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。     

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。     

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。     

胰岛移植早期血糖控制对大鼠胰岛移植效果的影响

目的　探讨胰岛移植早期血糖控制对大鼠胰岛移植效果的影响。方法　体外实验 :将新鲜制备的SD大鼠胰岛在 4种不同糖浓度中培养 5d后恢复至常规糖浓度中培养 3d ,观察其形态、存活时间及功能变化。胰岛移植实验 :将糖尿病SD大鼠随机分为 3组 :移植及胰岛素控血糖组 ;单纯移植组和未移植 (对照 )组。监测各组的血糖水平。结果　在体外 ,短期的高糖培养对SD大鼠胰岛的形态、存活时间无明显影响 ,但可降低其对高糖刺激的反应能力。在糖浓度恢复正常后 ,该功能可恢复。移植实验中 ,血糖控制组在移植后 14d时 ,受体鼠血糖 ( 2 7.0± 4.0 )mmol/L与对照组 ( 3 3 .1± 1.8)mmol/L间差异有显著性 (P <0 .0 5 ) ,而单纯移植组与对照组的血糖差异仅维持到移植后 8d。结论　移植早期的血糖控制有改善胰岛移植效果的作用。     

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.     

一种提高犬胰岛体外培养质量的方法

目的研究提高犬胰岛体外培养质量的方法。方法将犬胰岛分别进行普通培养 (A组)、常温(37℃)微重力培养(B组)和低温(26℃)微重力培养(C组),分别培养3、7、14和21 d, 并检测各组胰岛培养后收获率、存活率和功能。结果不同方法培养21 d后,B组和C组平均胰岛收获率为(66．2±4．1)％和(81．3±4．8)％高于A组的胰岛收获率(50．5±2．9)％,P<0．05。A组胰岛存活率降至(27．7±3．5)％,与B组的(49．7±3．2)％和C组的(64．8±4．7)％比较,P<0．05。 B组和C组DNA含量及胰岛素含量均维持较高的水平,B组和C组胰岛素储备能力较A组高,胰岛索刺激指数分别为(3．30±0．70)、(5．48±1．00)和(2．33±0．50),差异有统计学意义(P< 0．05)。结论低温微重力三维培养胰岛是提高胰岛培养质量的好方法。     

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。     

大鼠胰岛细胞转染Wee1Hu基因延长异种胰岛细胞移植后存活时间

目的 探讨大鼠胰岛细胞转染Wee1Hu基因对异种胰岛细胞移植后存活时间的影响。方法 以Wistar大鼠为供者，Balb／c糖尿病小鼠为受者，进行异种胰岛细胞移植。实验分为2组，每组20只。实验组：将转染Wee1Hu基因的1×10^6个供者胰岛细胞悬于0．5ml无菌生理盐水中，注射至受者的腹腔；空白对照组：将1×10^6个空载体胰岛细胞用与实验组相同的方法注射至受者腹腔。实验组和空白对照组的部分受者在移植前1周腹腔注射降植烷0．5ml，并于胰岛细胞移植后开始口服环孢素A30mg／kg，直至实验结束。监测2组胰岛细胞移植后的胰岛素释放功能及存活时间。结果 胰岛细胞移植后，空白对照组的受者均维持高血糖，且体重持续下降，平均生存时间为（18±2．5）d；实验组的受者血糖在2d内均降至正常，且体重增加，生存时间延长（38±3．5）d；两组相比较，差异有统计学意义（P〈0．05）。空白对照组维持正常血糖时间为（8±2．3）d，而实验组为（10±2．5）d。实验组中采用免疫抑制剂治疗的受者，长期维持正常血糖，平均维持时间超过30d，与空白对照组中采用免疫抑制剂治疗的受者比较，差异有统计学意义（P〈0．05）。结论 大鼠胰岛细胞转染Wee1Hu基因可延长异种胰岛细胞移植后的存活时间，与免疫抑制剂协同作用时效果更佳。     

血红素氧合酶-1基因转染对大鼠胰岛培养的影响

目的 探讨血红素氧合酶-1基因（HO-1）转染对培养的大鼠胰岛功能的影响。方法 用携带人HO-1基因和增强型绿色荧光蛋白基因（EGFP）的腺病毒载体转染大鼠胰岛，转染后48h，以EGFP及人HO-1蛋白的表达来确定转染结果，放免法测定各组胰岛在低糖（2．8mmol／L）、高糖（16．7mmol／L）刺激下胰岛素释放量，并计算刺激指数（SI）。结果 大鼠胰岛培养7d后，其低糖、高糖刺激下胰岛素释放量明显低于新鲜胰岛[（4．57±0．40比（6．47±0．55）mIU／L／30IEQ，（9．63±0．71）比（14．93±1．17）mIU／L／30IEQ，P〈0．05]；培养7d的各组胰岛在低糖刺激时胰岛素释放量差异无统计学意义（P〉0．05），但HO-1组胰岛在高糖刺激时胰岛素释放量明显高于EG．FP组和对照组[（12．50±2．17）mIU／L／30IEQ，（8．87±0．65）mIU／L／30IEQ，（9．63±0．71）mlU／L／30IEQ，P〈0．05]；HO-1组SI也高于EGFP组和对照组[（2．21±0．02），（2．08±0．05），（2．11±0．03），P〈0．05]。结论 HO-1基因转染能对体外培养的大鼠胰岛功能起到保护作用。     

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。     

Effect of Cryopreservation on the Survival and Function of Murine Islet Isografts and Allografts

We compared the efficacy of fresh and frozen/thawed islets by determining the minimum number required to consistently reverse diabetes in mice. Defined numbers of islets, isolated from Balb/c (H-2^d) and CBA/J (H-2^k) mice, were transplanted into streptozotocin-induced diabetic Balb/c mice. Frozen/thawed grafts were cooled slowly to −40°C, stored at −196°C, and thawed rapidly. At 100 days after transplantation, isografts were recovered for measurement of insulin content. Mean (±SD) recovery of cryopreserved islets after thawing was 80 ± 3% (range 67–89%). For both fresh and frozen/thawed isografts and allografts, 200 islets were required to establish normoglycemia. The degree of metabolic function provided by equivalent quantities of fresh and frozen/thawed grafts was similar; and all normoglycemic isograft recipients remained so until graft nephrectomy. The insulin content of fresh and frozen/thawed isografts containing 200 and 300 islets were 151 ± 25 and 126 ± 8 mU and 259 ±36 and 278 ± 20 mU, respectively. Among allograft recipients, median survival ranged from 15 to 20 days, and was not influenced by cryopreservation or graft size. The results of this study demonstrate a high rate of recovery of viable islets following cryopreservation. The function of equivalent quantities of fresh and cryopreserved islet isografts and allografts in nonimmunosuppressed recipients is similar.     

Transplantation of isolated islets of Langerhans in diabetic dogs. I. Results after allogeneic intraportal islet transplantation.

Twenty partially inbred German shepherd dogs were made diabetic by intravenous administration of streptozotocin (25 mg/kg) 1 and 3 days after partial pancreatectomy. Ten diabetic dogs received intraportal transplants of allogeneic islets of Langerhans isolated by collagenase digestion and Ficon gradient separation. After transplantation the mean fasting serum glucose level fell from 368 ± 74 to 108 ± 52 mg/dl. Normal glycemia was maintained for at least 3 weeks before rejection occurred. The mean survival time of the 10 recipients was 76 ± 30 days, while the 10 diabetic control dogs had a mean survival time of 30 ± 7 days. Intravenous glucose tolerance test curves were clearly better in recipient dogs than in the diabetic control dogs for at least 4 weeks after islet transplantation. Concentrations of immunoreactive insulin (IRI) in the portal vein in the superior vena cava were also significantly higher than in the diabetic control dogs 4 weeks after islet transplantation. The mean peak concentration of IRI in the superior vena cava of transplanted dogs after glucose administration was higher than that in the portal vein, indicating that the transplanted islets secreted insulin in response to glucose stimulation. Portal hypertension or disturbance of liver function did not occur after transplantation of isolated islets. These results show that the intrahepatic site is appropriate for transplantation of isolated islets in a large animal model of diabetes, and provide a basis for future application in man.     

Transplantation of isolated islets of Langerhans in diabetic dogs. II. The influence of histocompatibility.

By means of collagenase digestion and the Ficoll gradient separation technique, viable islets of Langerhans could be isolated from dog pancreata. The isolated islets were capable of secreting insulin after glucose stimulation. These islets of Langerhans were transplanted into the livers of diabetic dogs. We transplanted islets isolated from I donor pancreas for 1 kg body wt of the recipients. The dogs were made diabetic by subtotal pancreatectomy (80%) and two injections of 25 mg streptozotocin/kg body wt. In our experiments, we selected donor-recipient pairs by means of the mixed lymphocyte reaction macrophage electrophoretic migration inhibition (MLR-EM) test. In the group with a depression in the MLR-EM test of more than 15% (strong histoincompatibility), the mean survival time of the transplanted dogs was 46 ± 16 days. Another group of recipients with a depression of macrophage migration of less than 8% (weak histocompatibility) had a mean survival time of 135 ± 49 days after intraportal islet transplantation, and the blood glucose values could be normalized in this group for a period of 70 days. We found significant differences between both the groups in the glucose tolerance tests and in the IRI concentrations in the portal vein and in the vena cava superior. In the recipients, which received weakly histoincompatible islets, the IRI concentration in the superior vena cava was 64% of the normal value in the vena cava superior 4 weeks after intrahepatic islet transplantation. By means of the MLR-EM test, we were able to correlate inhibition with signs of rejection of the transplanted islets. Our experiments demonstrated that an allogeneic islet transplantation in diabetic dogs across a weakly histocompatible barrier can improve the results in comparison to the islet transplantation across a strongly histocompatible barrier. However, this typing maneuver alone is not able to prevent the islet rejection.     

Prolonged effect of troglitazone (CS-045) on xenograft survival of hybrid artificial pancreas

Troglitazone (CS-045), a thiazolidinedione derivative, is a new oral antidiabetic agent that enhances insulin sensitivity and improves insulin responsiveness. In this study we examined the effects of CS-045 on the survival of xenografted bioartificial pancreas. Isolated rat islets were microencapsulated with three-layer agarose microcapsules (polybrene, carboxymethyl cellulose, and an agarose-polystyrene sulfonic acid mixture). Diabetes was induced by intraperitoneal injection of streptozotocin 220 mg/kg. Recipient diabetic mice were separated into two groups. In the CS-045 treated group, the recipient mice were given feed mixed with CS-045 (0.2% w/w) starting from 1 wk before transplantation up to graft failure. The mice in the control group had feed without CS-045. Three hundred microencapsulated rat islets were xenotransplanted into the intraperitoneal cavity of each recipient mouse in both groups. One month after xenotransplantation, IVGTT was performed for all recipients. Xenotransplantation of 300 rat islets in microcapsules decreased the nonfasting blood glucose levels of both groups within 2 days. In the CS-045-treated group (n = 3), the normoglycemic period lasted for more than 1 mo without administration of immunosuppressive drugs (45 ± 4.3 days). However, in the control group (n = 4), the blood glucose levels of all recipients were already elevated on day 4. In the IVGTT study, the glucose assimilation was markedly and significantly better in the CS-045-treated group than in the control group (K = 1.7 ± 0.1 vs. 0.7 ± 0.28 respectively, p < 0.01). This study demonstrates that a newly developed oral antidiabetic agent, CS-045 could favorably ameliorate the diabetic state of the recipients xenotransplanted with the bioartificial pancreas, leading to an improved glucose tolerance and longer xenograft survival.     

Survival and function of syngeneic rat islet grafts placed within the thymus versus under the kidney capsule

The role of the thymus in the ongoing acquisition of tolerance to self antigens has made it an attractive site for islet transplantation. Several studies have reported survival of rodent islet allografts in the thymus without requiring the long-term use of immunosuppressive agents; however, the degree of glucose homeostasis in the intrathymic islet transplant recipients has not been examined. We transplanted 500, 1000, or 2000 syngeneic islets into the thymus of streptozotocin-induced diabetic Wistar-Furth rats, and compared the metabolic response of these recipients with animals receiving 2000 syngeneic islets under the kidney capsule. Three of four recipients which received 2000 islets under the kidney capsule achieved normoglycemia (≤8.4 mmol/L) within 1 wk and all animals became normoglycemic within 2 wk posttransplantation. In contrast, intrathymic implantation of 2000 islets induced normoglycemia in only one of six recipients during the same time interval, and when this number was reduced to 1000 or 500 islets, none of the recipients (n = 6) normalized within 1 wk posttransplantation. Animals that received an intrathymic transplant were glucose intolerant compared to normal controls and animals with subcapsular islet transplant. Removal of the graft-bearing organs resulted in hyperglycemia in all cases, and examination of the grafts revealed the presence of numerous well-granulated insulincontaining cells in both sites. The cellular insulin content of the subcapsular grafts (67.4 ± 12.1 μg; n = 4) was significantly higher (p ≤0.05) than what was extracted from intrathymic grafts (9.5 ± 1.2 μg from 1000 islets; n = 3 and 20.0 ± 4.6 μg from 2000 islets; n = 3). We conclude that 2000 syngeneic islets implanted either in the thymus or beneath the kidney capsule can normalize hyperglycemia in streptozotocin-diabetic rats; however, normal glucose tolerance was not established in intrathymic islet recipients, suggesting that a higher number of islets may be necessary to achieve normal glucose homeostasis.     

Rejection of islets versus immediately vascularized pancreatic allografts: a quantitative comparison

It has been suggested that free grafts of islets are rejected more vigorously than immediately vascularized intact organs grafts. However, the physiological manifestations of rejection depend, in part, upon the functional reserve of the transplanted tissue. If the number of islets transplanted is just adequate to maintain normoglycemia, the immune destruction of only a few islets will be manifested by hyperglycemia. Thus, differences in rejection time could be an artifact of the islet mass transplanted. We compared the onset of rejection of immediately vascularized segmental pancreatic grafts and of free grafts of islets under conditions in which the β cell mass transplanted, as determined by tissue insulin content, was equivalent. Lewis rats, made diabetic (plasma glucose > 400 mg/dl) by streptozotocin, received either free islet allografts by portal embolization or vascularized segmental pancreatic allografts derived from Fischer donors. Identical pancreatic segments that were not transplanted had a mean (± SE) total tissue insulin content of 33 ± 3 μg. The mean total insulin content of Fischer islets prepared by collagenase digestion in a quantity identical to that used for transplantation to single recipients was 35 ± 7 μg. Similar measurements were made in Fischer to Fischer and Lewis to Lewis isograft control groups. Recipients of both segmental pancreas and free islet grafts became normoglycemic after transplantation and this state was sustained indefinitely in recipients of syngeneic grafts. In rats receiving allografts, the day of rejection, defined as an elevation of plasma glucose to >200 mg/dl, occurred at a mean of 12.1 ± 0.3 days for recipients of pancreatic grafts (n = 17) and 5.2 ± 0.3 days in recipients of islet grafts (n = 17) (P < 0.001). The functional survival of free grafts of allogeneic islets is less than that of islets contained within immediately vascularized pancreatic grafts, even when the transplanted β cell mass is equivalent. However, this difference could still be due to nonimmunologic, quantitative factors that influenced the rate with which hyperglycemia occurred after initiation of the rejection process. The insulin content in the livers of islet isograft recipients showed that only 53 to 71% of the transplanted islets survived. Further experiments that compensate for this factor are needed to determine whether or not there are differences in susceptibility to rejection of the two types of grafts. Nevertheless, on the basis of the number of donors used per recipient, islet allotransplantation is less efficient than pancreas allotransplantation.     

Results of our first nine intraportal islet allografts in type 1, insulin-dependent diabetic patients

With the first demonstration of insulin independence following intraportal islet transplantation into a patient with type 1 diabetes, a new era of clinical islet transplantation will begin. This report provides our initial experience of clinical islet transplantation with a total of nine consecutive portal vein islet transplants in seven diabetic recipients. The first three transplants were done in nonrenal failure diabetics (NRFI) using 6319 +/- 2173 islets/kg body weight with islets processed from single pancreas and cultured for 7 days at 24 degrees C. Prednisone, azathioprine, and cyclosporine were initiated prior to transplant. While all three recipients demonstrated C-peptide function posttransplant, all three rejected their grafts at 2 weeks. Five days of OKT3 treatment failed to recover more than 10% of their rejecting islet grafts. The studies were then shifted to established kidney transplant recipients (EKI) maintaining their basal immunosuppression while adding 7 days of Minnesota antilymphoblast globulin (MALG) to the recipient using islets from single donor pancreas that had been cultured for 7 days at 24 degrees C. There were an average of 6161 +/- 911 islets transplanted intraportally into three EKI recipients. All three had C-peptide response from the transplant, but none achieved insulin independence. While the first patient rejected his graft at 2 weeks, two recipients demonstrated long-term islet function up to 10 months posttransplant. Sustacal challenge testing demonstrated C-peptide responsiveness, but in a delayed pattern suggesting insufficient islet mass had been transplanted. The next three kidney transplant recipients received islets from more than one donor pancreas averaging 13,916 +/- 556 islets/kg body weight. The first of these was the first to achieve insulin independence from 10 to day 25 posttransplant when she appeared to have a rejection episode. The second and third recipients were retransplanted with islets from multiple donors having achieved partial islet function from single pancreas donor. The first patient on triple immunosuppression is demonstrating long-term partial function at 184 days but is not insulin independent. The third patient on prednisone and azathioprine received one half his islets after 7-day culture and the other half after 7-day culture combined with cryopreservation. He is continuing to demonstrate insulin independence for 154 days post-transplant with a glycated hemoglobin value of 5.6%. Sustacal challenge data demonstrate a total stimulated C-peptide response of 155 rhomol/ml at 4 months post-transplant compared with 148 +/- 12 rhomol/ml for normal controls (NC) and 425 rhomol/ml for nondiabetic, established kidney transplant recipients on triple immunosuppression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of glucagon-like peptide-1 gene expression on graft function in mouse islet transplantation

This study investigated the effect of local glucagon‐like peptide‐1 (GLP‐1) production within mouse islets on cytoprotection in vitro and in vivo by gene transfer of GLP‐1. Transduction of recombinant adenovirus vector expressing GLP‐1 (rAd‐GLP‐1) induced a significant increase in bioactive GLP‐1 in the mouse islet culture, whereas transduction with adenovirus vector expressing β‐galactosidase (rAd‐LacZ), as a control, had no effect on GLP‐1 secretion. Islets transduced with rAd‐GLP‐1 were protected from H₂O₂‐induced cell damage in vitro. In addition, glucose‐stimulated insulin secretion was significantly increased in rAd‐GLP‐1‐transduced islets. When transplanted under the kidney capsule of diabetic syngeneic mice, islet grafts retrieved 4 or 7 days after transplantation revealed that the rAd‐GLP‐1‐transduced group had significantly more Ki67‐positive cells as compared with the rAd‐LacZ‐transduced group. Regarding blood glucose control, diabetic mice transplanted with a marginal mass of rAd‐GLP‐1‐transduced islets became normoglycemic more rapidly and 78% of the recipients were normoglycemic at 35 days post‐transplant, whereas only 48% of the mice transplanted with rAd‐LacZ‐transduced islets were normoglycemic (P < 0.05). In conclusion, delivery of the GLP‐1 gene to islets enhanced islet cell survival during the early post‐transplant period, and preserved islet mass and functions over time in the transplants.     

Beneficial effects of hyperbaric oxygen therapy on islet transplantation

We envisage that hyperbaric oxygen (HBO) would ameliorate islet anoxia, preventing early graft failure. Thus, treatment of HBO to diabetic recipients should improve the outcome of islet transplantation. We tested this hypothesis by syngeneically transplanting insufficient number of islets (150 islets) into streptozotocin-diabetic C57BL/6 mice, each followed by HBO (2.4 ATA, 100% O2) therapy for 1.5 h from day 0 to 28, once daily (group A) or twice daily (group B), or from day 5 to 28, once daily (group C) or twice daily (group D), 6 days/week. Recipients without HBO treatment served as controls. At day 28 after transplantation, groups B, C, and D gained weight and had lower blood glucose compared with their baseline values. In addition, groups B and D had higher insulin content of the graft than the controls. To determine the optimal timing of HBO therapy, groups B and D were compared with recipients treated with HBO twice daily, 6 days/week, from day -14 to 0 (group E) and from day -14 to 28 (group F). At day 28 after transplantation, groups B, D, E, and F had significantly lower blood glucose than their individual baseline values and higher insulin content of the graft than controls. But only group F had more beta-cell mass of the graft than controls. These findings lend credence to the expectation that peritransplant application of adequate frequency of HBO to diabetic recipients would enhance the performance and growth of the islet graft, resulting in an improvement of the outcome of the transplantation.