ细胞鉴别染色，丫啶橙－碘丙啶和双醋酸荧光素－溴乙锭双色荧光染色，鉴别活细。     

An Isolated Venous Sac as a Novel Site for Cell Therapy in Diabetes Mellitus

BACKGROUND: Transplanting pancreatic islets is of significant interest for type 1 diabetes mellitus. After intraportal injection of islets, inferior engraftment and eventual loss of transplanted islets constitute major limitations. Therefore, alternative approaches will be helpful. Here, we evaluated in animals whether an isolated venous sac would support survival of transplanted islets, along with correction of hyperglycemia. METHODS: Pancreatic islets isolated from adult Lewis rats were transplanted either into an isolated venous sac made from lumbar vein or into the portal vein of syngeneic rats. The integrity and vascular organization of the venous sac was determined by studies of the local microcirculation. The engraftment, survival, and function of transplanted islets were analyzed by histology, including endocrine function in situ and by glycemic control in rats with streptozotocin-induced diabetes. RESULTS: Transplanted islets showed normal morphology with insulin expression in isolated venous sac during the long term. Transplanted islets received blood supply from vasa vasorum and had access to drainage through venous tributaries in the venous sac. This resulted in restoration of euglycemia in diabetic rats. Removal of islet graft-bearing venous sac in diabetic rats led to recurrence of hyperglycemia. By contrast, euglycemia was not restored in rats treated by intraportal transplantation of islets. CONCLUSIONS: We demonstrated that pancreatic islets successfully engrafted and functioned in the isolated venous sac with ability to restore euglycemia in diabetic rats. Therefore, the isolated venous sac offers a new site for transplantation of pancreatic islets. This would be clinically beneficial as an alternative to intrahepatic islet transplantation.     

Repeated isologous transplantation of islets of Langerhans in the diabetic rat.

The metabolic response to repeated isologous transplantation of isolated pancreatic islets was studied in 11 streptozotocin-diabetic AGUS rats. The islets were isolated with collagenase and transplanted intra-portally in batches previously found to be quantitatively insufficient for reversal of the diabetic state. Primary transplantation caused a slight improvement whereas retransplantation with the same number of islets three weeks later was followed by normalization of blood glucose, plasma insulin, urine volume and urine glucose per 24 hours during a three-month observation period. Repeated isologous transplantation of isolated pancreatic islets has an additive effect on the metabolism in that two insufficient tissue doses correct streptozotocin induced diabetes in the rats.     

Porcine Pancreatic Islets: Isolation, Microencapsulation, and Xenotransplantation

Abstract: To provide a plentiful source of pancreatic islets for future clinical transplants into diabetic patients, we have developed a simple and reliable method to isolate porcine islets of a high degree of purity. Porcine pancreata were perfused and digested with collagenase, and the islets were then purified on dextran density gradients. In order to avoid any damage to the islets, no mechanical devices nor any strenuous treatment was employed. As many as 5 times 10⁵ islets were isolated from a single porcine pancreas. Islets were encapsulated in alginate-polylysine-alginate membranes with the aid of an electrostatic droplet generator. In vitro studies demonstrated that the isolated islets secreted insulin in response to glucose and 3-isobutyl-L-methylxanthine (IBMX) challenge for at least 4 weeks. Perifusion studies showed that the kinetics of insulin release from the encapsulated islets was similar to that exhibited by free islets. In in vivo studies, 18 diabetic BALB-c mice were transplanted with 1,500-2,500 encapsulated islets each. In 13 recipients, the diabetic condition was reversed for at least 85 days. When capsules were removed from 2 transplant recipients, their diabetic condition quickly recurred.     

The use of non-heart-beating donors for isolated pancreatic islet transplantation

Recent improvements in isolated islet transplantation indicate that this therapy may ultimately prove applicable to patients with type I diabetes. An obstacle preventing widespread application of islet transplantation is an insufficient supply of cadaveric pancreata. Non-heart-beating donors (NHBDs) are generally not deemed suitable for whole-organ pancreas donation and could provide a significant source of pancreata for islet transplantation. Isolated pancreatic islets prepared from 10 NHBDs were compared with those procured from 10 brain-dead donors (BDDs). The success of the isolation for the two groups was analyzed for preparation purity, quality, and recovered islet mass. The function of NHBD and BDD islets was evaluated using in vitro and in vivo assays. On the basis of the results of this analysis, an NHBD isolated islet allograft was performed in a type I diabetic. The recovery of islets from NHBDs was comparable to that of control BDDs. In vitro assessment of NHBD islet function revealed function-equivalent BDD islets, and NHBD islets transplanted to non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice efficiently reversed diabetes. Transplantation of 446,320 islet equivalents (IEq) (8,500 IEq/kg of recipient body weight) from a single NHBD successfully reversed the diabetes of a type I diabetic recipient. Normally functioning pancreatic islets can be isolated successfully from NHBDs. A single donor transplant from an NHBD resulted in a state of stable insulin independence in a type I diabetic recipient. These results indicate that NHBDs may provide an as yet untapped source of pancreatic tissue for preparation of isolated islets for clinical transplantation.     

Xenotransplantation of Microencapsulated Canine Islets into Diabetic Rats

Abstract: Islets of Langerhans were isolated in high yields from canine pancreata. In the procedure, the pan-creata were perfused and digested with collagenase, and the islets were then purified on histopaque density gradients. As many as 60,000 islets were isolated from a single pancreas. Islets were encapsulated in alginate-polylysinealginate membranes with the aid of an air-jet droplet generator. In vitro studies demonstrated that the isolated and encapsulated islets secreted insulin in response to glucose and IBMX challenge for at least 9 weeks. In in vivo studies 6 diabetic Wistar rats were transplanted with 5,000 to 8,000 encapsulated islets each. The diabetic condition was reversed in all recipients for up to 112 days. In control animals, which received free, unencapsulated islets, the xenografts remained functional for fewer than 21 days. Microcapsules retrieved from normoglycemic transplant recipients 1 and 2 months posttransplantation were shown to contain viable islet tissue, and no cellular overgrowth was observed on capsular surfaces. The results of the study indicate a considerable clinical potential of microencapsulated canine islet xenografts.     

Evaluation of energy state of islet independent of size using a newly developed ATP bioluminescence assay

ATP content or energy charge (EC) of islets may be a good parameter to assess viability. In this study, we examined adenine nucleotides and EC of freshly isolated or 24-hour cultured rat islets of various diameters using a novel bioluminescent enzymatic cycling assay system. For freshly isolated islets, ATP content and islet diameter showed a high correlation (r = 0.842, P < .001), but a significant correlation was not observed for cultured islets (r = 0.284) when all islets were included for the analysis. When only the cultured islets with a diameter <350 microm were included for analysis, a significant correlation was observed (r = 0.719). EC of freshly isolated islets fluctuated widely irrespective of diameter, in contrast with results of 24-hour cultured islets, which showed a stable, high EC, regardless of diameter. These data suggest that the ATP content of islets correlates with the islet size and that EC of islets widely fluctuates following isolation, indicating a significant role of monitoring ATP and EC of islets before transplantation.     

A model to evaluate toxic factors influencing islets during collagenase digestion: the role of serine protease inhibitor in the protection of islets

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.