提高冻存胚胎胰岛移植效果的实验研究

目的探讨三维组织细胞旋转培养系统(RCCS)对胚胎胰岛冻存复苏后质量的影响。方法将胚胎胰岛平均分为3组,实验组1、2为胚胎胰岛冻存前后分别用RCCS培养和普通培养,对照组新鲜胰岛经RCCS培养。切取胚胎胰腺,胶原酶V消化,纯化。然后进行标准冻存步骤,复苏后继续培养。并检测各组胰岛数量、活性、胰岛素刺激实验结果。结果纯化后收获胰岛最多每个胚胎5012.73IEQ,最少2432.68IEQ,平均(3548.07±273.46)IEQ。微重力培养组胰岛细胞存活率、胰岛素释放量、胰岛素刺激指数等均高于普通培养组。移植经过微重力培养的(2000±1)%IEQ新鲜胚胎胰岛或冻存胚胎胰岛在移植后1周内可达100%纠正糖尿病。结论微重力旋转培养有利于胰岛细胞的生长繁殖,使胰岛具有更好的胰岛素分泌能力,该方法同胰岛冻存相结合,可以进一步提高胰岛的冻存效果,为胰岛库的成功建立探索出一条新的途径。     

高压氧微重力培养提高冻存胰岛质量和移植效果的实验研究

目的 探讨胰岛冻存前后经高压氧细胞旋转培养系统(HORCCS)培养后移植入糖尿病大鼠能否提高胰岛移植的效果.方法 将分离纯化的大鼠胰岛分为:A.体外实验组:将各组大鼠胰岛经HORCCS培养或普通培养30 d,检测细胞内DNA和胰岛素含量,胰岛存活率,胰岛素分泌水平.B.胰岛移植实验组:将各组大鼠胰岛经HORCCS培养或普通培养7d,然后移植,观察移植受体血糖和胰岛素水平.电镜观察各胰岛移植实验组中培养7 d时的胰岛的超微结构改变.结果 经高压氧 RCCS培养14 d的胰岛存活率及胰岛素分泌水平高于普通培养组(P<0.05).移植了经HORCCS培养的胰岛后,受体血糖在移植后2周即恢复为正常值,并维持到移植后10周.全部受体维持正常血糖耐受曲线.电镜下可见经HORCCS培养后,冻存复苏胰岛表面形成微小的孔道.结论 胰岛冻存前后经高压氧RCCS培养后可以建立营养输送管道,不但有利于氧和营养物质的运输,更有利于胰岛内细胞的均匀一致的冻存,从而减少冻存对胰岛的损害,提高胰岛的分泌活性和成活率.     

微重力培养提高冻存大鼠胰岛移植的效果

目的观察大鼠胰岛冻存后经三维微重力细胞旋转培养系统(RCCS)培养后移植给受者能否明显提高胰岛移植的效果。方法将分离纯化的大鼠胰岛分为3组。实验1组:即将胰岛在RCCS中培养3d后,悬浮于4℃低温保存液(HTS)中,放置30min,然后进行冻存步骤;冻存1个月后进行复苏,继续在RCCS中培养30d;实验2组:新鲜大鼠胰岛在RCCS中培养30d;对照组:大鼠胰岛在冻存前后均在普通培养基中培养,培养步骤和时间同实验1组。上述3组胰岛复苏后再按各组应用的培养体系培养7d,然后分别以2000IEQ冻存或新鲜胰岛移植入糖尿病大鼠体内,并观察10周。结果每个胰腺纯化后最多收获胰岛1058.47IEQ,最少411.88IEQ,平均(826.95±93.42)IEQ。实验1组、实验2组的胰岛收获率、细胞内胰岛素和DNA含量以及胰岛素刺激指数均高于对照组(P<0.05);实验2组和实验1组的2000IEQ新鲜胰岛或冻存胰岛在移植后1周内可100%纠正糖尿病。结论大鼠胰岛冻存前后经微重力培养后移植可以明显提高1型糖尿病大鼠的治疗效果。     

一步法联合冻存提高大鼠胰岛冻存质量的研究

目的探讨提高大鼠胰岛冻存质量的方法,以期为建立胰岛库奠定基础。方法将受体鼠分为实验组A组:即一步法联合冻存的胰岛移植组;对照组B组:新鲜的胰岛移植组;对照组C组:逐步法冻存的胰岛移植组。观察3组之间胰岛收获率,活性及移植后效果等方面的差异。结果纯化后收获大鼠胰岛每只(826.87±93.24)IEQ。A组平均胰岛收获率为(87±4)%,高于C组的胰岛收获率(81±4)%。A组和C组经过胰岛刺激素释放实验后均有较B组高的基础胰岛素释放量,但刺激指数则明显低于B组的637.3±39.5。胰岛移植入糖尿病鼠受体后,A组和B组在移植后1周即恢复血糖为正常值,并维持到观察结束。而C组中则需要较长时间纠正血糖到正常。移植(2011.14±114.22)IEQ胰岛在A组和B组可达到100%纠正糖尿病。结论采用全新的细胞内低温保存液(HTS)结合细胞冻存液(DMSO)对大鼠胰岛进行一步法冻存取得了明显优于用DMSO逐步冻存的效果。     

低温微重力培养对大鼠胰岛细胞质量影响的实验

目的:研究低温微重力对大鼠胰岛移植质量的影响，以期减少体外培养对胰岛活性和数量的影响，提高胰岛移植质量。方法:将分离纯化的大鼠胰岛分为3组：大鼠新鲜胰岛移植组（于移植前在普通培养基中培养21 d,对照组）；实验1组（大鼠新鲜胰岛在37 ℃ RCCS中培养21 d）；实验2组（新鲜大鼠胰岛在26 ℃ RCCS中培养14 d后复温至37 ℃继续在37 ℃ RCCS中培养7 d）。　观察各种培养液中的胰岛素含量。并将上述3个实验组不同条件培养的胰岛分别以2000IEQ胰岛移植量植入糖尿病大鼠体内，并观察10周。结果:实验2组的胰岛存活率、胰岛素分泌水平及胰岛素刺激指数均高于对照组和实验1组，差异有统计学意义(P＜0.05)。21 d时实验2组胰岛存活率高达(67.4±4.6)%，而对照组和实验1组胰岛存活率分别降至(28.1±3.3)%和(50.3±3.5)%，3组间差异有统计学意义(P＜0.05)。给糖尿病大鼠移植实验1，2组的胰岛后均能控制血糖至正常水平，但实验2组胰岛移植对糖尿病大鼠7 d内血糖控制优于实验1组；对照组血糖控制差，3组间两两比较均有统计学差异（均P＞0.05）。结论:大鼠胰岛经低温微重力培养后移植，可以明显提高1型糖尿病大鼠的治疗效果。     

微重力培养提高大鼠胰岛冻存质量的研究

本研究应用三维组织细胞旋转培养系统（the rotary cell culture system，RCCS）对冻存前后的大鼠胰岛细胞进行三维微重力组织培养，以期能提高胰岛复苏率，从而为人类胰岛的建库、进行胰岛移植奠定基础。     

提高胚胎胰岛冷冻保存质量的实验研究

目的：研究三维组织细胞旋转培养系统（RCCS）对胚胎胰岛冷冻保存复苏后质量的影响。方法：将胚胎胰岛平均分为实验组1、2和对照组,实验组1、2冷冻保存胰岛,冷冻保存前后分别用RCCS培养和普通培养,对照组经RCCS培养新鲜胰岛。切取胚胎胰腺,经胶原酶V消化、纯化,培养3d,然后进行标准冷冻保存,复苏后继续培养,并检测各组胰岛数量及活性。结果：纯化后每个胚胎收获胰岛最多5115.43IEQ,最少2331.98IEQ,平均（3551.27&#177;253.76）IEQ。实验组1胰岛细胞存活率、胰岛素释放量、胰岛素刺激指数均高于实验组2。结论：RCCS培养有利于胰岛细胞的生长繁殖,使其具有更好的分泌胰岛素能力,该方法同胰岛冷冻保存相结合,可以进一步提高胰岛的冷冻保存效果,为胰岛库的成功建立探索出一条新的途径。     

大鼠胎脑组织冻存与同种异体移植

目的 探讨冻存大鼠胎脑细胞体外培养的活性及其移植后的存活情况。方法 对经冷冻保存1－24周的WK大鼠胎脑细胞进行体外培养,观察其存活情况,并将保存24h后培养14d的胎脑细胞行脑内定位移植。结果 冻存1－24周的胎脑组织其活细胞率在90％以上,复苏后培养14d的胎离细胞活性最强;胎脑细胞移植后可在受者体内存活至少30d以上。结论 冷冻保存后培养的胎脑细胞级够存活,并可用于移植。     

长期冻存对微囊化肝细胞形态结构功能的影响及在急性肝功能衰竭中的治疗作用

目的　研究长期液氮冻存对微囊化肝细胞结构和功能的影响。方法　将３ 组 Ｓ Ｄ 大鼠微囊化肝细胞分别用液氮冻存１５ 、３０ 、６０ 天后,检测肝细胞活率、结构、功能及其对急性肝衰的初步治疗效果。结果　冻存后３ 组的细胞活率分别为（７２ ．７５ ±３ ．９） ％ 、（７０ ．０７ ±５ ．６８） ％ 、（７２ ．３９ ±１３ ．７５） ％ ;肝细胞的形态结构正常;肝细胞葡萄糖６磷酸酶、糖原及白蛋白染色正常,培养４ 天后各组的总蛋白和尿素氮的合成分泌功能与新鲜微囊化肝细胞差异无显著性（ Ｐ＜ ０ ．０１） 。冻存组（１５ 天组） 腹腔移植能明显提高急性肝功能衰竭模型大鼠的存活率５５ ％ （１１／２０） ,与裸细胞组（３３ ％ ,８／２４） 及空囊组（１６ ．６ ％ ,５／３０） 相比差异有显著性（ Ｐ＜ ０ ．０１） 。结论　液氮可良好的保存微囊化肝细胞的形态结构及功能;冻存微囊化肝细胞腹腔移植有良好的肝功能支持作用。     

丹参对大鼠胰岛获取和冻存及体外培养影响的实验研究

胰岛移植是治疗I型糖尿病较理想方法，移植成功的关键是胰岛足够数量和良好功能。细胞冻存是收集足够胰岛、建立胰岛库的可行手段。但冻存复苏过程可引起细胞的破坏和死亡，使细胞数量减少、活性及功能降低。本实验探讨丹参能否有益于胰岛获取及冻存前后的存活和功能，及其作用的可能机制。     

Fresh islets are more effective for islet transplantation than cultured islets

For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.     

Influence of a current style of culture on the quality of isolated pancreatic islets

OBJECTIVE: Comparable outcomes of islet transplantation with short periods of culture may be achieved with various culture media. To clarify the influence of a style of culture on isolated pancreatic islets, islet quality of fresh islets was compared with those cultured in several different fashions including not only for viability but also for inflammatory mediators. MATERIALS AND METHODS: Wistar rat islets were cultured for 48 hours with CMRL including 10% allogeneic serum; CMRL including 0.5% human serum albumin (HSA); and Miami medium including 0.5% HSA. The influence of culture conditions on islet integrity was evaluated by survival rate of islets during culture and visual scoring. The influence of culture conditions on islet function and viability was examined by ADP/ATP tests, insulin/DNA content, and glucose stimulation tests. RESULTS: Although the survival rates were similar for all groups, the visual scoring was lower in Miami medium. The stimulation index in glucose challenge tests was higher for fresh islets than the media (P = .02). Insulin/DNA ratios revealed the same tendency as glucose challenge tests (P = .0005). ADP/ATP ratio was lower in both the fresh and serum groups than in the others (P = .38), suggesting that apoptotic islets are relatively fewer in both fresh and serum groups. Most importantly, the expression of tissue factor (TF) on the islets was considerably lower in the fresh group, suggesting that a current style of culture could enhance TF-dependent instant blood-mediated inflammatory reactions after transplantation. CONCLUSION: In conclusion, Isolated islets without prior culture shows characteristics beneficial for transplantation using current modes of culture.     

Immunoisolation effect of polyvinyl alcohol (PVA) macroencapsulated islets in type 1 diabetes therapy

Islet transplantation has shown great success in the treatment of type 1 diabetes since the Edmonton protocol was established. However, it still has two major problems to overcome: the lack of organ donors and the side effects of immunosuppression. Encapsulated islets have emerged as a potential option for islet transplantation because it can, at least partly, overcome these two problems. Wistar rat islets suspended in 3% polyvinyl alcohol (PVA) hydrogel were frozen-thawed to make macroencapsulated islets (MEIs). The recovery rate, insulin content, and morphological change in culture medium with/without fresh human plasma (FHP) were measured in MEIs and free islets in vitro. In vivo, MEIs of either Wistar or Lewis rats were transplanted into the peritoneal cavity of streptozotocin (STZ)-induced diabetic Lewis rats and nonfasting blood glucose (NFBG), body weight, and histological evaluations were processed. FHP destroyed rat free islets but did not affect the islet morphology, islet recovery rate, or insulin content of rat MEIs. The transplantation of MEIs decreased the NFBG level and prevented body weight loss without a significant difference between the donor strains. Insulin-positive islets were observed in PVA MEIs 24 weeks after allotransplantation. These results suggest that PVA MEIs may be used as a cure for type 1 diabetes.     

Pretransplant islet culture: a comparison of four serum-free media using a murine model of islet transplantation

INTRODUCTION: Human islet transplant protocols frequently incorporate a brief period of islet culture before transplantation. The optimal medium for pretransplant islet culture is unknown. METHODS: We compared four serum-free media formulated for human islets: Miami (MM1), Memphis (M-SFM), Edmonton (EDM), and hCell OCZEM-SF/AF (hCell). Islets isolated from a single human pancreas with purity >80% were cultured in 2500-islet-equivalent (IE) fractions using the media listed. After 7 days, each 2500-IE fraction was grafted under the kidney capsule of a streptozocin-diabetic rag1 mouse (n = 4 per group). Mice were evaluated with serum glucose monitoring, stimulated C-peptide release, and glucose tolerance tests. Islet fractions transplanted immediately after isolation (n = 4 mice) served as controls. In vitro islet function was assessed on days 0 and 3 and included insulin release (after static glucose stimulation), total cellular C-peptide content, cell count, and viability. RESULTS: Glucose control was improved in all cohorts of mice after transplant, but only islet grafts cultured in MM1 were statistically indistinguishable from fresh islets. MM1- and hCell-cultured islet grafts showed improved glucose tolerance compared with fresh islets; C-peptide release was similar among the four cohorts. In vitro, only islets cultured in MM1 had similar stimulation index to fresh islets, whereas only hCell- and MM1-cultured islets demonstrated recovery of C-peptide content and insulin release. CONCLUSIONS: Media choice before transplant can influence islet quality, even when culture periods are short. Miami MM1 and hCell media may provide better islet protection than alternative media.     

Clinical use of donation after circulatory death pancreas for islet transplantation

Due to a shortage of donation after brain death (DBD) organs, donation after circulatory death (DCD) is increasingly performed. In the field of islet transplantation, there is uncertainty regarding the suitability of DCD pancreas in terms of islet yield and function after islet isolation. The aim of this study was to investigate the potential use of DCD pancreas for islet transplantation. Islet isolation procedures from 126 category 3 DCD and 258 DBD pancreas were performed in a 9-year period. Islet yield after isolation was significantly lower for DCD compared to DBD pancreas (395 515 islet equivalents [IEQ] and 480 017 IEQ, respectively; p = .003). The decrease in IEQ during 2 days of culture was not different between the two groups. Warm ischemia time was not related to DCD islet yield. In vitro insulin secretion after a glucose challenge was similar between DCD and DBD islets. After islet transplantation, DCD islet graft recipients had similar graft function (AUC C-peptide) during mixed meal tolerance tests and Igls score compared to DBD graft recipients. In conclusion, DCD islets can be considered for clinical islet transplantation.     

新生猪Sertoli细胞与大鼠胰岛细胞联合移植延长大鼠移植胰岛的存活时间

目的 探讨新生猪Sertoli细胞(NPSCs)与大鼠胰岛细胞联合移植对延长大鼠同种异体胰岛移植物存活时间的作用及其主要的机制.方法 将糖尿病Wistar大鼠随机分为3组.(1)单纯移植组(n=6):单纯移植1500个胰岛当量(IEQ)的SD大鼠胰岛细胞至糖尿病大鼠的左肾包膜下;(2)分侧移植组(n=4):将1500个IEQ的SD大鼠胰岛细胞移植到糖尿病大鼠的左肾包膜下,同时将1×10~7个NPSCs移植到糖尿病大鼠的右肾包膜下;(3)混合移植组(n=6):将1500个IEQ的SD大鼠胰岛细胞和1×10~7个NPSCs混合移植到糖尿病大鼠的左肾包膜下.移植后每天监测各组的血糖水平,以了解移植物的存活时间;移植后发生排斥反应时获取移植物标本,行HE和免疫组织化学染色,观察移植物中CD3~+T淋巴细胞浸润情况及细胞凋亡调控基因(Bcl-2)和血红素氧合酶1(HO-1)基因的表达水平.结果 单纯移植组、分侧移植组和混合移植组胰岛移植物存活时间分别为(5.7±1.0)d、(5.3±0.5)d和(16.3±1.4)d,混合移植组存活时间较其他两组显著延长(P<0.05).单纯移植组移植区可见大量淋巴细胞浸润,主要为CD3+T淋巴细胞;混合移植组移植区Bcl-2基因呈高表达;各组移植区HO-1基因均有表达,差异不明显.结论 NPSCs与大鼠胰岛细胞联合移植可以延长大鼠同种异体胰岛移植物的存活时间,其机制可能与NPSCs抑制移植物淋巴细胞浸润、促进Bcl-2基因高表达的局部免疫调节作用有关.     

Superiority of Fresh Islets Compared With Cultured Islets

Introduction

It has recently been reported that the outcomes of islet transplantation with short periods of culture are comparable with those of freshly isolated islets. To clarify the influence of culture, fresh islets were compared with cultured islets in terms of quality.

Materials and Methods

The quality of freshly isolated islets was compared with that of cultured islets with CMRL 1066 including 10% allogeneic serum, CMRL 1066 including 0.5% human serum albumin, or Miami medium. We evaluated static glucose stimulation tests, insulin/DNA contents, ADP/ATP ratios, and an intraportal transplantation model into syngeneic diabetic rats. The expression of inflammatory mediators in the islets was examined using Western blotting for tissue factor (TF), which is the initiator of detrimental instant, blood-mediated, inflammatory reactions (IBMIR).

Results

Although the survival rate was similar in all groups, the stimulation index upon glucose challenge and the insulin/DNA ratio were significantly higher among fresh islets. Most importantly, the expression of TF on islets was significantly lower in fresh islets, suggesting that culture enhanced TF-dependent IBMIR after transplantation. In an in vivo transplantation model, the curative rate and insulin production by the recipient liver was considerably greater in the fresh islet group.

Conclusions

Isolated islets without prior culture showed results superior to cultured islets.     

肾移植联合成人胰岛细胞移植治疗糖尿病肾病七例报告

目的 建立新型成人胰岛细胞分离纯化方法和无激素免疫抑制方案.观察.肾移植联合胰岛细胞移植治疗1型糖尿病肾病的安全性与有效性.方法 全氟化碳液与威斯康星大学器官保存液双层冷藏胰腺,Liberase酶消化,COBE 2991型专用胰岛细胞分离机分离及连续密度梯度纯化,获取高纯度与高活性的胰岛细胞.常规方法行尸体肾移植,次El采用外科方法将短期培养的胰岛细胞经门静脉移植到肝脏内,采用无激素免疫抑制治疗.术后定期监测血糖与胰岛素用量、C肽与糖化血红蛋白水平以及肝肾功能.结果 23个胰腺均成功分离胰岛细胞,平均数量30万胰岛当量(IEQ)、纯度92%、活率95%、刺激指数3.16,病原学结果均阴性.7例1型糖尿病肾病患者共行胰岛细胞移植12次(移植1次3例、2次3例、3次1例).每次移植胰岛数量平均为11 820 IEQ/kg.采用阿来佐单抗诱导、西罗莫司和小剂量他克莫司、无激素免疫抑制治疗.随访1.5～3.0年,4例完全撤除胰岛素,3例胰岛素用量较术前减少＞70%.术后血糖稳定维持在正常水平,C肽均＞0.166nmol/L,糖化血红蛋白正常,肝肾功能正常.结论 新型成人胰岛细胞分离纯化方法可靠,胰岛细胞联合肾移植治疗1型糖尿病肾病安全、有效.