胸腺内胰岛移植的实验研究

目的观察胸腺内和肾包膜下胰岛移植对移植物存活时间的影响。方法以Ｃ５７ＢＬ／６小鼠为受体,ＢＡＬＢ／ｃ小鼠为供体,肾包膜下胰岛移植分为单纯移植和移植的同时加腹腔内一次性注射兔抗小鼠胸腺细胞血清（ＡＴＳ）两组,胸腺内移植组亦分为单纯移植和移植加用ＡＴＳ两组。结果单纯胸腺内移植组其移植物的存活期为（１９．５±１０．１）天,长于单纯肾包膜下移植组（１４．０±２．１）天;移植的同时加用ＡＴＳ,则肾包膜下移植组移植物的存活期为（４３．０±１１．７）天,胸腺内移植组可达（９３．８±２５．５）天以上,其中６只（６／８）胸腺内移植物获长期存活,并且诱导了受体对供体的特异无反应性。结论胸腺可能为胰岛移植的理想部位,而且在诱导免疫耐受中具有重要作用。     

糖尿病小鼠胰岛移植部位的实验研究

目的探讨小鼠肝内、肾包膜下等不同部位胰岛移植物生存时间及免疫学功能的差异。方法采用滤网法分离纯化胰岛,供体胰岛植入受体肝内及肾包膜下。用酶联免疫吸附试验 (ELISA)测定INF-γ的含量。3H-胸腺嘧啶脱氧核苷(3H-TdR)掺入法测定单向混合淋巴细胞反应。结果肾包膜下移植组胰岛生存时间(12．18±1．25)d明显长于肝内移植组[(7．09±0．70)d,P< 0．01]。单项混合淋巴细胞反应显示,肝内移植组淋巴细胞较肾包膜下移植组增殖明显(P<0．01)。上清液测IFN-γ含量,肝内移植组明显升高,差别有统计学意义(P<0．05)。结论不同移植部位, 胰岛的生存时间及免疫功能差异存在统计学意义,肾包膜下是胰岛移植的理想部位。     

大鼠胚胎器官原基异种移植的形态与功能检测

目的 观察大鼠胚胎器官原基(后肾、胸腺)异种移植后的生长发育,作移植后肾、胸腺的形态检查,并检测移植后肾的功能.方法 取受孕第15天(E15)的Lewis大鼠胚胎后肾或胸腺分别植入Balb/c裸小鼠腹腔大网膜及肾包膜下,观察至移植术后第16周.移植术后第5周作移植后肾、胸腺的形态检查,并检测移植后肾的功能.结果 E15 Lewis大鼠胚胎后肾及胸腺在受体裸小鼠腹腔内发育增大明显,后肾伴有包裹性积液囊形成.移植后肾及胸腺行光镜和电镜检查可见发育完好的肾及胸腺的形态结构.移植后肾积液的肌酐、尿素氮浓度明显高于血清,但低于膀胱尿液中的浓度.后肾移植组双肾切除后的存活时间明显长于未作后肾移植组.至移植术后第16周,移植后肾出现萎缩硬化.结论 E15 Lewis大鼠胚胎后肾或胸腺原基异种移植至Balb/c裸小鼠,移植后肾及胸腺能够在宿主体内生长发育成近似正常器官的形态结构,移植后肾具有一定的泌尿功能.     

同种胎儿组织移植的存活率

本实验旨在研究取自不同胎龄的大鼠胎儿器官组织植入同种大鼠体内的效果。用杂交系Sprague-Dawley大鼠,受体为10周龄雌性大鼠,重225～250 g。怀孕11～13天的大鼠选为供体。移植组织:成熟大鼠肾组织(组Ⅰ)的采取,系切除双侧肾脏,立即置于改良的Eagle培养液中(4℃),在解剖显微镜下切成直径小于1 mm的组织块。胎肾组织(组Ⅱ)、胎性腺组织(组Ⅲ)和胎肝组织(组Ⅳ)的采取,是在大鼠受孕后第15～21天,分别剖腹切开子宫取胎,摘取胎儿肝、肾和性腺组织,置于上述培养液中,切成同样大小碎片,此外还采取胎龄为13天的胎肝。采用肾包膜下埋藏技术,将组织移植于受体之左肾包膜下的二个不同部     

骨髓间充质干细胞移植途径对胰岛移植物免疫排斥反应的影响

目的:探讨骨髓间充质干细胞(MSC)与胰岛共移植对诱导胰岛移植物免疫耐受的作用,并比较MSC不同途径移植的效果。方法:SD大鼠和Lewis大鼠分别作为供、受体。取SD大鼠股骨,贴壁培养法分离和扩增MSC,胶原酶V分离胰岛。应用链脲佐菌素制备Lewis大鼠糖尿病模型后,将其随机均分为A组(将BrdU标记的MSC与胰岛经门静脉混合输入),B组(将胰岛经门静脉输入,BrdU标记的MSC经尾静脉输入),C组(胰岛经门静脉输入,联合应用环孢素A)和D组(单纯胰岛门静脉移植)。观察各组术后血糖变化,比较各组胰岛移植物存活时间。术后第7天切取各组部分存活大鼠肝脏、胸腺、脾脏行免疫组化染色观察MSC归巢位置。结果:A,B两组大鼠术后正常血糖维持时间最长,C组次之,D组最短;各组胰岛存活时间A组为(12.1±2.3)d,B组为(8.6±1.4)d,C组为(13.2±1.9)d,D组为(2.2±0.6)d;MSC归巢部位观察显示,A组BrdU阳性的MSC主要分布于肝脏,并在植入胰岛周围形成"类微囊化效应",B组BrdU阳性的MSC主要分布于胸腺、脾脏。结论:MSC与胰岛共移植能诱导胰岛移植物免疫耐受,且MSC和胰岛混合经门静脉移植效果优于胰岛门静脉移植联合MSC外周静脉移植。     

黑地黄丸改善5/6肾切除大鼠肾衰竭模型微炎症状态的免疫调节机制研究

目的：通过黑地黄丸对5/6肾切除肾衰竭大鼠（ CRF大鼠）的免疫器官胸腺、脾脏（指数）及血清IL-1β、IL-6、TNF-α、IL-10、CRP水平的影响，探讨黑地黄丸改善CRF大鼠“微炎症状态”的免疫调节机制，并寻求最佳剂量。方法：将50只Wistar大鼠随机分为空白组，模型组，黑地黄丸高、中、低剂量组5组，除空白组外其他各组大鼠均行5/6肾切除术，建立CRF大鼠模型，连续给药12周后测定免疫器官胸腺、脾脏指数，采用ELISA法测定血清中IL-1β、IL-6、TNF-α、IL-10、CRP的含量。结果：黑地黄丸各剂量组均能明显升高 CRF大鼠胸腺、脾脏指数，同时降低血清 IL -1β、TNF -倩、IL-6、CRP的水平，升高IL-10水平，提示黑地黄丸对CRF大鼠胸腺和脾具保护作用，并可有效调节CRF大鼠细胞因子紊乱。结论：黑地黄丸对CRF大鼠“微炎症状态”改善机制与其调节大鼠的免疫功能密切相关，具体表现在保护CRF大鼠胸腺、脾脏，抑制炎性因子IL-1β、TNF-倩、IL-6的合成与分泌，下调CRP水平，上调IL-10水平等方面。且以黑地黄丸高剂量组效果最佳。     

不阻断腹主动脉及下腔静脉的大鼠胰腺移植

实验以３０只大鼠为受体进行胰腺移植的两种手术方式对比研究，第１组阻断受体腹主动脉、下腔静脉后分别与供胰腹主动脉、门静脉吻合；第２组不阻断腹主动脉、下腔静脉，而是将供腹腔动脉、门静脉分别与体左肾动、静脉吻合。结果发现，不阻断受体腹主动脉、下腔静脉的术式并发症少，提高了移植的成功率。     

自体原位肝移植逆行灌注法对大鼠急性肾损伤的影响

目的探讨自体原位肝移植术中经下腔静脉逆行灌注对大鼠肾功能的影响,为临床肝移植应用经下腔静脉逆行灌注法提供实验依据。方法 60只自体原位肝移植大鼠随机分为逆行灌注组、门静脉灌注组与假手术组各20只。前两组建立自体肝移植模型,其中逆行灌注组采用经下腔静脉逆行灌注法,先开放下腔静脉,再开放门静脉,最后开放肝动脉。门静脉灌注组采用常规经门静脉正向灌注法,先开放门静脉,再开放下腔静脉,最后开放肝动脉。假手术组开腹后游离肝门处门静脉、肝动脉及肝上、下下腔静脉,不予阻断,17min后关腹。分别检测3组术前1h、术后1h、8h及术后1d、5d的血清肌酐(Scr)、血尿素氮(BUN)水平;无肝期结束后1h、8h、1d取左肾组织行光镜检查观察肾组织病理形态学变化。结果术前1h,各组肾功能指标比较差异均无统计学意义(均为P>0.05);与假手术组比较,逆行灌注组、门静脉灌注组术后1h、8h及1d的Scr、BUN水平显著增高,而且逆行灌注组上述两指标明显低于门静脉灌注组(均为P<0.05),但术后5d3组比较差异均无统计学意义(均为P>0.05)。无肝期结束后1h,逆行灌注组和门静脉灌注组肾组织病理学检查发现肾间质充血,8h出现明显的肾小管上皮细胞水肿及肾间质充血,逆行灌注组明显轻于门静脉灌注组;无肝期结束后1d两组肾组织损伤呈现好转趋势,且逆行灌注组明显优于门静脉灌注组。结论自体原位肝移植术中实施逆行灌注可减轻大鼠急性肾损伤,改善大鼠早期肾功能。     

大鼠胸腺内注射同种抗原对甲状旁腺移植物存活的影响

目的　改善甲状旁腺移植物的存活时间。方法　用SDLewis及DA大鼠进行甲状旁腺移植实验。由供体Lewis大鼠的脾细胞提取抗原。按照不同的抗原注射途径（尾静脉、门静脉及胸腺内）,是否合用抗淋巴细胞血清及第3品系大鼠的甲状旁腺移植共分为9组。结果　胸腺内注射抗原结合抗淋巴细胞血清的应用，使甲状旁腺移植物的平均存活期达到（196.00±3.96）d，与其他各组相比差异有非常显著性（P＜0.01）。结论　大鼠胸腺内注射抗原结合抗淋巴细胞血清的应用成功地诱导受体产生了供体特异性免疫耐受。     

同种大鼠胚胎胰组织移植后在异体内的增殖和分化

目的 研究同种大鼠胚胎胰组织移植后在异体内重新血管化并再生长发育的可能性。方法 将妊娠14．5d（E14．5）和妊娠15．5d（E15．5）的Lewis大鼠胚胎胰移植到成年健康Lewis大鼠的肾包膜下，分别在移植后3周和6周开腹观察移植胰的发育情况，并取标本作病理切片观察，采用免疫组织化学染色半定量和定位。结果 （1）E14．5和E15．5的胚胎胰移植入肾包膜下3周后，体积均增大约10～15倍，外分泌部分腺泡导管分化发育，B细胞大量增殖，形成胰岛，组织周围有新生血管，且胰岛素分泌功能正常。（2）胚胎胰植入肾包膜下6周后，E14．5的胚胎胰内分泌部分进一步增殖；E15．5的胚胎胰则未见增殖，外分泌部分均有纤维化表现，内分泌部分与外分泌部分有分离趋势。结论 （1）E14．5与E15．5的胚胎胰移植于同种大鼠肾包膜下均可在异体内再血管化，并发育为近似正常的胰腺组织。（2）E14．5和E15．5胎龄的大鼠胚胎胰都是比较适合移植的。     

Transplantation of fetal rat islets into the cerebral ventricles of alloxan-diabetic rats. Amelioration of diabetes by syngeneic but not allogeneic islets

Syngeneic fetal rat islets were isolated and transplanted into alloxan-diabetic inbred male Lewis rats. The effect of transplantation of islets into the cerebral ventricles on the diabetic state of the recipients was compared with that of the conventional transplantation of islets homeotypically into the liver via the portal vein. Fourteen of fourteen rats surviving after stereotaxic implantation of islets into the ventricles returned to normoglycemia; normoglycemia has been maintained for up to 34 wk. Glucose tolerance tests revealed an improved, although not completely normalized, pattern. Histologic examination of the brains of these recipients revealed clusters of intact islets in the ventricle. These data provided a physiologic basis for further investigation of the immunologically privileged nature of the intraventricular space as a site for implantation of allogeneic pancreatic islets. Islets from Wistar-Furth rats (major histocompatibility difference) or Fischer 344 rats (minor histocompatibility difference) were transplanted into the ventricles of alloxan-diabetic Lewis rats. There were only small and unsustained changes in body weight and blood and urine glucose of any of the rats receiving the allogeneic islets. Histologic examination of the ventricles of these rats 3 wk after transplantation revealed only glial scar tissue. These data suggest that the cerebral ventricles cannot serve as a privileged site for islet transplantation, at least using the type of islet preparation employed in these experiments.     

Insulin-releasing activity and successful transplantation of pancreatic islets preserved by tissue culture.

Preserving pancreatic islets for 7 days by a making use of the organ culture, we studied the insulin-releasing activity at the time of administration of glucose and various digestive tract hormones for the purpose of clarifying the function of preserved pancreatic islets. Furthermore, we transplanted pancreatic islets preserved for 3 to 5 days into the portal vein of rats with streptozotocin-induced diabetes and reached the following conclusions: (1). The islets of Langerhans of the pancreas responded well to glucose up to the seventh day of preservation and showed patterns similar to those of fresh pancreatic islets with respect to both the dose response and the time response. (2). Preserved pancreatic islets of the pancreas had insulin-releasing activity almost equal to that of fresh pancreatic islets against stimulation by glucagon, tolbutamide, and various digestive tract hormones. (3). Rats with streptozotocin-induced diabetes showed a marked improvement in blood glucose and urine glucose following transplantation of preserved pancreatic islets into the portal vein, and this effectiveness persisted for 6 to 8 weeks.     

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。     

Influence of the numbers of islets on the models of rat syngeneic-islet and allogeneic-islet transplantations

One of the main barriers to widespread application of islet transplantation is the limited availability of human pancreatic islets. The reduction of graft islet mass for transplantation to a recipient is one of the strategies in islet transplantation. However, transplantation of only a small number of islets may result in primary nonfunction. To optimize the sites and numbers of islets for transplantation, we analyzed these factors using pancreatic islets from Lewis or F344 rats transplanted into rats rendered diabetic by streptozotocin (50 mg/kg IV) and confirmed as such prior to transplantation (>300 mg/dL blood glucose). Approximately 500 to 1500 islets were injected via the portal vein or under the renal capsule into the diabetic F344 rats. The blood glucose level of all animals bearing 1500 syngeneic or allogeneic islets transplanted to the liver or under the kidney capsule exhibited restored normoglycemia (<200 mg/dL) at 1 day after transplantation. Graft function deteriorated after only 3 days in three animals (5.8%). The loss of graft function after 3 days occurred in 10 of 28 rats transplanted with 1000 to 1200 syngeneic islets, 4 of 19 rats transplanted with 800 to 900 syngeneic islets, and 7 of 17 rats transplanted with 500 to 600 syngeneic islets. There was no significant difference in the loss of graft function between the sites of transplantation via portal vein or under the kidney capsule. In conclusion, higher frequencies of primary nonfunction occurred with less than 1500 islets transplanted. They were independent of the sites in the rat-islet transplantation model.     

Allotransplantation of rat islets into the cisterna magna of streptozotocin-induced diabetic rats.

Islets were isolated from the pancreata of Sprague-Dawley rats and transplanted into streptozotocin-induced diabetic outbred Wistar rats. The effect of transplantation of islets into the cisterna magna on the diabetic state of the recipients was compared with that of the conventional transplantation of islets into liver via the portal vein. After successful intraportal (IP) transplantation, rejection took place between days 7 and 15 in all diabetic recipients. All of the eleven rats surviving after stereotaxic implantation of islets into the cisterna magna returned to normoglycemia within 7 days after transplantation. Nine of the recipients with intra-cisterna magna (IM) islet allografts were still normoglycemic at 210 days after transplantation. The glucose disappearance rate of the IM transplant rats was slower than that of the IP transplant rats, and blood glucose returned to the normal basal level within 5 hr following glucose administration. Although the insulin levels were almost undetectable in cerebrospinal fluid before IM transplantation, the insulin levels were markedly increased after IM transplantation and twice as great in CSF than blood. Thus, these findings indicate that the cisterna magna can serve as an immunologically privileged site for implantation of allogeneic pancreatic islets, and islets in CSF can regulate and maintain normal glucose homeostasis via secretion of insulin across the blood-brain barrier.     

Long-term survival and function of intraportal porcine and human islet xenografts in nondiabetic nude mice

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study "glucose toxicity" and the role of IBMIR in the fate of intraportal islet grafts.     

异体大鼠骨髓单个核细胞胰岛细胞移植治疗糖尿病

目的 观察异体骨髓单个核细胞和胰岛细胞通过肝脏和静脉途径移植后对糖尿病大鼠的治疗作用.方法 密度梯度离心法分离胰岛细胞,淋巴细胞分离液分离骨髓单个核细胞,28只糖尿病大鼠模型随机分为A、B、C、D组,A组在肝脏被膜下多点注射1000个胰岛细胞,B组在体外将1000个胰岛细胞和1×107个骨髓单个核细胞混合后在肝脏被膜下多点注射,C组通过尾静脉注射1000个胰岛细胞,D组在体外将1000个胰岛细胞和1×107个骨髓单个核细胞混合后通过尾静脉注射,移植后于不同时间点尾静脉测定随机血糖,比较不同细胞组合和移植途径之间对糖尿病的治疗作用.结果 A、B组血糖于术后3 d内开始下降,A组血糖可降至正常水平(7.98±2.28)mmol/L,血糖维持正常水平(3.71±0.95)d,B组降至(7.72±1. 75)mmol/L可维持(4.86±1.06)d,静脉移植组血糖于术后4 d内降至正常(7.35±1.40)mmol/L,可维持(7.85±1.46)d,D组静脉注射胰岛于4 d起效(7.00±0.83)mmol/L,血糖可降至正常水平可维持(14.10±1.21)d,各组间血糖随时间变化的趋势及维持正常水平的时间具有统计学意义(P＜0.05).结论 骨髓单个核细胞和胰岛混合细胞通过尾静脉移植对大鼠血糖维持正常时间最长,血糖控制水平最理想.     

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.     

Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase injection

Pancreatic distension with collagenase solution followed by stationary in vitro digestion yields large numbers of intact islets. We compared in rats two routes of collagenase injection, pancreatic ductal (PD) and portal venous (PV), for islet yield, in vitro insulin secretory capacities, and in vivo functional viability. The islet yield in the PD method (n = 11) was greater than that in the PV method (n = 8) (682 +/- 27 vs. 417 +/- 39 per pancreas, P less than 0.025). The insulin release from the PD islets in response to 16.7 mM glucose increased gradually following culture, 3.2 +/- 0.8 ng/10 islets/30 min (fresh) to 12.3 +/- 2.1 (24-hr culture). In contrast, insulin release from the PV islets increased during the first 6 hr of culture, but decreased after 24 hr in culture. Under electronmicroscopic examination, the PD islets revealed a well preserved structure with healthy endocrine cells, while the PV islets showed a dilated capillary network and distorted endocrine cell continuity. When 100 PD islets were transplanted into streptozotocin-induced diabetic B6AF1 mice (n = 8), all the recipient mice restored normoglycemia (less than 200 mg/dl) within 1-4 days following transplantation and maintained it until rejection. However, the recipient mice given 100 PV islets showed a significant delay in restoring normoglycemia, and 3 of 8 mice given 100 PV islets were still hyperglycemic on day 4 postgrafting. In summary, pancreatic ductal collagenase injection followed by stationary in vitro digestion reproducibly yields higher numbers of intact and viable islets when compared with portal venous collagenase injection, indicating the superiority of this method to portal venous injection.