The role of donor hepatocytes and/or splenocytes pre-injection in reducing islet xenotransplantation rejection

OBJECTIVE: To observe the effect of tail vein injection with donor hepatocyte and/or splenocyte on islets xenotransplantation rejection. METHODS: New-born male pigs and BALB/C mice were selected as donors and recipients respectively. Islets xenotransplantation was performed in recipients just after the third time of tail vein injection with donor hepatocytes and/or splenocytes. Macrophage phagocytosis, NK killing activity, T lymphocyte transforming function of spleen cells, antibody forming function of B lymphocytes, and T lymphocyte subsets were taken to monitor transplantation rejection. The effects of this kind of transplantation were indicated as variation of blood glucose and survival days of recipients. RESULTS: Streptozotocin (STZ) succeeded in inducing diabetes mellitus models of mice. Pre-injection of donor hepatocytes, and splenocytes or their mixture via tail vein was effective in preventing donor islets transplantation from rejection, which was demonstrated by the mentioned immunological marks. And each group of transplantation could decrease the blood glucose of recipients and prolong the survival days. Pre-injection of mixture of donor hepatocytes and splenocytes was more effective in preventing rejection than pre-injection of donor hepatocytes or splenocytes separately. CONCLUSION: We propose that pre-injection of donor hepatocytes, splenocytes separately or their mixture before donor islets transplantation is a good way to prevent rejection.

Intracerebral xenotransplantation of semipermeable membrane- encapsuled pancreatic islets

AIM： To identify the decreasing effect of xenotransplantion in combination with privileged sites on rejection and death of biological semipermeable membrane-（BSM） encapsulated implanted islets. METHODS： After the BSM experiment in vitro, BSM- encapsulated SD rat＇s islet-like cell clusters （ICCs） were xenotransplanted into normal dog＇s brain. Morphological changes were observed under light and transmission electron microscope. The islets and apoptosis of implanted B cells were identified by insulin-TONEL double staining. RESULTS： The BSM used in our study had a favorable permeability, some degree of rigidity, lighter foreign body reaction and toxicity. The grafts consisted of epithelioid cells and loose connective tissue. Severe infiltration of inflammatory cells was not observed. The implanted ICCs were identified 2 mo later and showed typical apoptosis. CONCLUSION： BSM xenotransplantation in combination with the privileged site can inhibit the rejection of implanted heterogeneous ICCs, and death of implanted heterogeneous B cells is associated with apoptosis. 2005 The WJG Press and Elsevier Inc. All rights reserved.

A humanized mouse model to study human immune response in xenotransplantation

BACKGROUND: A major barrier to the clinical application of xenotransplantation as a treatment option for patients is T cell-mediated rejection. Studies based on experimental rodent models of xenograft tolerance or rejection in vivo have provided useful information about the role of T cell immune response in xenotransplantation. However not all observations seen in rodents faithfully recapitulate the human situation This study aimed to establish a humanized mouse model of xenotransplantation, which mimics xenograft rejection in the context of the human immune system. METHODS: NOD-SCID IL2rγ -/- mice were transplanted with neonatal porcine islet cell clusters (NICC) followed by reconstitution of human peripheral blood mononuclear cells (PBMC). Human leukocyte engraftment and islet xenograft rejection were confirmed by flow cytometric and histological analyses. RESULTS: In the absence of human PBMC, porcine NICC transplanted into NOD-SCID IL2rγ -/- mice revealed excellent graft integrity and endocrine function. Human PBMC demonstrated a high level of engraftment in NOD-SCID IL2rγ -/- mice. Reconstitution of NICC recipient NOD-SCID IL2rγ -/- mice with human PBMC led to the rapid destruction of NICC xenografts in a PBMC number-dependent manner. CONCLUSIONS: Human PBMC-reconstituted NOD-SCID IL2rγ -/- mice provide an ideal model to study human immune responses in xenotransplantation. Studies based on this humanized mouse model will provide insight for improving the outcomes of clinical xenotransplantation.

Use of genetically-engineered pig donors in islet transplantation

Type 1 diabetes(T1D) is an autoimmune disease wherein the pancreas does not produce enough insulin due to islet beta cell destruction. Despite improvements in delivering exogenous insulin to T1 D patients, pancreas or islet transplantation remains the best way to regulate their glycaemia. Results from experimental islet transplantation have improved dramatically in the last 15 years, to the point where it can be comparable to pancreas transplantation, but without the accompanying morbidity associated with this procedure. As with other transplants, the limiting factor in islet allotransplantation is the relatively small number of organs made available by deceased human donors throughout the world. A strong case can be made for islet xenotransplantation to fill the gap between supply and demand; however, transplantation across species presents challenges that are unique to that setting. In the search for the most suitable animal for human xenotransplantation, the pig has many advantages that make it the likely animal of choice. Potentially one of the most beneficial advantages is the ability to genetically engineer porcine donors to be more compatible with human recipients. Several genetic manipulations have already proven useful in relation to hyperacute rejection and inflammation(instant blood mediated inflammatory reaction), with the potential of even further advancement in the near future.

Percutaneous transhepatic portal catheterization guided by ultrasound technology for islet transplantation in rhesus monkey

BACKGROUND:Pig islet xenotransplantation has the potential to overcome the shortage of donated human islets for islet cell transplantation in type 1 diabetes.Testing in nonhuman primate models is necessary before clinical application in humans.Intraportal islet transplantation in monkeys is usually performed by surgical infusion during laparotomy or laparoscopy.In this paper,we describe a new method of percutaneous transhepatic portal catheterization(PTPC) as an alternative to current methods of islet transplantation in rhesus monkeys.METHODS:We performed ultrasound-guided PTPC in five adult rhesus monkeys weighing 7-8 kg,with portal vein catheterization confirmed by digital subtraction angiography.We monitored for complications in the thoracic and abdominal cavity.To evaluate the safety of ultrasound-guided PTPC,we recorded the changes in portal pressure throughout the microbead transplantation procedure.RESULTS:Ultrasound-guided PTPC and infusion of 16 000 microbeads/kg body weight into the portal vein was successful in all five monkeys.Differences in the hepatobiliary anatomy of rhesus monkeys compared to humans led to a higher initial complication rate.The first monkey died of abdominal hemorrhage 10 hours post-transplantation.The second suffered from a mild pneumothorax but recovered fully after taking only conservative measures.After gaining experience with the first two monkeys,we decreased both the hepatic puncture time and the number of puncture attempts required,with the remaining three monkeys experiencing no complications.Portal pressures initially increased proportional to the number of transplanted microbeads but returned to preinfusion levels at 30 minutes post-transplantation.The changes in portal pressures occurring during the procedure were not significantly different.CONCLUSIONS:Ultrasound-guided PTPC is an effective,convenient,and minimally invasive method suitable for use in non-human primate models of islet cell transplantation provided that care is taken with hepatic puncture.Its advantages must be weighed against the risks of procedure-related complications.

Pig islets for islet xenotransplantation： current status and future perspectives

Objective To review the current status and progress on pig islet xenotransplantation.Data sources Data used in this review were mainly from English literature of Pubmed database.The search terms were ＂pig islet＂ and ＂xenotransplantation＂.Study selection The original articles and critical reviews selected were relevant to this review＇s theme.Results Pigs are suggested to be an ideal candidate for obtaining available islet cells for transplantation.However,the potential clinical application of pig islet is still facing challenges including inadequate yield of high-quality functional islets and xenorejection of the transplants.The former can be overcome mainly by selection of a suitable pathogen-free source herd and the development of isolation and purification technology.While the feasibility of successful preclinical pig islet xenotranplantation provides insights in the possible mechanisms of xenogeneic immune recognition and rejection to overwhelm the latter.In addition,the achievement of long-term insulin independence in diabetic models by means of distinct islet products and novel immunotherapeutic strategies is promising.Conclusions Pig islet xenotransplantation is one of the prospective treatments to bridge the gap between the needs of transplantation in patients with diabetes and available islet cells.Nonetheless,further studies and efforts are needed to translate obtained findings into tangible applications.

Optimal pig donor selection in islet xenotransplantation: current status and future perspectives

Islet transplantation is an attractive treatment of type 1 diabetes mellitus. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited supply of islet grafts. Published studies demonstrated that pig islets could function in diabetic primates for a long time （〉6 months）. However, pig-islet xenotransplantation must overcome the selection of an optimal pig donor to obtain an adequate supply of islets with high-quality, to reduce xeno- antigenicity of islet and prolong xenograft survival, and to translate experimental findings into clinical application. This review discusses the suitable pig donor for islet xenotransplantation in terms of pig age, strain, structure/function of islet, and genetically modified pig.

新生猪胰岛移植治疗糖尿病病人的临床研究(英文)

目的:评估新生猪胰岛移植治疗1型糖尿病方法的安全性和有效性。方法:所有22例病人均接受经肝动脉新生猪胰岛移植治疗,移植后病人接受多种免疫抑制治疗方案;第1组14例病人使用环孢菌素、骁息和甲基强的松龙,没有接受猪C肽检测;第2组2例病人只使用环孢菌素和骁息,第3组6例病人的免疫抑制方案是OKT3、他克莫司、西罗莫司和甲基强的松龙。在移植治疗前和移植后1年,病人进行了血糖、外源性胰岛素用量、糖化血红蛋白、猪内源性反转录病毒(PERV)和肝功能的评估,第2组和第3组的8例病人接受了血清猪C肽检测。6例病人在移植后4～6年进行了复查。结果:第1组14例病人移植后胰岛素用量减少和HbA1c水平降低。第2组2例病人在移植后代谢指标没有变化,猪C肽检测阴性。第3组的6例病人在移植3个月以后,胰岛素用量均减少,HbA1c正常,6例病人血清均检测到有意义的猪C肽。其中2例病人接受了第2次新生猪胰岛移植,1例病人短暂脱离胰岛素治疗7 d。所有病人在移植后均未出现严重不良反应,没有PERV感染的证据。6例病人在新生猪胰岛移植4～6年后接受检查,此6例病人均在接受移植治疗1年后停止免疫抑制治疗,复查时6例病人均接受胰岛素治疗。其中4例病人严格限制糖的摄入,2例病人为自由饮食;这2例病人中,有1例出现2次酮症,有轻度糖尿病视网膜病变;1例出现1次由于急性胃肠炎导致的酮症;其余4例均未出现任何并发症。6例病人再次接受PERV检测均为阴性。结论:异种胰岛可以在人体内存活并发挥其功能,没有发现严重不良反应。

多种分离纯化大鼠胰岛细胞的实验方法比较

【目的】采用不同的分离或消化方法 ,探讨如何提高大鼠胰岛细胞分离纯化后的收获量和质量。【方法】将 2 5只供体SD雄性大鼠依胰腺分离或消化的方法不同随机地分为 5组 :①A组 :胆总管灌注胶原酶P ;②B组 :胆总管灌注Hanks液 ;③C组 :胆总管不灌注液体 ;A、B、C 3组胰腺直接分次消化 ;④D组 :胆总管不灌注 ,胰腺剪碎后分次消化 ;⑤对照组 :胆总管灌注胶原酶P ,胰腺直接单次消化。将分离的胰岛沉淀物用Ficoll 4 0 0纯化并培养 ,再把胰岛细胞移植于糖尿病裸鼠受体腹腔内。【结果】纯化后的胰岛细胞在收获量上A和B组明显多于对照组或C组 ,而D组最低 ;A、B和D 3组的纯度均高于对照组或C组 ;在胰岛成活率方面 ,A、B、C 3组均高于对照组或D组。移植于糖尿病裸鼠受体内的大鼠胰岛细胞均可逆转高血糖状态。【结论】经胆总管灌注胶原酶后的大鼠胰腺用分次消化的方法可提高胰岛细胞的收获量、纯度和活性。

微囊化大鼠胰岛异种移植于糖尿病小鼠的实验观察

目的：观察海藻酸钠－聚赖氨酸－海藻酸钠（ＡＰＡ）微胶囊对异种胰岛组织移植的免疫隔离作用。方法：采用ＳＵＮ氏ＡＰＡ微胶囊技术包裹大鼠胰岛细胞团，移植于链脲霉素（ＳＴＺ）诱发的糖尿病模型小鼠腹腔。结果：糖尿病组小鼠及植入空囊或注射生理盐水的对照组小鼠血糖无明显变化；用微胶囊包裹及未包裹的大鼠胰岛异种移植于糖尿病小鼠腹腔内，移植后血糖明显下降９～１２ｍｍｏｌ／Ｌ，未包囊胰岛移植组受试鼠血糖降低仅维持２～３ｄ，微囊化胰岛移植组血糖下降可持续１１０ｄ。移植后１个月从腹腔回收的微囊胰岛呈圆型，表面光滑，无明显的纤维组织包绕，囊内胰岛ＤＴＺ染色呈桔黄色。结论：采用ＳＵＮ氏静电ＡＰＡ微胶囊制作技术制作的胶囊，可明显延长大鼠胰岛作为异种移植物在小鼠体内的存活时间，表明这种微囊具有较好的免疫隔离作用和生物相容性。

低温保存微胶囊化胰岛的实验研究

胰岛移植治疗糖尿病需要足够数量的高质量胰岛，低温保存是积累胰岛的有效方法。本文以 ＳＤ大鼠为实验对象，采用胶原酶消化分离， Ｆｉｃｏｌｌ溶液间断密度梯度法获得纯度为９０％的胰岛；利用高压静电成囊装置，能制成粒径为（０．３～０．４）ｍｍ的高质量微囊化胰岛；比较研究分析了微囊化鼠胰岛与非微囊化胰岛低温冻存后的效果；微囊化胰岛低温冻存后，胰岛回收率为９０．２％，对葡萄糖刺激反应敏感，异种移植能逆转糖尿病，并维持血糖正常９０天；而未微囊化胰岛冻存后，胰岛回收率为 ６８． ５％，对葡萄糖刺激反应降低，能逆转高血糖有效期不足７天。微胶囊能够保护胰岛免受冻害，保持免疫隔离作用，延长移植存活期。

糖尿病犬经肝动脉肝内移植微囊化猪胰岛细胞的安全性和有效性

目的评估移植于I型糖尿病犬肝脏中的微囊化新生猪胰岛细胞(NPI)的生物相容性、免疫学特性及生理学特性。方法I型糖尿病犬分为A、B两组,每组15只,A组每只犬分别经肝动脉灌注微囊化新生猪胰岛细胞40～60万个,B组每只犬分别经肝动脉灌注未微囊化新生猪胰岛细胞40～60万个,两组动物移植后均不使用免疫抑制治疗。移植前后分别测量移植受体的肝脏功能及淋巴细胞CD4+/CD8+比值。移植6个月后所有移植受体的肝脏均进行病理学检查。结果移植后A组外源性胰岛素用量从移植前的22 u逐渐降至5 u,B组所需外源性胰岛素从移植前的24 u下降至10 u。移植后2～3周B组胰岛素用量恢复到移植前的水平,而A组的部分动物的胰岛素剂量继续减至8 u。B组受体移植后血的CD4+较移植前升高,而A组的CD4+和CD8+细胞移植后无明显变化。移植后所有受体的肝功能及组织结构未见异常。结论微囊化的新生猪胰岛细胞在受体犬的肝脏中有很好的生物相容性。微囊可以延长移植物的存活,且异种移植微囊化的新生猪胰岛细胞能够纠正糖尿病犬的高血糖状态。

猪胰岛与大鼠睾丸支持细胞共同移植对糖尿病大鼠胰岛生长的影响

目的 研究胰岛异种移植的方法。 方法 用SD大鼠睾丸支持细胞 (Sertoli细胞 )与新生猪胰岛样细胞团 (ICCs)共同培养后进行异种移植。 结果 对照组胰岛细胞存活率及存活时间分别为 (63 .6%± 4.2 %和 3 .4d± 1.2d) ,较共同培养组 (87.2 %± 2 .7%和 13 .5d± 4.6d)显著为低(P <0 .0 1) ;对照组大部分胰岛细胞超微结构破坏 ,而共同培养组胰岛细胞超微结构基本正常 ;对照组胰岛素 2 4h累积分泌量和对葡萄糖刺激的反应性下降 ,共同培养组胰岛素分泌量始终处于高水平状态 (P <0 .0 1)。 结论 猪胰岛细胞与Sertoli细胞共同培养移植可以促进胰岛生长 。

裸鼠糖尿病模型的建立及皮下异种胰岛移植研究

目的探讨链脲左菌素(STZ)建立BALB/c nu/nu裸鼠Ⅰ型糖尿病模型的方法,及将大鼠胰岛分离纯化后移植至糖尿病裸鼠皮下,对移植后裸鼠进行疗效评价。方法选用Balb/c裸鼠分为三组:糖尿病且胰岛移植组、糖尿病胰岛未移植组和正常组;用STZ对裸鼠进行糖尿病建模,大鼠胰岛分离、纯化后移植至糖尿病裸鼠背部皮下,进行血糖、体重和HE染色考察,以糖尿病未移植组和正常组作对照。结果STZ浓度175 mg/kg时,腹腔注射裸鼠糖尿病造模成功率高,纯化的大鼠胰岛活性较好,但移植后疗效平均维持时间约5 d,移植第5 d的OGTT结果显示胰岛皮下移植组和糖尿病组血糖均较正常组高,移植3 d时HE染色显示胰岛在皮下存活,但胰岛中心部位细胞有坏死。结论本研究建立了一种免疫排斥缺陷宿主异种胰岛皮下移植治疗糖尿病的方法,为进一步探索改进胰岛移植研究提供参考。

大鼠胰岛与生长因子共微囊提高囊内胰岛存活率

目的探讨微胶囊胰岛皮下移植治疗化学性糖尿病小鼠和生长因子(growth factor,GF)与大鼠胰岛共微囊对提高囊内胰岛存活率的作用。方法选取C57BL/6雄性小鼠皮下预血管化糖尿病模型25只,行微胶囊大鼠胰岛皮下移植。随机分为5组,每组5只。①A组:GF与大鼠胰岛共微囊移植组;②B组:微胶囊大鼠胰岛移植组;③C组:GF微囊移植组;④D组:大鼠胰岛移植组;⑤E组:空囊移植组。观察受体小鼠的血糖和体重变化,移植后8周比较微胶囊内胰岛存活数。结果移植后1周至移植后3周A组与B组间血糖均有非常显著差异(P<0.01),移植后4周至移植后8周A组与B组间血糖无显著差异(P>0.05),移植后1周至移植后4周B组与C组间血糖均有非常显著差异(P<0.01)。C组血糖异常升高,移植4周后小鼠陆续死亡。D组和E组移植无效。A组和B组间各实验点小鼠体重均无显著差异(P>0.05),A组小鼠体重成模时下降,移植2周后开始上升。A组和B组胰岛存活数有非常显著差异(F=36.84,P<0.01)。结论GF与大鼠胰岛共微胶囊移植,GF可促进胰岛的细胞增殖和再生,显著提高囊内胰岛的存活率。

大囊包被异种胰岛免疫保护机理初探

继实验证明琼脂糖大囊包被异种胰岛移植能减缓排斥反应之后，作者又对琼脂糖大囊包被异种胰岛的免疫保护机理进行了初步探讨。实验分为琼脂糖大囊包被异种（大鼠）胰岛组、未包被异种（大鼠）胰岛组、大囊无胰岛组及同系小鼠胰岛组，每组５个样本。采用淋巴细胞胰岛混合培养（ｍｉｘｅｄｌｙｍｐｈｏｃｙｔｅｉｓｌｅｔｃｕｌｔｕｒｅ，ＭＬＩＣ），测定并计算各组淋巴细胞刺激指数（ｓｔｉｍｕｌａｔｉｎｇｉｎｄｅｘ，ＳＩ）及培养上清液中ＩＬ－２活性。结果：琼脂糖大囊包被异种胰岛组及大囊无胰岛组的ＳＩ和ＩＬ－２活性与同系小鼠胰岛组比较无显著性差异；未包被异种胰岛组的ＳＩ和ＩＬ－２活性分别为１４．２２±３．３８及１７．８±２．１７ＩＵ／ｍｌ，均显著高于同系小鼠胰岛组及琼脂糖大囊包被异种胰岛组。上述结果证明琼脂糖大囊包被技术可明显降低异种胰岛移植物的免疫原性。推测其机理可能与琼脂糖形成的人工膜能将异种胰岛与宿主免疫系统隔离。

异种胰岛细胞移植治疗1型糖尿病临床观察

目的猪作为人异种胰岛细胞移植的供体被认为可能是解决人类供体短缺的办法。本研究目的是观察异种胰岛细胞移植对1型糖尿病的治疗效果。方法本研究共3名1型糖尿病自愿受试者,随访6个月。3名患者均在内分泌专科医生的指导下坚持胰岛素治疗,但是长期血糖控制欠佳,血糖波动大,多次出现严重低血糖。供体是新生猪,采用胶原酶消化法分离胰岛细胞,经过体外培养后注入受试者肝内。移植细胞数分别为80万、100万、60万。免疫抑制剂:2例选用环孢霉素(cyclosporine,CSA)+骁悉(mycophenolatemofetil,MMF),1例选用他克莫司(tacrolimus,FK506)+MMF。结果所有病人在移植后1周内均有低热和轻度的肝区疼痛,没有严重的并发症。移植术后仍需胰岛素治疗。术后20d左右,胰岛素用量较术前逐渐减少。6个月后观察,3名受试者血糖均能较好控制,血糖大幅度波动的情况得到改善,HbA1c明显下降,C肽水平显著升高,每天胰岛素用量分别减少了46.7%、25%、20%。结论异种(猪)胰岛细胞移植治疗1型糖尿病,可以使血糖得到理想控制,改善血糖的大幅度波动,低血糖发生率降低,胰岛素用量减少,短期内无明显副反应。

经腹腔镜微囊化猪胰岛异种移植术

目的 探讨腹腔镜技术应用于海藻酸钠 -聚赖氨酸 -海藻酸钠 (APA)微囊包裹猪胰岛小网膜腔内异种移植术的可行性。 方法 采用腹腔镜技术对 5例 6人次Ⅰ型糖尿病患者进行了APA微囊新生猪胰岛小网膜腔内异种移植治疗 ,观察患者移植前后空腹血糖、C肽和胰岛素用量变化。 结果 全部病例均未出现手术并发症。术后受体C肽较术前升高 3- 2 3倍 ,胰岛素用量减少 ,基中 1例完全停用胰岛素 ,成为胰岛素不依赖者达 31个月。 结论 腹腔镜技术用于临床微囊化新生猪胰岛小网膜腔移植术治疗Ⅰ型糖尿病安全可行。

猪胰岛细胞异种移植治疗糖尿病的研究进展

猪胰岛细胞移植是一种极具潜力治疗糖尿病的方法.目前仍以实验室研究为主,其临床应用主要面临两大挑战:高质量的活性胰岛短缺与异种免疫排斥反应.近年来随着胰岛分离纯化技术的改进、新型免疫耐受诱导策略的出现及移植技术的发展等,猪胰岛细胞异种移植取得了巨大的进展并进入临床前期研究.本文就猪胰岛移植物的制备、排斥反应与防治措施、移植安全性及临床研究等方面的进展作一综述.

异种胰岛移植的免疫耐受诱导

胰岛移植能够纠正IDDM的高血糖并减少糖尿病相关并发症 ,临床广泛应用将造成尸源性胰腺供体严重不足 ,动物来源的胰岛将成为今后胰岛的主要供源。异种移植的最大障碍是移植排斥反应 ,目前控制异种胰岛移植的免疫抑制治疗无法在临床推广。啮齿动物实验研究证明异种胰岛移植不需要长期重度免疫抑制治疗就能获得生存 ;杂交系动物实验研究提示异种胰岛移植的临床应用是可行的。随着对大动物杂交种系间异种移植排斥机理和动物病原体传播能力的深入了解 ,异种胰岛移植将进入临床。