1型糖尿病小鼠胰岛微血管内皮细胞超微结构受损

目的:探讨1型糖尿病小鼠胰岛微血管内皮细胞的超微结构改变。方法:BALB/c小鼠随机分为糖尿病组和对照组,每组6只。应用免疫组化染色检测胰岛素及胰岛微血管血小板内皮细胞黏附分子-1(CD31)表达水平;应用透射电镜观察糖尿病小鼠胰岛β细胞及胰岛微血管超微结构变化。结果:糖尿病组小鼠胰岛β细胞数量、β细胞/α细胞数量比、β细胞分泌颗粒数量及胰岛素阳性染色面积百分比显著降低(P0.01)。糖尿病组小鼠胰岛微血管数量,CD31表达水平显著降低(P0.01);微血管内皮细胞及胰岛周细胞线粒体肿胀变性;微血管基膜厚度显著升高(P0.01)。结论:1型糖尿病小鼠胰岛微血管内皮细胞超微结构受损。     

糖尿病小鼠皮肤与胰腺微循环功能受损

目的 探讨糖尿病小鼠微循环功能变化.方法 BALB/c小鼠随机分为对照组(n=10)和糖尿病组(n=20).常规STZ制备糖尿病模型,心脏灌流后取材,HE染色观察胰腺组织及胰岛微血管形态学及病理改变.用免疫组织化学染色,观察胰岛及胰岛微血管内皮细胞的形态学及病理改变、胰岛素及胰岛微血管PECAM-1的表达.用MoorLDLS检测皮肤微循环血流灌注水平;用Moor VMS-LDF检测胰腺微循环血流灌注及微血管自律运动功能.结果 糖尿病小鼠胰岛形态不规则,完整性破坏;胰岛微血管内皮细胞PECAM-1表达失去连续性;胰岛中胰岛素染色阳性面积显著低于对照组(P＜0.05).糖尿病组小鼠皮肤微循环总血流灌注显著降低(P＜0.01).糖尿病组小鼠胰腺微血管自律运动频率和振幅均显著低于对照组(P＜0.01,P＜0.001).结论 STZ诱导的BALB/c糖尿病小鼠微循环功能受损.     

大鼠胰岛微血管内皮细胞分离、纯化与培养的改进

目的：建立稳定可靠的大鼠胰岛微血管内皮细胞分离培养方法。方法：分离纯化大鼠胰岛后进行内皮细胞选择性培养，使用UEA-1包被的免疫磁珠对胰岛微血管内皮细胞进行纯化。免疫荧光法检测经典的内皮细胞标志物第Ⅷ因子相关抗原（vWF）和CD34的表达和吞噬Dil标记的乙酰化低密度脂蛋白（Dil-Ac-LDL）能力。结果：胰岛培养4-5 d后，可见内皮细胞从贴壁的胰岛内爬出，通过UEA-1包被的免疫磁珠筛选出胰岛微血管内皮细胞接种后24 h细胞开始贴壁。该细胞具有单层生长、接触抑制的特性。大鼠胰岛微血管内皮细胞表达vWF和CD34，可摄取Dil-Ac-LDL。结论：本研究方法是一种较为高效的分离、纯化和培养大鼠胰岛微血管内皮细胞的方法。     

2型糖尿病胰岛β细胞氧化应激的研究进展

目的:探讨2型糖尿病(T2DM)胰岛β细胞氧化应激的研究进展.方法:从氧化应激的概念、胰岛β细胞发生氧化应激的因素、氧化应激损伤胰岛β细胞的机制三方面来探讨T2DM胰岛β细胞氧化应激的研究进展.结果:T2DM胰岛β细胞内含有较低水平的抗氧化系统, 在高糖、高脂等作用下,容易发生氧化应激反应, 氧化应激通过多种途径损伤胰岛β细胞, 使胰岛素合成分泌减少,加重T2DM.结论:T2DM胰岛β细胞容易发生氧化应激是多因素多途径共同作用的复杂过程,积极应用抗氧化剂治疗,能保护胰岛β细胞功能,预防和治疗T2DM的发生与发展.     

内质网应激对胰岛β细胞凋亡的损伤效应

胰岛β细胞具有高度发达的内质网,是对内质网应激最敏感的细胞之一.内质网应激是胰岛β细胞在受到应激条件下,细胞的适应性反应.内外环境中的多种因素：氧化损伤、脂毒性、细胞因子作用等均可打破内质网的稳态,导致蛋白质折叠障碍或错误折叠,进而触发内质网应激.严重而持久的应激将导致β细胞的凋亡,参与多种代谢性疾病乃至多种疾病的发生.综述多种因素诱导的内质网应激对胰岛β细胞的损伤效应.     

共表达sCD40L-Ig和CTLA4-Ig双基因修饰原代人内皮细胞包被大鼠胰岛模型的建立

目的:建立以分离培养的原代人脐静脉内皮细胞包被大鼠胰岛细胞模型。方法:分离培养原代人脐静脉内皮细胞,并分别通过免疫组化试验检测人Ⅷ因子相关抗原,透射电镜观察Weible-palade小体,荧光显微镜观察DiI-Ac-LDL的吞噬效果,对所分离的人脐静脉内皮细胞进行鉴定。分离大鼠胰岛细胞,然后在肝素化培养皿中进行共表达sCD40L-Ig和CTLA4-Ig双基因的内皮细胞包被。对包被后的胰岛细胞进行形态学观察并检测其包被前后的功能。结果:分离培养的内皮细胞表达人Ⅷ因子相关抗原,电镜下可以观察到Weible-palade小体,荧光显微镜下可以观察到内皮细胞对DiI-Ac-LDL的吞噬。共表达sCD40L-Ig和CTLA4-Ig双基因的内皮细胞包被胰岛模型可以观察到内皮细胞的绿色荧光,未包被胰岛和内皮细胞包被胰岛的胰岛素释放指数分别为4.09和3.94,与未包被胰岛实验组相比,内皮细胞包被胰岛对高糖刺激反应无明显差异,但胰岛素释放量有所减少。结论:成功分离培养并鉴定了原代人脐静脉内皮细胞,并成功建立内皮细胞包被胰岛细胞模型,包被后胰岛功能正常。     

骨髓微环境中微血管内皮细胞作用的研究进展

骨生长和骨重建依赖于骨内微环境中成骨细胞、破骨细胞以及其他细胞之间的相互作用。正常情况下，成人骨骼系统具有精细的调节功能，使骨形成和骨吸收过程处于平衡，但在老化或各种病理状态下，这种平衡状态可能就会打破。现已认识到起源于器官组织微循环系统的血管内皮细胞在形态和功能上是不同于起源于作为通道血管的血管内皮细胞。不同器官和组织起源的微血管内皮细胞在形态、基因、表型和功能方面都有差别，这些差别与细胞所起源的器官和组织的功能特性密切相关。近年来研究表明骨内微环境中存在一个复杂的由多种细胞组成的调控网络。而骨髓微血管内皮细胞可能是这个网络中的重要一员。现就骨髓微血管内皮细胞在骨微环境中的作用综述。     

CTLA4Ig基因修饰BMSCs对异种胰岛移植排斥反应的影响

目的 探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白( CTLA4Ig)基因修饰骨髓间充质干细胞(BMSCs)在异种胰岛移植排斥反应中的作用.方法 构建携带CTLA4Ig基因的重组腺病毒载体,用其感染BMSCs并和人胰岛细胞移植到糖尿病大鼠肾包膜下,建立人-大鼠异种胰岛移植模型.观察胰岛移植后糖尿病大鼠血糖变化、生存情况以...     

微循环障碍相关细胞信号通路的研究进展

<正>微循环是直接参与组织、细胞的物质、信息和能量的传递系统。充足的微循环血流灌注对维持组织和器官的正常功能以及调节外周血管阻力具有重要作用。许多研究表明微循环障碍通过影响组织灌注以及血流阻力,从而在各系统疾病的发生发展中发挥重要作用。正常细胞内信号通路网络有利于微血管内皮细胞与组织细胞、炎症细     

促代谢因子betatrophin与糖尿病的研究进展

促代谢因子(betatrophin)是一种新发现的影响糖类及脂类代谢的因子,主要表达在肝脏和脂肪组织中。Betatrophin不仅影响糖脂代谢,而且能在体内外对胰岛β细胞复制起一定调控作用。糖脂代谢紊乱和胰岛β细胞数量减少是糖尿病及糖尿病大血管病变等并发症的重要危险因素,因此betatrophin水平与糖尿病有着密切关系。     

Islet Mass Plays a Critical Role in Initiation, but not Progression, of the Diabetogenic Process in NOD Mice

Pancreatectomy (90%) at a preinsulitis age (7 weeks) protects NOD mice from diabetes while pancreatectomy at a mid-insulitis age (13 weeks) has no such protective effect. The present study examined the effects of islet transplantation in pancreatectomized diabetes-free NOD mice. Transplantation of syngeneic NOD islets as well as allogeneic C3H/He and C57BL/6 islets 3 weeks after pancreatectomy-induced spontaneous diabetes whereas transplantation of xenogeneic Sprague-Dawley rat islets or allogeneic C3H/He skin failed to induce diabetes, demonstrating that the diabetogenic antigen(s) of NOD islets is also expressed by islets of diabetes-resistant mouse strains but not by xenogeneic rat islets. Removal of NOD islet grafts by nephrectomy 7-14 days after transplantation had no effect on the subsequent chronic development of diabetes, while graft removal 3 days after transplantation completely abolished the diabetogenic effect of islet transplantation. Thus, activation of the diabetogenic response by islet isografting takes less than 7 days and the continuous presence of a large islet mass is not required for progression to diabetes. While islet transplantation at 10 and 15 weeks of age caused diabetes, delayed islet transplantation at 23 and 35 weeks of age failed to induce diabetes in pancreatectomized diabetes-free NOD mice, suggesting that initiation of the diabetogenic autoimmune process must take place within a certain window of time. The pancreatectomy/islet transplantation model is excellent for studying the immunological events surrounding activation and progression of the diabetogenic autoimmune process and for identifying the diabetogenic islet antigen(s).     

No Evidence for an Association of Coxsackie Virus Infections during Pregnancy and Early Childhood with Development of Islet Autoantibodies in Offspring of Mothers or Fathers with Type 1 Diabetes

Recent case-control studies reported an increased frequency of antibodies against Coxsackie virus (CV) antigens in patients with newly diagnosed type 1 diabetes and during pregnancy in mothers of diabetic offspring, suggesting a role for CV infections in the pathogenesis of type 1 diabetes (T1D). However, it is not known whether CV infections are causally related to the development of islet autoantibodies or merely represent secondary events in subjects already affected with established islet autoimmunity. Therefore we have prospectively evaluated CV infections from birth, prior to and in parallel with the appearance of islet autoantibodies in offspring of parents with T1D. Using indirect ELISAs, IgG-antibodies (abs) against a panel of CV, and IgG- and IgM-abs to CVB3, CVB4, and CVB5 were measured at 9 months, 2, 5, and 8 years in 28 offspring of mothers or fathers with T1D or of mothers with gestational diabetes who developed persistent islet antibodies (IAA, GADA, IA-2A), and compared to 51 islet autoantibody-negative offspring matched for place and date of birth. CV infections were also determined at delivery in 16 mothers whose offspring developed islet autoantibodies later in life and compared to 110 mothers (matched for HLA-DR, place and date of birth) whose offspring remained islet autoantibody-negative during early childhood. CV-antibodies were detected in only 2/28 (7.1%) offspring who developed islet autoantibodies during follow up and in 7/51 (13.7%) offspring without islet autoantibodies (median follow up time 3.0 years, range 2.0-8.7). CV-IgG abs were detected in one mother (6.3%), whose offspring developed islet autoantibodies during early childhood, compared to 15 mothers (13.6%) with islet autoantibody-negative offspring (P=0.5). Also, partum levels of CV-IgG and CVB3-, -4-, and -5-IgM abs were similar in both groups (median 35 U, 0.08 index (I), 0.08, 0.05 vs. 35 U, 0.06 I, 0.11, and 0.06, resp., P> 0.35 in each case). These data make it unlikely that CV infections during pregnancy or in early childhood play a major role in the induction of islet autoimmunity in offspring of mothers or fathers with T1D or of mothers with gestational diabetes.     

酮症倾向的2型糖尿病研究进展

以自发性酮症起病的2型糖尿病具有1型和2型糖尿病的某些临床特征,其发生与重度胰岛素抵抗及胰岛β细胞功能不足有关。在中止胰岛素治疗后,部分患者可获得正常血糖缓解。测定胰岛自身免疫抗体和胰岛β细胞功能对自发性酮症起病的2型糖尿病的临床管理具有指导意义。     

甜菊糖甙对db/db小鼠胰岛素抵抗和胰岛炎症的影响

目的：大量证据显示,胰岛的炎症反应和胰岛素抵抗是db/db小鼠发生2型糖尿病（DM2）的两个主要因素。甜菊糖甙是一种从菊科植物中提取的天然化合物对于糖尿病病人有许多益处。本实验试图观察甜菊糖甙是否能够改善db/db小鼠的胰岛素抵抗和胰岛炎症反应,从而改善糖代谢。方法：给予db/db小鼠甜菊糖甙或者空白溶剂处理2个月。在处理结束时观察空腹血糖和胰岛素耐量,同时分离胰岛行葡萄糖刺激的胰岛素分泌试验,并行胰腺组织免疫荧光观察胰岛内巨噬细胞浸润情况。结果：甜菊糖甙干预2个月不影响db/db小鼠体重,但显著降低小鼠的空腹血糖,明显改善胰岛素抵抗。同时葡萄糖刺激的胰岛素分泌试验结果提示,甜菊糖甙可显著提高db/db小鼠胰岛在体外分泌胰岛素的能力。此外,免疫荧光提示,甜菊糖甙能显著减轻db/db小鼠胰岛内巨噬细胞浸润程度明显减轻和改善胰岛炎症反应。结论：甜菊糖甙能通过改善胰岛素抵抗和胰岛炎症反应来改善db/db小鼠的糖代谢和胰岛素分泌能力。     

Different islet protein expression profiles during spontaneous diabetes development vs. allograft rejection in BB-DP rats

Type 1 diabetes (T1D) is characterized by selective autoimmune destruction of the insulin producing β-cells in the islets of Langerhans. When the β-cells are destroyed exogenous administration of insulin is necessary for maintenance of glucose homeostasis. Allogeneic islet transplantation has been used as a means to circumvent the need for insulin administration and has in some cases been able to restore endogenous insulin production for years. However, long life immunosuppression is needed to prevent the graft from being rejected and destroyed. Changes in protein expression pattern during spontaneous diabetes development in the diabetes prone BioBreeding rat (BB-DP) have previously been described. In the present study, we have investigated if any of the changes seen in the protein expression pattern during spontaneous diabetes development are also present during allograft rejection of BB-DP rat islets.

Two hundred neonatal islets were syngeneically transplanted under the kidney capsule of 30 day old BB-DP rats and removed prior to and at onset of diabetes. Allogeneically transplanted islets from BB-DP rats were removed before onset of allograft rejection and at maximal islet graft inflammation (rejection). The protein expression profiles of the transplants were visualised by two-dimensional gel (2-DG) electrophoresis, analysed and compared.

In total, 2590 protein spots were visualised and of these 310 changed expression (p < 0.01) in syngeneic islet transplants in the BB-DP rats from 7 days after transplantation until onset of diabetes. In BB-DP islets transplanted to WK rats 53 protein spots (p < 0.01) showed changes in expression when comparing islet grafts removed 7 days after transplantation with islet grafts removed 12 days after transplantation where mononuclear cell infiltration is at its maximum. Only four protein spots (1%) were significantly changed in both syngeneic (autoimmune) and allogeneic islet destruction. When comparing protein expression changes in syngeneic BB-DP islet transplants from 37 days after transplantation to onset of diabetes with protein expression changes in allografts from day 7 to 12 after transplantation only three spot were found to commonly change expression in both situations.

In conclusion, a large number of protein expression changes were detected in both autoimmune islet destruction and allogeneic islet rejection, only two overlaps were detected, suggesting that autoimmune islet destruction and allogeneic islet rejection may result from different target cell responses to signals induced by the cellular infiltrate. Whether this reflects activation of distinct signalling pathways in islet cells is currently unknown and need to be further investigated.     

糖尿病小鼠胰岛β细胞结构的光镜和电镜研究

目的观察2型糖尿病模型db/db小鼠胰岛β细胞的超微结构、胰岛素表达及数量变化,探讨β细胞的病理改变与2型糖尿病病因的关系。方法分别选取3、5、8月龄尾静脉空腹血糖高于10.1mmol/L,且肥胖的db/db自发性糖尿病小鼠,每组8只,作为糖尿病组;选取相应年龄段尾静脉空腹血糖低于6.0mmol/L,体重正常的db/+m表型正常小鼠,每组8只,作为对照组。于相应年龄段取胰尾,用于透射电镜观察、免疫组织化学观察和图像分析。结果电镜下随病情进展,db/db小鼠胰岛β细胞内的分泌颗粒数量明显减少,有的细胞甚至缺如,致密芯电子密度降低,β细胞可见凋亡的早期改变以及细胞核和细胞器的病理改变,细胞间髓样小体增多。免疫组织化学显示同月龄糖尿病组小鼠胰岛β细胞阳性率和胰岛素蛋白平均光密度值(OD值)低于相应对照组(p<0.05),且随着病程的进展,db/db小鼠胰岛β细胞阳性率和胰岛素表达呈现递减趋势(p<0.05)。结论2型糖尿病β细胞的超微结构遭到破坏,引起β细胞合成分泌胰岛素障碍和数量减少,与2型糖尿病病情的轻重有关,反映了2型糖尿病病程不同阶段的病机特点。     

Reduced Food Intake is the Major Contributor to the Protective Effect of Rimonabant on Islet in Established Obesity-Associated Type 2 Diabetes

Purpose

Although the presence of cannabinoid type 1 (CB1) receptor in islets has been reported, the major contributor to the protective effect of rimonabant on islet morphology is unknown. We determined whether the protective effect of rimonabant on pancreatic islet morphology is valid in established diabetes and also whether any effect was independent of decreased food intake.

Materials and Methods

After diabetes was confirmed, Otsuka Long-Evans Tokushima Fatty rats, aged 32 weeks, were treated with rimonabant (30 mg/kg/d, rimonabant group) for 6 weeks. Metabolic profiles and islet morphology of rats treated with rimonabant were compared with those of controls without treatment (control group), a pair-fed control group, and rats treated with rosiglitazone (4 mg/kg/d, rosiglitazone group).

Results

Compared to the control group, rats treated with rimonabant exhibited reduced glycated albumin levels (p<0.001), islet fibrosis (p<0.01), and improved glucose tolerance (p<0.05), with no differences from the pair-fed control group. The retroperitoneal adipose tissue mass was lower in the rimonabant group than those of the pair-fed control and rosiglitazone groups (p<0.05). Rimonabant, pair-fed control, and rosiglitazone groups showed decreased insulin resistance and increased adiponectin, with no differences between the rimonabant and pair-fed control groups.

Conclusion

Rimonabant had a protective effect on islet morphology in vivo even in established diabetes. However, the protective effect was also reproduced by pair-feeding. Thus, the results of this study did not support the significance of islet CB1 receptors in islet protection with rimonabant in established obesity-associated type 2 diabetes.     

Antibody combination therapy targeting CD25, CD70 and CD8 reduces islet inflammation and improves glycaemia in diabetic mice

Destruction of pancreatic islets in type 1 diabetes is caused by infiltrating, primed and activated T cells. In a clinical setting this autoimmune process is already in an advanced stage before intervention therapy can be administered. Therefore, an effective intervention needs to reduce islet inflammation and preserve any remaining islet function. In this study we have investigated the role of targeting activated T cells in reversing autoimmune diabetes. A combination therapy consisting of CD25-, CD70- and CD8-specific monoclonal antibodies was administered to non-obese diabetic (NOD) mice with either new-onset diabetes or with advanced diabetes. In NOD mice with new-onset diabetes antibody combination treatment reversed hyperglycaemia and achieved long-term protection from diabetes (blood glucose <13·9 mmol/l) in >50% of mice. In contrast, in the control, untreated group blood glucose levels continued to increase and none of the mice were protected from diabetes (P < 0·0001). Starting therapy early when hyperglycaemia was relatively mild proved critical, as the mice with advanced diabetes showed less efficient control of blood glucose and shorter life span. Histological analysis (insulitis score) showed islet preservation and reduced immune infiltration in all treated groups, compared to their controls. In conclusion, antibody combination therapy that targets CD25, CD70 and CD8 results in decreased islet infiltration and improved blood glucose levels in NOD mice with established diabetes.     

骨髓间充质干细胞促进胰岛再生的作用与研究现状

背景：研究发现,骨髓间充质干细胞移植入糖尿病大鼠后能够降低其血糖。 目的：综述骨髓间充质干细胞在促进胰岛再生方面的作用与研究现状。 方法：应用计算机检索2003年7月至2011年12月PubMed数据库相关文章,检索词为“bone marrow derive mesenchymal stem cell,islet cells”,并限定文章语言种类为English。同时计算机检索2003年7月至2011年12月万方数据库相关文章,检索词为“骨髓间充质干细胞,胰岛细胞”,并限定文章语言种类为中文。最终纳入符合标准的文献25篇。 结果与结论：目前,移植胰岛治疗糖尿病已取得良好疗效,但由于胰岛来源匮乏和异种或异体来源的胰岛引起免疫排斥反应而难以使众多糖尿患者受益。骨髓间充质干细胞取材方便,容易进行体外分离、培养和纯化,且具有多向分化潜能。若将骨髓间充质干细胞诱导分化为胰岛细胞,可望解决胰岛细胞来源和免疫排斥问题。文章对骨髓间充质干细胞分化为胰岛细胞治疗糖尿病的研究进展进行综述,并指出了存在问题和今后的研究方向。