微囊化大鼠胰岛细胞移植治疗糖尿病小鼠的实验研究

观察微囊化和未囊化大鼠胰岛细胞移植治疗糖尿病小鼠的疗效。选用SD大鼠经胆总管原位灌注胶原酶液,分离消化胰腺组织,用淋巴细胞分离液(Histopaque-1077)纯化胰岛细胞,将微囊化和未囊化大鼠胰岛细胞移植于糖尿病小鼠腹腔。分离的胰岛细胞对刺激反应良好,微囊化和未囊化胰岛细胞移植后均可纠正糖尿病小鼠的高血糖状态,但未囊化胰岛细胞移植组只能维持血糖正常2~3d,而微囊化胰岛细胞移植组可持续降低血糖30d以上。微囊化胰岛细胞移植治疗糖尿病小鼠具有良好的效果,微囊具有较好的免疫隔离作用。     

微囊胰岛研究进展

本综述了微囊胰岛移植治疗糖尿病研究现状，重点介绍了胰岛分离、纯化、微囊化方法及海藻酸钠－聚赖氨酸－海藻酸钠微囊胰岛的体外研究与动物试验，对微囊胰岛体现内外功能进行了较全面的评价。展望了微囊胰岛的发展方向。作认为微囊胰岛移植有望应用于临床治疗胰岛素依赖型糖尿病。     

胰岛细胞移植治疗糖尿病

胰岛细胞移植是治疗糖尿病的一个有效方法,但供胰的极度缺乏阻碍了这个方法的应用。因此很多科学家在探索胰岛β细胞的其它来源途径。目前出现的热点途径有成体干细胞和胚胎干细胞,基因工程改造的β细胞,异种胰岛细胞等。尽管这些方法都还需进一步完善,但无疑给 1型和 2型糖尿病的治愈带来了希望。     

微囊胰岛研究进展

本文综述了微囊胰岛移植治疗糖尿病研究现状，重点介绍了胰岛分离、纯化、微囊化方法及海藻酸钠－聚赖氨酸－海藻酸钠微囊胰岛的体外研究与动物试验，对微囊胰岛体内外功能进行了较全面的评价。展望了微囊胰岛的发展方向。作者认为微囊胰岛移植有望应用于临床治疗胰岛素依赖型糖尿病。     

改良微囊胰岛细胞制备技术及性能研究

为快速制备符合实用要求的微囊化乳猪胰岛细胞，研究了关键因素对制备微囊的影响。结果表明，海藻酸钠浓度以及胰岛细胞和海藻酸钠混合的比例对微囊有较大影响，浓度为１．５－２％、比例为１：８－１０为制备微囊化乳猪胰岛细胞的适宜条件，所得微囊化胰岛细胞具有生物活性。     

胰岛细胞移植进展

近年来,随着在供体胰腺的获取与处理、胰岛的分离与纯化、新型免疫抑制剂的应用等领域不断取得突破,胰岛细胞移植已经公认为是一种治疗1型糖尿病安全有效的治疗方法。胰岛细胞移植治疗的理想目标在于获得比目前治疗方案更符合生理的代谢控制,便患者摆脱对胰岛素的依赖,并保持移植物长期有功能存活和改善患者的生活质量。然而,大量的临床试验表明目前的胰岛移植治疗糖尿病方案仍然存在较多难题,距离临床推广应用仍需时日。本文简要介绍了胰岛细胞移植的历史,并对胰岛分离技术、移植程序、免疫抑制剂的应用、异种胰岛细胞移植、干细胞移植、基因技术在胰岛细胞移植中的应用、羊膜上皮细胞移植、胰岛细胞移植所面临的挑战及将来发展方向等方面作了简要综述。     

微囊化细胞的低温保存

背景：微胶囊是一种有效的免疫隔离工具,低温冷冻方便了微囊的保存与运输,有利于微囊化技术在临床的推广应用。 目的：综述微囊低温保存技术中微囊低温保存特点、不同保存方法优缺点、研究方法及微囊低温保存实验现状。 方法：检索1980/2010 PubMed数据库,1991/2010万方数据库、维普数据库有关微囊低温保存研究,低温保存工艺理论及专用仪器,微囊低温保存技术医学应用的文献。 结果与结论：微囊化细胞在低温保存过程中的损伤特点与细胞、组织有很大不同。微囊相对较大的尺寸与复杂的囊壁半透膜结构,使得微囊结构与囊内细胞更容易受到溶质损伤与冰晶损伤,因此不能简单套用单细胞悬液的低温保存方案。慢速冷却法与玻璃化法是两种常见的微囊化细胞低温保存方法,各有利弊。微囊化细胞低温保存技术尚未成熟,在开发新型微囊低温保存设备,优化设计微囊低温保存方案的同时,需进一步加强对微囊冻存机制更深层次的探索。     

微囊杂种猪胰岛移植治疗Ⅰ型糖尿病的实验研究

目的 :建立成年杂种猪胰岛分离、纯化和包埋的方法 ,并对微囊胰岛的体外分泌功能及体内移植降糖功能进行研究。方法 :采用经胰管灌注胶原酶 ,葡聚糖间断密度梯度离心 ,分离和纯化猪胰岛 ,用海藻酸钠 聚赖氨酸 海藻酸钠 (APA)包埋猪胰岛。用双硫腙染色鉴别胰岛 ,用荧光染色计数胰岛存活率。对微囊胰岛进行体外培养 ,用不同浓度葡萄糖刺激微囊胰岛考察其功能 ,并进行大鼠糖尿病模型移植。结果 :每个猪胰腺可得到的胰岛数量平均为 98,5 5 0± 13,80 4个 ,纯化后胰岛纯度达 80 %以上 ,平均胰岛回收率 43.9%。体外试验显示微囊胰岛对葡萄糖刺激发生应答 ,分泌胰岛素。微囊胰岛在 16天的培养期间可持续分泌胰岛素 ,囊内胰岛细胞生长良好 ,微囊无溶解和破碎。培养 2天的微囊胰岛对高糖刺激有明显反应 ,其胰岛素释放量分别为低糖的 1.49倍 (静态 )和 2 .45倍 (灌流 ) ,P <0 .0 5。糖尿病大鼠体内移植微囊杂种猪胰岛 5 ,0 0 0～ 6 ,0 0 0 ,可明显降低动物模型血糖 ,并持续 7～ 11d。结论 :APA微囊膜及微囊化杂种猪胰岛在体外功能良好。短期培养及体内移植降糖功能确切 ,将此法制备的微囊胰岛用于治疗I型糖尿病有良好的应用前景。     

实验性糖尿病大鼠胰岛移植的初步研究

实验研究证明,胰岛移植不仅能纠正实验性糖尿病动物的代谢异常,并能防止糖尿病性微血管病变的发生和发展,因此,对胰岛移植的研究具有重要的临床意义。我们自1983年开始,用大白鼠进行胰岛移植的实验研究,初步取得成效,现将观察结果报告如下。     

微囊化细胞移植的研究进展

微囊技术为组织／细胞移植开辟了新途径,它有产地避免了移植后的免疫排斥反应,并解决了移植物来源稀少的问题。微囊的包裹材料有多种,以海藻酸钠－多聚赖氨酸－海藻酸钠（APA）应用最为广泛,可通过提高其生物相容性,从而减弱免疫排斥反应。微囊具有良好的免疫隔离作用。体现在对免疫活性细胞及部分细胞因子的阻挡作用,使移植物能存活下来并能发挥其功能,目前对微囊化人工细胞的研究取得了很大的进展,特别是基因工程细胞日益成为研究的焦点。微囊技术是一新兴的,尚需进一步改进的技术,它在异体组织／细胞移植等方面必将有广阔的应用前景。     

胰岛移植治疗糖尿病的进展和有待解决的问题

 摘要 糖尿病是严重危害人类健康的疾病之一，已经成为继心脑血管疾病和肿瘤之后的人类健康第三大杀手。目前全世界的糖尿病患者总数已经超过 2 亿，并且以每年 200 万人的速度增加。我国糖尿病的患者总数已超过 6000 万，仅次于印度列世界第 2 位。糖尿病患者由于胰岛素产生障碍或胰岛素不能发挥生理作用而引起糖代谢紊乱，导致血管病变，可发生肢体坏死、失明和肾功能衰竭等一系列严重的并发症。采用胰岛素治疗虽然在一定程度上能够改善患者的糖代谢紊乱，然而并不能有效地防止或逆转糖尿病引起的微血管病变及其并发症。因此，寻找新的糖尿病治疗手段一直是医学研究的前沿课题。......     

Heterografting cultures of human pancreatic islet cells in rats with experimental diabetes mellitus

Cultures of human fetal pancreatic islet cells were transplanted into the liver of rats with diabetes induced by alloxan. This heterografting led to a prolonged fall of the blood sugar. Histological examination of the recipients'liver revealed groups of implanted islet cells.Laboratory of Pathomorphology, Research Institute of Transplantation and Artificial Organs, Ministry of Health of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 1, pp. 48–50, January, 1980.     

骨髓间充质干细胞及与胰岛细胞共移植治疗糖尿病

背景：药物是目前糖尿病治疗最主要的方法,但病情的进展以及相关并发症的发生使药物的作用受到挑战。 目的：探讨骨髓间充质干细胞与胰岛细胞共移植治疗糖尿病的应用效果以及可行性。 方法：分离、纯化大鼠骨髓间充质干细胞并进行体外培养,建立糖尿病模型,对糖尿病模型大鼠分别注射骨髓间充质干细胞、骨髓间充质干细胞和胰岛细胞共培养混合物以及生理盐水或磷酸盐缓冲液作为对照。通过观察监测各糖尿病模型大鼠血糖水平的变化、胰岛素分泌情况以及胰腺组织的病理学变化评估移植治疗效果。 结果与结论：骨髓间充质干细胞移植治疗糖尿病模型大鼠,移植后C-肽值明显升高,血糖水平明显下降,但仍未降至正常范围内,且随着时间的延长,血糖水平再次升高。骨髓间充质干细胞与胰岛细胞共移植治疗糖尿病模型大鼠,血糖水平亦明显下降,且下降程度大于单纯骨髓间充质干细胞移植治疗,可下降至正常水平,并在一定时间内能够维持。骨髓间充质干细胞与胰岛细胞共移植治疗糖尿病具有一定的效果,具有应用的可行性。     

HLA antigens and islet cell antibodies in gestational diabetes

The HLA antigens of 136 patients with gestational diabetes are compared with control populations. No significant variations are observed in their frequencies, particularly for those antigens associated with Type 1 diabetes mellitus. Islet cell antibodies have also been studied in the serum of 52 of these patients and 20 of them were positive, whereas only one of 37 pregnant nondiabetic women had such antibodies (χ² = 15.2). A very high association between ICA and DR3 and DR4 was encountered (χ² = 17, with two df); half of the patients positive for either one of these antigens were ICA positive. These results indicate that ICA associates equally with DR3 and DR4, against the hypothesis that this expression of autoimmunity is more a characteristic of DR3- than of DR4-associated genetic susceptibility. These patients will be followed to determine if the ICA + individuals are at increased risk for the development of insulin-dependent diabetes.     

Analysis of B cell subsets following pancreatic islet cell transplantation in a patient with type 1 diabetes by cytometric fingerprinting

Manual gating of bivariate plots remains the most frequently used data analysis method in flow cytometry. However, gating is operator-dependent and cumbersome, particularly with the increasing complexity of modern multicolor immunophenotyping data. A method that can remove operator bias, enable systematic and thorough analysis of complex high-dimensional data, correlate temporal changes in different subsets and lead to biomarker discovery is needed. Here we apply such a method, called cytometric fingerprinting (CF), to data obtained on peripheral blood B cells from an adult patient with type-1 diabetes who underwent pancreatic islet transplantation. We establish that CF can be used to analyze longitudinal trends in immunophenotypic data, and show that results from CF are comparable to those obtained with traditional gating methods. Both methods reveal the appearance of transitional B cells and subsequent accumulation of more mature B cells following immunosuppression and transplantation. This pattern is consistent with a temporally ordered process of B cell auto-reconstitution. We also show the comparative efficiency of fingerprinting in recognizing relative changes in B cell subsets with respect to time, its ability to couple the data with statistical methods (agglomerative clustering) and its potential to define novel subsets.     

Immunological aspects of pancreatic islet cell transplantation

Type 1 diabetes mellitus (T1DM) is one of the most common diseases of childhood. Insulin discovery changed the clinical course of T1DM from an acutely fatal disease to a chronic disease, but this discovery was later found to be inefficient to control its long-term complications. Whole-pancreas and islet cell transplantation seem to provide a potential solution by restoring the normal physiology of glucose–insulin homeostasis. Although islet transplantation is less invasive than whole-pancreas transplantation, the insulin-free state after islet transplantation remained low (10%) at 5 years after surgery. Here, we will present the specific immunologic challenges that are specific to islet cell transplantation, including instant blood-mediated inflammatory reaction and the recurrence of autoimmunity. We will also briefly discuss the immunosuppressive regimens used and the recent radiologic techniques in the detection of engraftment and early rejection of islet cells.     

Immune modulation of co-transplantation mesenchymal stem cells with islet on T and dendritic cells

Allogeneic pancreatic islet transplantation theoretically represents a cure for type 1 diabetes. However, current immune suppressive therapies are often associated with undesired side effects. Given this problem, and the shortage of human islet donors, the majority of type 1 diabetes patients cannot currently be offered an islet transplant. However, it has been found that mesenchymal stem cells (MSCs) could exert unique immunosuppressive effects both in vitro and in vivo. Herein we transplanted allogeneic 200 islets alone or in combination with MSCs (3 × 10⁶ cells) under the kidney capsules of diabetic C57LB/6 mouse. We found that the ratios of T helper type 1 (Th1) to Th2 and Tc1 to Tc2 were reduced, and the numbers of naive and memory T cells were down‐regulated in peripheral blood after transplantation. In addition, the maturation, endocytosis and interleukin‐12 secretion of dendritic cell (DCs)‐derived bone marrow cells (BMCs) from receptor mice were suppressed. Rejection reaction was alleviated by MSCs which exerted suppressive effects through T lymphocyte subsets and DCs.     

SD大鼠胰岛的分离纯化及培养

目的探讨分离纯化大鼠胰岛素分泌细胞的方法,并评价细胞的生物学特性。方法选用3～4周龄健康成年SD大鼠,常规外科手术显露SD大鼠胰腺和胆总管,在胆总管内插入4.5～5号头皮针后固定,逆行注入预冷的0.5mg/ml的胶原酶V溶液8～10ml,使胰腺膨胀后迅速取出胰腺放入6mlHanks液中38℃消化10min,用含10%胎牛血清的Hanks液终止消化,60目筛网过滤后用Ficoll400非连续梯度离心纯化胰岛。纯化后的细胞用DTZ染色和胰岛素释放试验来分析胰岛素的分泌情况。结果 DTZ染色后β细胞胞浆着色,为均一的猩红色。胰岛细胞分布于Ficoll400浓度为23%～20%和20%～11%的界面之间,纯度高达90%,并且细胞的胰岛素分泌情况良好。结论胶原酶V消化,Ficoll400纯化是一种简单高效的胰岛素分泌细胞分离纯化的方法,可以获取数量多,活性和纯度好的胰岛素分泌细胞。     

Effect of a new drug releasing system on microencapsulated islet transplantation

Objective: This study aimed to develop a novel release system for grafted islets. Materials and methods: A graphene oxide-FTY720 release system was constructed to test the drug loading and releasing capacity. The recipient rats were divided into four groups as following: Experiment group A (EG A) and B (EG B); Control group A (CG A) and B (CG B). In each group, (2000±100) IEQ microencapsulated islets were implanted into the abdominal cavity of the recipients with oral FTY720, local graphene oxide-FTY720 injection, without immunosuppressants, and with graphene oxide-saturated solution respectively. We detected the immunological data, the blood glucose level, and pericapsular overgrowth to show the transplantation effect. Results: 31% of adsorptive FTY720 was released within 6 h, and 82% of FTY720 was released within 48 h. From day 5 to 8, the amount of PBL in EG B was significantly less than those in EG A (P<0.01). The CD3+ and CD8+ T lymphocytes were suppressed 3 days longer in EG B than in EG A. On day 19 posttransplantation, the blood glucose level in EG B was much lower than that in EG A (P<0.01). On the same day, pericapsular overgrowth was grade I in EG B, grade II in other groups. Conclusions: Graphene oxide-FTY720 complex showed a drug releasing effect. Local application of graphene-FTY720 releasing system could decrease the amount of peripheral blood lymphocytes (PBL) and the percentage of CD3 and CD8 T lymphocytes in blood for longer time than oral drug application. This releasing system could achieve a better blood glucose control.