胰岛细胞移植过程中影响细胞活性的相关因素

目的：探讨胰岛细胞移植过程中胰腺的切取方法、冷缺血时间、组织相容性对于胰岛细胞活性的影响。 方法：实验于2001-09／2005-01在南方医科大学附属珠江医院完成。高渗枸橼酸盐嘌呤溶液经主动脉原位灌注后，探讨肝、肾、胰腺联合及分别切取情况；测定不同的冷缺血时间条件下胰岛细胞活性的变化；体外培养胰岛细胞和血液供受者的组织相容性和胰岛细胞存活的关系。 结果：④对于胰腺、肾脏联合切取，肝脏、胰腺、肾脏联合切取及各器官的单独切取顺利，在冷缺血时间5h以内胰岛细胞活性率都在80％，各方法之间无明显差别。②胰腺与肾脏经高渗枸橼酸盐嘌呤溶液原位灌注满意，胰岛细胞的活性与冷缺血时间呈负相关，冷保存指数和胰岛细胞活性呈正相关。③人类胰岛暴露于未经抗凝的人类血中，胰岛将诱发一个迅速血小板激活和消耗，中性粒细胞和单核细胞也被消耗，加入肝素后这种反应明显减轻。 结论：腹主动脉进行原位灌注后，用于胰岛细胞移植的胰腺和其他器官的切取不会影响胰岛细胞的活性；经高渗枸橼酸盐嘌呤溶液保存的胰腺的冷缺血时间不易超过5．0～6，0h；胰岛细胞移植入血后，都将引发血细胞的类似炎症反应；HIA配型有助于提高移植后胰岛细胞的存活。     

冷缺血时间及血液相容性与移植胰岛细胞活性的关系

背景:在胰岛移植过程中,胰岛细胞的数量和胰岛细胞的活性是非常重要的。胰岛细胞移植中冷缺血时间和组织相容性是关系胰岛细胞移植能否成功的关键问题。目的:观察冷缺血时间及血液相容性不同对于胰岛细胞数量和活性的影响。设计:观察性实验。单位:广西中医学院附属瑞康医院。材料:采用脑死亡自愿捐赠者的器官(已知血型和HLA配型),在其他器官切取后或同时进行胰腺的获取;血型相同、HLA配型相符的血液;HLA配型高致敏(错配3个以上位点)或群体反应性抗体>50%或淋巴毒阳性受者的血液;分离纯化后的胰岛细胞悬浊液,经过70μm的筛网滤过后,保留直径大于70μm的胰岛细胞,配置成浓度1.20×105L-1左右活性胰岛细胞悬浊液。方法:高渗枸橼酸盐嘌呤溶液经过腹主动脉原位灌注后,参照文献进行肝、肾、胰腺联合及分别切取。用于切取的时间:胰腺肾脏的联合切取、肝脏胰腺肾脏的联合切取方法平均需时15min,肝脏、胰腺、肾脏的单独切取平均需时20min,用于胰岛分离的胰腺12份,冷缺血时间2.5～8h。冷却血时间在5h以内的9份,温缺血时间0～3min,消化的时间15±2.4min。采用胶原霉P进行消化分离胰岛细胞,双硫腙、丫腚橙染色测定不同的冷缺血时间条件下活性胰岛细胞活性,分析体外胰岛细胞和血液与供受者的组织相容性和胰岛细胞存活的关系。主要观察指标:胰岛细胞活性率和血小板、中性粒细胞和单核细胞计数。结果:肝、肾、胰腺联合切取及各器官的单独切取顺利,在冷却血5h以内胰岛细胞活性率都在80%以上,用于胰岛细胞移植的胰腺和其他器官的切取不会影响胰岛细胞的活性;人类胰岛暴露于未经抗凝的人类血中,胰岛将诱发一个迅速的血细胞消耗;进行血小板、中性粒细胞和单核细胞计数,HLA错配组和HLA匹配组与对照组比较血细胞都发生明显的消耗;加入肝素后血细胞计数与对照组比较差异明显(P<0.05),反应明显减轻。HLA匹配组和HLA错配组胰岛细胞体外培养24h活性胰岛细胞数量较,差异明显(P<0.05)。结论:在冷缺血时间<5h的情况下获取的胰腺可以用于临床胰岛细胞的移植;血液相容性好能够明显提高胰岛细胞移植的成功率。     

胰岛细胞移植过程中影响细胞活性的相关因素

目的:探讨胰岛细胞移植过程中胰腺的切取方法、冷缺血时间、组织相容性对于胰岛细胞活性的影响。方法:实验于2001-09/2005-01在南方医科大学附属珠江医院完成。高渗枸橼酸盐嘌呤溶液经主动脉原位灌注后,探讨肝、肾、胰腺联合及分别切取情况;测定不同的冷缺血时间条件下胰岛细胞活性的变化;体外培养胰岛细胞和血液供受者的组织相容性和胰岛细胞存活的关系。结果:①对于胰腺、肾脏联合切取,肝脏、胰腺、肾脏联合切取及各器官的单独切取顺利,在冷缺血时间5h以内胰岛细胞活性率都在80%,各方法之间无明显差别。②胰腺与肾脏经高渗枸橼酸盐嘌呤溶液原位灌注满意,胰岛细胞的活性与冷缺血时间呈负相关,冷保存指数和胰岛细胞活性呈正相关。③人类胰岛暴露于未经抗凝的人类血中,胰岛将诱发一个迅速血小板激活和消耗,中性粒细胞和单核细胞也被消耗,加入肝素后这种反应明显减轻。结论:腹主动脉进行原位灌注后,用于胰岛细胞移植的胰腺和其他器官的切取不会影响胰岛细胞的活性;经高渗枸橼酸盐嘌呤溶液保存的胰腺的冷缺血时间不易超过5.0～6.0h;胰岛细胞移植入血后,都将引发血细胞的类似炎症反应;HLA配型有助于提高移植后胰岛细胞的存活。     

人胰岛细胞移植中胰岛细胞活性研究

目的：器官联合切取中的不同胰腺获取方法是否对于胰岛细胞的活性有影响。方法：实验于2001—08／2004—08在南方医科大学珠江医院器官移植中心和南方医科大学解剖教研室完成，12例自愿捐赠器官供，采用高渗枸橼酸盐嘌呤溶液经过腹主动脉进行原位灌注后，进行胰腺、肾脏联合切取，肝脏、胰腺、肾脏联合切取及各器官的单独切取。取后采用胶原酶P消化胰腺，分离并纯化胰岛细胞，进行胰岛细胞活性染色．然后对比不同切除方法对于胰岛细胞活性率的影响。结果：胰腺、肾脏联合切取，肝脏、胰腺、肾脏联合切取及各器官的单独切取，如顺利，无污染、在冷却血时间5h以内，胰岛细胞活性率都在80％，经原位灌注后各方法之间无明显差别。结论：经腹主动脉进行原位灌注后，各种方法切取都符合无污染，冷却时间5h以内，不会影响胰岛细胞的活性。     

人胰岛细胞移植中胰岛细胞活性研究

目的:器官联合切取中的不同胰腺获取方法是否对于胰岛细胞的活性有影响。方法:实验于2001-08/2004-08在南方医科大学珠江医院器官移植中心和南方医科大学解剖教研室完成,12例自愿捐赠器官供者,采用高渗枸橼酸盐嘌呤溶液经过腹主动脉进行原位灌注后,进行胰腺、肾脏联合切取,肝脏、胰腺、肾脏联合切取及各器官的单独切取。取后采用胶原酶P消化胰腺,分离并纯化胰岛细胞,进行胰岛细胞活性染色,然后对比不同切除方法对于胰岛细胞活性率的影响。结果:胰腺、肾脏联合切取,肝脏、胰腺、肾脏联合切取及各器官的单独切取,如顺利、无污染、在冷却血时间5h以内,胰岛细胞活性率都在80%,经原位灌注后各方法之间无明显差别。结论:经腹主动脉进行原位灌注后,各种方法切取都符合无污染,冷却时间5h以内,不会影响胰岛细胞的活性。     

胰岛细胞移植治疗1型糖尿病的护理

总结3例胰岛细胞移植治疗1型糖尿病的护理体会。护理重点为术前做好心理卫生宣教,重视移植受体的适应证和禁忌证的评估和供体的胰岛细胞的处理,同时加强术前准备;术后严密观察生命体征变化,预防并发症发生,做好血糖监测。3例患者手术顺利,术后血糖稳定,无移植并发症以及低血糖发生。临床观察1年,2例完全脱离胰岛素治疗,1例胰岛素用量较前减少60%以上。     

大鼠胰岛细胞的分离提纯及移植效果观察

背景:胰岛细胞移植已成为治疗1型和部分2型糖尿病的有效途径,但供体不足的问题限制了其发展。目的:改进胰岛细胞的分离和纯化方法,并观察其移植效果。设计:实验方法改进。单位:中国医科大学实验动物部和细胞生物研究室。材料:实验于2006-01/10在中国医科大学实验动物部和细胞生物研究室完成。供体为普通级封闭群Wistar大鼠,雌雄不限,体质量250～300g;受体为普通级封闭群SD大鼠,雄性,(Frompage2388)体质量180～220g,两种大鼠均购自中国医科大学实验动物部(许可证号:SYXK(LIAO)2003-0013)。方法:①胰岛细胞的分离纯化及胰岛功能评估:取未禁食的Wistar大鼠,麻醉后处死。于胆总管起始处剪一小口,将连着装有胶原酶的注射器的1mm直径的腰麻管顺行插入,将冷的胶原酶溶液(1.5g/L)注入胰管内,使胰腺充分膨胀。切取胰腺,将装有胰腺的离心管水浴消化,水浴期间间断加入1mol/L的NaOH使消化液的pH值保持在7.8±1.0,经Ficoll密度梯度离心法纯化胰岛。应用双硫腙法评估胰岛的纯度,应用丫啶橙/碘丙啶法评估胰岛的存活率,存活率=活细胞总数/(活细胞总数 死细胞总数)×100%。通过胰岛素释放实验检测胰岛活性:胰岛素释放指数=第3小时(高糖环境)的胰岛素含量/第2小时(低糖环境)的胰岛素含量。②胰岛移植效果的观察:受体SD大鼠腹腔内注射链脲霉素,非禁食状态下2次血糖>16.7mmol/L确定为糖尿病大鼠。将糖尿病大鼠按随机数字表法分为两组,即胰岛移植组和糖尿病组,每组8只。胰岛移植组于肾被膜下注射约1000个胰岛,糖尿病组注射相应体积的1640培养液,再随机取8只血糖浓度≤5.5mmol/L的SD大鼠作为正常对照组。术后每天检测大鼠血糖,以非禁食血糖小于11.1mmol/L者视为胰岛移植存活。胰岛移植组大鼠血糖正常3d后对胰岛移植组、糖尿病组和正常对照组大鼠分别进行静脉糖耐量试验,测定0,15,30,60,90及120min血糖。主要观察指标:胰岛的纯度、存活率及活性。结果:纳入受体SD大鼠24只,全部进入结果分析,无脱失。①平均每只大鼠胰腺可获得(1150±141)个胰岛,纯度>95%,存活率>98%,胰岛形态完好。②体外胰岛素释放实验低、高糖组的胰岛素释放量为(70.5±6.9)mIU/L,(321.4±11.6)mIU/L,胰岛素释放指数为4.60±0.52,提示所提取的胰岛β细胞有良好的功能。③胰岛移植组大鼠胰岛移植当天血糖既开始下降,3d后血糖下降至正常,维持正常血糖水平(6±2)d;而糖尿病组大鼠血糖均在16.7mmol/L以上。胰岛移植组静脉葡萄糖耐量试验曲线与正常对照组相似。结论:经原位灌注法分离、Ficoll密度梯度离心纯化的胰岛制备技术可以获得高纯度和活力良好的大鼠胰岛细胞。     

异种胰岛细胞移植进展

近年来随着胰岛细胞移植技术在欧美地区的逐渐成熟和临床应用的发展[1,2],胰岛移植有望成为治疗糖尿病的根本方案,但困扰胰岛移植的两个主要问题仍然存在：胰岛细胞来源不足与移植后的免疫排斥反应。现就当前异种胰岛细胞移植的研究进展简要综述如下：     

胰岛移植:寻找新的β细胞来源

<正>胰岛移植被认为是1型糖尿病最有前景的治疗方法之一。若以生活质量改善及血糖控制为目标,胰岛移植的效果令人满意;但若以脱离外源胰岛素或预防并发症为目标,则前者差强人意,后者效果不明[1]。为此,美国糖尿病协会仍将胰岛移植定为实验阶段,距离临床应用尚有距离[2]。胰岛移植及面临的问题胰岛移植最早可以追溯至1972年的大鼠实验[3],此后逐渐开展,但成功率很低。一项对1990年至1999     

胰岛细胞移植的新进展

胰岛素虽然很大程度改善了糖尿病患者的糖代谢紊乱及延迟了相关并发症的发生，但存在低血糖、胰岛素抵抗以及需反复注射的缺点。胰岛细胞移植简单、安全、有效，免除了胰腺移植术后血管并发症等的发生，是治疗糖尿病最有前景的治疗方法。本文就近几年胰岛细胞移植的免疫耐爱诱导、免疫抑制药物的应用、移植物微循环的建立等最新进展作一综述。     

Langerhans islet preparation in cell transplantation

Islet cell transplantation is potentially one of the best techniques for the almost perfect symptomatic treatment of Type 1 diabetes mellitus. Thanks to the considerable progress achieved in isolating and purifying human islet cells, highly encouraging results have recently been reported. However, due to the need for immunosuppression treatment, the indications are presently as limited as those for transplantation of the vascularized pancreas, namely cotransplantation. The ultimate goal of transplantation is to allow long-term function of the grafted tissues using no or transitory host immunosuppression. When compared to solid organ grafts, cellular transplants are more amenable to creative strategies to accomplish this goal. We review the current status of islet transplantation in man and then the major steps before islet transplantation and their prospects.     

Immunoisolation techniques for islet cell transplantation

Diabetes remains a devastating disease, with tremendous cost in terms of human suffering and healthcare expenditures. The burden of diabetes is primarily related to the multiple complications, including retinopathy, nephropathy, neuropathy and cardiovascular disease that can develop as the disease progresses. It has been shown that these complications can be prevented, and in some cases, reversed by islet cell transplantation, which, until recently, had remained elusive as a viable routine treatment modality. In recent studies, islet cell transplantation has shown great promise as a viable alternative to solid pancreas transplantation. However, severe shortage of human pancreases and the need to use immunosuppressive drugs to prevent transplant rejection, remain major obstacles to routine use of islet cell transplants for the treatment of patients with Type 1 diabetes. In the attempt to overcome these barriers, many procedures have been designed to immunoisolate islet cells for transplantation. The ultimate goal in islet cell transplantation is the availability of unlimited supply of cells to be transplanted in a simple procedure performed with little or no use of immunosuppressive drugs. The development of reliable procedures to immunoisolate islets by microencapsulation prior to transplantation has a great deal of potential to accomplish this objective.     

Immunoisolation techniques for islet cell transplantation

Diabetes remains a devastating disease, with tremendous cost in terms of human suffering and healthcare expenditures. The burden of diabetes is primarily related to the multiple complications, including retinopathy, nephropathy, neuropathy and cardiovascular disease that can develop as the disease progresses. It has been shown that these complications can be prevented, and in some cases, reversed by islet cell transplantation, which, until recently, had remained elusive as a viable routine treatment modality. In recent studies, islet cell transplantation has shown great promise as a viable alternative to solid pancreas transplantation. However, severe shortage of human pancreases and the need to use immunosuppressive drugs to prevent transplant rejection, remain major obstacles to routine use of islet cell transplants for the treatment of patients with Type 1 diabetes. In the attempt to overcome these barriers, many procedures have been designed to immunoisolate islet cells for transplantation. The ultimate goal in islet cell transplantation is the availability of unlimited supply of cells to be transplanted in a simple procedure performed with little or no use of immunosuppressive drugs. The development of reliable procedures to immunoisolate islets by microencapsulation prior to transplantation has a great deal of potential to accomplish this objective.     

从胰岛细胞移植到胰腺干细胞移植，治愈糖尿病还有多远？

背景：胰岛细胞移植和胰腺干细胞移植是近年来糖尿病治疗的研究热点,也是治愈糖尿病最有希望的途径。目的：探讨胰岛细胞移植和胰腺干细胞移植治疗糖尿病的可行性、优势、面临的问题及解决的办法。方法：收集胰岛细胞移植和胰腺干细胞移植治疗糖尿病的相关实验和临床研究,进行实验数据分析两种细胞移植途径治疗糖尿病的影响因素,从细胞分子生物学水平认识胰岛细胞移植和胰腺干细胞移植治疗糖尿病的优势和缺点。结果与结论：胰岛细胞移植治疗糖尿病受供体不足的制约,胰腺干细胞移植解决了胰岛细胞供体短缺的有效途径,但胰腺干细胞移植的研究还停留在动物实验阶段,需要进行广泛的临床研究。首先要明确胰腺干细胞的特异性标志物,其次要掌握将相关干细胞诱导分化为胰腺干细胞的方法和技术。     

胰岛移植过程中胰岛培养的研究现状

目的:总结胰岛移植过程中胰岛培养的研究现状,为利用胰岛培养准备更多更好的胰岛提供理论依据和技术支持。资料来源:以计算机检索Medline和Pubmed数据库1994-01/2007-01与胰岛培养相关的文章,检索词为“isle,tculture”,限定文章语言种类为English。同时计算机检索CNKI中国全文数据库1994-01/2007-01与胰岛培养相关的文章,检索词为“胰岛,培养”,限定文章语言种类为中文。资料选择:对资料进行初审,纳入标准:①与胰岛培养相关的文章。②1994年以后发表的文章。排除标准:Meta分析类文章。资料提炼:共检索到1384篇与胰岛培养相关的文章,入选33篇。对所检索文章的相关信息加以综合概括,其中关于胰岛培养中基础培养基、培养添加物、培养温度、密度等方面的文章18篇,关于胰岛培养新尝试的文章12篇。资料综合:将胰岛培养后再行移植有一定优势,包括降低免疫原性和为术前准备赢得时间等。目前对于挑选合适培养基及其添加物,调整合适培养温度、培养密度以及保护胰岛的活性等都有了比较深入细致的研究;同时很多学者开始尝试一些新的方法延长胰岛培养时间,改善胰岛功能,包括加入细胞外基质、与其他细胞共培养、选用微重力系统培养以及利用胶囊包裹培养等均显示良好的效果。结论:虽然胰岛培养可能会影响胰岛的活性和功能,但随着对胰岛生理和病理生理特性研究的深入,新的技术方法可将这些影响降到最低。胰岛培养给临床实践带来的便利是其最大的优势,加强胰岛培养的研究与应用有着重要的意义。     

Allogeneic islet transplantation

Significant progress has been made in the field of beta-cell replacement therapies by islet transplantation in patients with unstable Type 1 diabetes mellitus (T1DM). Recent clinical trials have shown that islet transplantation can reproducibly lead to insulin independence when adequate islet numbers are implanted. Benefits include improvement of glycemic control, prevention of severe hypoglycemia and amelioration of quality of life. Numerous challenges still limit this therapeutic option from becoming the treatment of choice for T1DM. The limitations are primarily associated with the low islet yield of human pancreas isolations and the need for chronic immunosuppressive therapies. Herein the authors present an overview of the historical progress of islet transplantation and outline the recent advances of the field. Cellular therapies offer the potential for a cure for patients with T1DM. The progress in beta-cell replacement treatment by islet transplantation as well as those of emerging immune interventions for the restoration of self tolerance justify great optimism for years to come.     

Allogeneic islet transplantation

Significant progress has been made in the field of β-cell replacement therapies by islet transplantation in patients with unstable Type 1 diabetes mellitus (T1DM). Recent clinical trials have shown that islet transplantation can reproducibly lead to insulin independence when adequate islet numbers are implanted. Benefits include improvement of glycemic control, prevention of severe hypoglycemia and amelioration of quality of life. Numerous challenges still limit this therapeutic option from becoming the treatment of choice for T1DM. The limitations are primarily associated with the low islet yield of human pancreas isolations and the need for chronic immunosuppressive therapies. Herein the authors present an overview of the historical progress of islet transplantation and outline the recent advances of the field. Cellular therapies offer the potential for a cure for patients with T1DM. The progress in β-cell replacement treatment by islet transplantation as well as those of emerging immune interventions for the restoration of self tolerance justify great optimism for years to come.     

Immunosuppression in islet transplantation

Islet transplantation can temporarily cure type 1 diabetes mellitus (T1DM) but requires simultaneous immunosuppression to avoid allograft rejection. In this issue of the JCI, Monti et al. report that immune conditioning via use of the Edmonton protocol - a treatment approach in which T1DM patients infused with pancreatic islets from multiple cadaveric donors simultaneously receive immunosuppressive drugs - results in lymphopenia that is associated with elevated serum levels of the homeostatic cytokines IL-7 and IL-15, which causes in vivo expansion of the autoreactive CD8(+) T cell population (see the related article beginning on page 1806). Reemergence of autoreactivity is likely the main culprit underlying long-term islet graft failure, and new strategies will need to be tested to circumvent this homeostatic expansion and recurrent autoreactivity.