干细胞移植定向分化为胰岛β细胞治疗糖尿病

糖尿病是由于胰岛β细胞破坏或胰岛素抵抗所致的胰岛素绝对或相对缺乏.干细胞具备的增殖能力和分化潜能使其成为胰岛素分泌细胞的潜在米源,还可以解决免疫排斥的难题.胰腺干细胞、胚胎干细胞、骨髓干细胞、脐血干细胞等可定向诱导分化为胰岛β细胞,或使用药物增加胰岛β细胞再生进而发挥治疗糖尿病作用.干细胞治疗糖尿病研究已取得了一定进展,部分实验已纠正糖尿病动物的高血糖状态.但尚需深入研究胰岛的发育和分化机制,这样才能从中获得信息用于诱导胚胎干细胞向β细胞分化,程序性、针对性应用诱导因子以取得更高的分化率,获得更成熟的胰岛素分泌细胞.     

EGF 与 Gastrin 联合应用对糖尿病大鼠胰岛β细胞再生的影响

表皮生长因子（EGF）是肽类生长因子,许多研究证明,EGF 参与体外诱导干细胞向胰岛样细胞分化。已有文献证实 Gastrin 是一种介导胰岛新生的生长因子［1］,观察转导胰岛素启动子调控 Gastrin 基因的小鼠,虽然没有观察到实验组小鼠胰岛细胞数量的变化,但表达 TGF、Gastrin 的胰岛细胞数增多。在大鼠的胰腺导管结扎模型中发现 Gastrin 可促进胰岛β细胞再生,推测可能在该模型中,外源性的 Gastrin 可能是促进胰岛β细胞再生必要因子［2］。本文通过联合应用 Gastrin、EGF,观察 Gastrin、EGF 对糖尿病大鼠胰岛β细胞增殖作用影响。     

骨髓移植后糖尿病小鼠胰岛再生的途径

目的：观察骨髓来源细胞（BMDCs）移植后新生胰岛B细胞的来源。方法：建立糖尿病小鼠模型并分成两组：实验组小鼠（n=10）通过尾静脉移植BMDCs;对照组小鼠（n=10）通过尾静脉注射磷酸盐缓冲液。观察两组小鼠血糖的变化、胰岛β细胞数量、胰腺组织相关标记物的表达。结果：与对照组比较,实验组小鼠移植后第4周血糖出现明显下降（18．7±2．3mmol·L^-1比26．3±2．3mmol·L^-1,P〈0．01）,并持续下降至第6周（17．5±6．7mmol．L^-1比27．6±0．3mmol．L^-1,P〈O．01）,胰岛B细胞数目显著增加（527．9±206．0个比92．8±34．8个,P〈0．01）;实验组小鼠胰岛内发现BrdU和Insulin双阳性细胞,表明有0细胞的自身增殖。同时也发现部分BrdU阳性细胞不表达Insulin,但表达PDX-1;另外,实验发现胰岛内存在Ngn3阳性细胞以及Insulin和G1ucagon双阳性细胞,表明在胰岛内存在可以分化成β细胞的千细胞。结论：BMDCs移植能促进糖尿病小鼠胰岛β细胞的再生,新生的胰岛β细胞来源于胰岛口细胞的自身增殖,也来源于胰岛干细胞的增殖分化。     

胰岛干细胞的研究与进展:诱导产生功能性β细胞的路还有多远?

背景:胰岛素替代治疗是目前最常用于治疗糖尿病的方法,然而这种治疗方法有很多缺陷.胰岛移植作为治疗糖尿病的一种有效方法已经得到公认.胰岛供体的缺乏和移植排斥反应的存在限制了胰岛移植的临床应用.胰岛干细胞可以有效解决这个难题.目的:文章对胰岛β干细胞的来源、诱导分化效率,目前存在的问题和应用前景进行综述.方法:应用计算机检索Pubmed 数据库1990-01/2009-12有关于糖尿病干细胞研究的相关文献,检索关键词:diabetes, stem cell, treatment, islets.通过阅读标题和文摘进行初筛,排除重复性研究、Meta分析类文献,保留23篇文献进行分析总结.结果与结论:由干细胞诱导分化得到的胰腺β细胞可以发挥调节血糖的作用.目前用于诱导分化为胰腺β细胞的干细胞来源包括胚胎干细胞、胰腺干细胞、骨髓间充质干细胞等.在不同的干细胞诱导分化为胰腺β细胞的研究中,分为体内和体外两种诱导分化方法,而体外诱导法多数采用分步诱导的方式,也有部分实验利用基因技术的方法进行诱导.目前胰岛干细胞的研究仍处于初步阶段,如何诱导产生大量的功能性β细胞仍是一个巨大挑战.     

体外诱导胰腺干细胞向胰岛样细胞团的分化

背景：目前由于胰岛来源匮乏,使得胰岛细胞移植治疗糖尿病无法满足临床需求,故体外将胰腺干细胞诱导分化为胰岛成为研究焦点。目的：于体外将小鼠胰腺干细胞诱导成胰岛样细胞团并对其进行相关检测,探寻一种胰腺干细胞体外诱导分化成胰岛及鉴定的技术和方法。方法：体外获得纯化的小鼠胰腺干细胞,采用联合诱导剂对其进行成胰岛方向的诱导分化,并对诱导形成的胰岛样细胞团进行形态学观察、双硫腙染色、RT-PCR和Western blot检测。结果与结论：实验通过细胞形态学和细胞生长特性的观察以及免疫细胞化学染色证实体外成功获得了小鼠胰腺干细胞,采用联合诱导剂将其诱导成胰岛样结构,呈球形,以较细长的蒂部与瓶底连接,双硫腙染色将其染成铁红色。RT-PCR和Western blot法可分别检测到胰岛样细胞团的胰岛素mRNA和胰岛素蛋白。结果证实小鼠胰腺干细胞可体外诱导分化成含β细胞的胰岛样细胞团。     

干细胞诱导分化为胰腺β细胞的研究现状

学术背景：干细胞体外诱导分化成的胰岛细胞可以发挥对血糖的生理性调节作用。大量研究证明可以从胚胎干细胞、胰腺干细胞、骨髓间充质干细胞、神经干细胞、肝脏干细胞或脐血干细胞等诱导分化出胰腺β细胞。目的：深入认识干细胞诱导分化为胰腺β细胞的研究现状。检索策略：由该论文的研究人员应用计算机检索Pubmed数据库1990—01，2006—07的相关文献，检索词“stem cell，differenliation，culture，pancreas”，并限定文章语言种类为English。同时计算机检索维普数据库2000-01，2006—07的相关文献，检索词“干细胞，胰岛，诱导分化”，并限定文章语言种类为中文。共检索到142篇文献，对资料进行初审，纳入标准：①与干细胞和干细胞向胰腺β细胞诱导分化相关的文章。②若内容相似，选取首次实验报告及近5年较权威杂志发表的文章。排除标准：重复或类似的研究。文献评价：文献的来源主要是通过对干细胞向胰腺β细胞诱导分化方面内容进行汇总分析。所选用的30篇文献中，1篇为综述，其余均为临床或基础实验研究。资料综合：胰岛移植是目前治疗Ⅰ型糖尿病和部分Ⅱ型糖尿病效果最理想的方法。由干细胞诱导分化得到的胰腺β细胞可以发挥调节血糖的作用，在许多小鼠糖尿病模型研究中起到了降低血糖的作用。目前用于诱导分化为胰腺β细胞的干细胞来源包括胚胎干细胞、胰腺干细胞、骨髓间充质干细胞、神经干细胞、肝脏干细胞或脐血干细胞等。在不同的干细胞诱导分化为胰腺β细胞的研究中，分为体内和体外两种诱导分化方法，而体外诱导法多数采用分步诱导的方式，也有部分实验利用基因技术的方法进行诱导。结论：不同来源的干细胞可以在体外通过多种方法诱导分化得到胰腺β细胞，但得到的胰腺β细胞数量及其诱导分化方法还有待进一步研究。     

自体胰岛移植的进展及现状研究

部分或全部胰腺切除术后, 患者常有糖耐量异常甚至糖尿病的发生。针对此类胰腺手术, 将切除组织中的胰岛提取后自体移植以保护胰腺内分泌功能的方法逐渐受到重视。本文总结自体胰岛移植自1977年开展首例至今40余年的进展历程, 包括胰岛分离提纯及移植技术的进步、术后效果特别是术后胰岛素不依赖率的改善以及该术式适应证的不断拓展, 并对当前自体胰岛移植现状和所面临的困难进行论述, 对其发展前景作出展望。     

胰岛素瘤患者六例的围术期护理

胰岛素瘤为胰岛β细胞组成的肿瘤,是因为β细胞分泌大量的胰岛素,胰岛素释放入血,引起以低血糖为主的一系列症状的疾病。手术切除肿瘤是根治疾病的最有效方法,且应尽早施行。肿瘤切除后,正常胰岛细胞的分泌功能尚未恢复,加上手术刺激,容易出现反跳性高血糖;另外,胰腺手术后易出现胰瘘、出血等并发症,所以应重视围术期护理。2007年3月至2009年3月,我院成功对6例胰岛素瘤患者施行手术,现将围术期护理报告如下。     

链脲佐菌素对鼠胰腺病理学的影响

目的:观察链脲佐菌素致糖尿病鼠胰腺病理学变化,探讨链脲佐菌素致糖尿病的机制。方法:采用腹腔注射链脲佐菌素建立糖尿病大鼠、小鼠模型并检测血糖、C肽水平,取胰腺HE染色,观察其病理形态变化。结果:大鼠、小鼠腹腔注射链脲佐菌素后血糖均明显升高,C肽水平下降。成模大鼠、小鼠胰岛缩小,β细胞数量减少。结论:链脲佐菌素直接破坏胰岛β细胞,使β细胞数量减少,导致糖尿病的发生。     

1型糖尿病小鼠模型的构建

目的构建1型糖尿病小鼠模型,为进一步研究1型糖尿病发病机制及治疗方法提供依据。方法模型的诱导采用多次小剂量链脲佐菌素(STZ)给药法(MLDSTZ),雄性BALB/c小鼠64只,随机分为3组:模型组、阴性对照组、正常对照组。模型组小鼠连续5 d腹腔注射STZ溶液,阴性对照组腹腔注射柠檬酸缓冲液,正常对照组不注射。给药后观察小鼠一般状况,检测血糖、尿糖,同时以石蜡切片观察小鼠的胰腺组织病理变化。结果 STZ诱导的糖尿病模型鼠与对照相比存在明显的多饮、多食、多尿和体质量减轻等典型的糖尿病表现。在末次注射STZ 3周后,模型组小鼠尿糖均为阳性,对照组均为阴性。在4周后,模型组小鼠空腹血糖水平明显高于对照组,差异有统计学意义(P<0.01)。模型鼠均有不同程度的胰岛萎缩、胰岛β细胞空泡变性、数目减少及不同程度的胰岛炎改变。结论采用MLDSTZ构建的1型糖尿病小鼠模型稳定,方法可靠。     

抗氧化微量营养素对糖尿病小鼠胰岛细胞形态影响

目的　研究抗氧化微量营养素对糖尿病小鼠胰岛细胞形态的影响。方法　使用链脲佐菌素 (STZ)制备糖尿病小鼠模型 ,经每日灌胃补充硒 (Se)、钒 (V)、铬 (Cr)、维生素E(VE)等抗氧化微量营养素 ,每周监测血糖和体重 ,补充 7周后处死动物 ,石蜡包埋胰腺组织 ,制备HE染色切片及免疫组织化学检测。结果　补充抗氧化微量营养素可明显降低糖尿病小鼠血糖水平 ,改善胰岛形态结构。结论　抗氧化微量营养素可调节糖尿病小鼠糖代谢水平 ,对胰岛 β细胞有一定的保护作用。     

Recovery from diabetes in mice by beta cell regeneration

The mechanisms that regulate pancreatic beta cell mass are poorly understood. While autoimmune and pharmacological destruction of insulin-producing beta cells is often irreversible, adult beta cell mass does fluctuate in response to physiological cues including pregnancy and insulin resistance. This plasticity points to the possibility of harnessing the regenerative capacity of the beta cell to treat diabetes. We developed a transgenic mouse model to study the dynamics of beta cell regeneration from a diabetic state. Following doxycycline administration, transgenic mice expressed diphtheria toxin in beta cells, resulting in apoptosis of 70%-80% of beta cells, destruction of islet architecture, and diabetes. Withdrawal of doxycycline resulted in a spontaneous normalization of blood glucose levels and islet architecture and a significant regeneration of beta cell mass with no apparent toxicity of transient hyperglycemia. Lineage tracing analysis indicated that enhanced proliferation of surviving beta cells played the major role in regeneration. Surprisingly, treatment with Sirolimus and Tacrolimus, immunosuppressants used in the Edmonton protocol for human islet transplantation, inhibited beta cell regeneration and prevented the normalization of glucose homeostasis. These results suggest that regenerative therapy for type 1 diabetes may be achieved if autoimmunity is halted using regeneration-compatible drugs.     

Islet Transplantation for Type 1 Diabetes, 2015: What Have We Learned From Alloislet and Autoislet Successes?

The therapeutic potential of pancreatic islet allotransplantation, in which human donor islets are used, as a treatment for type 1 diabetes (T1D) has fascinated diabetes researchers and clinicians for decades. At the same time, the therapeutic potential of total pancreatectomy and islet autotransplantation (TPIAT) (in which one’s own islets are used) as a preventive treatment for diabetes in patients who undergo total pancreatectomy for chronic, painful pancreatitis has received relatively less attention. This is ironic, since the latter has been much more effective than the former in terms of successful glucose management and duration of efficacy. The reasons for this disparity can be partially identified. TPIAT receives very little attention in textbooks of internal medicine and general surgery and surprisingly little print in textbooks of endocrinology and transplantation. T1D is much more predominant than TPIAT as a clinical entity. Provision of insulin or replacement of islets is mandatory and a primary goal in T1D. Provision of pain relief from chronic pancreatitis is the primary goal of total pancreatectomy in TPIAT, whereas treatment of diabetes, and certainly prevention of diabetes, has been more of a secondary consideration. Nonetheless, research developments in both fields have contributed to success in one another. In this Perspective, I will provide a brief history of islet transplantation and contrast and compare the procedures of allo- and autoislet transplantation from three major points of view 1) the procedures of islet procurement, isolation, and transplantation; 2) the role and complications of immunosuppressive drugs; and 3) the posttransplant consequences on β- as well as α-cell function.     

Androgen treatment prevents diabetes in nonobese diabetic mice

The nonobese diabetic (NOD) mouse strain provides a model system for human autoimmune diabetes. This disease model is extensively used not only to examine the etiology and pathogenesis of diabetes, but also as a means to evaluate therapies. In NOD mice, the disease progresses from insulitis to islet destruction and clinical diabetes in a high percentage of female mice. In this study, androgen therapy, begun after the onset of insulitis, was found to prevent islet destruction and diabetes without eliminating the islet inflammation in female NOD mice. However, diabetes can be adoptively transferred into such hormone-treated recipients. The prevention of disease onset by androgen is likely due to the hormonal alteration of the development or function of the immune cells necessary for islet destruction.     

Regeneration of pancreatic beta-cell mass for the treatment of diabetes

INTRODUCTION: The study of the endocrine compartment of the pancreas (the islets of Langerhans) is of great translational interest, as strategies aimed at restoring its mass could become therapies for glycemic dysregulation in type 1 and 2 diabetes mellitus, drug-related diabetes following diabetogenic therapies or hyperglycemic disturbances following the treatment of cancer and nesidioblastosis. Such strategies generally fall under one of the 'three Rs,' namely, replacement (islet transplantation and stem cell differentiation), reprogramming (chiefly from the exocrine compartment of the pancreas) and regeneration (replication and induction of endogenous stem cells). AREAS COVERED: This expert opinion focuses on the latter, as islets are known to regenerate under specific circumstances of physiological (e.g., pregnancy), pathological (e.g., obesity, hyperglycemia, mutations in the glucose-sensing pathway) or experimental (e.g., partial pancreatectomy, cellophane wrapping, partial duct ligation) nature. This review presents the different models of pancreatic regeneration, which encompass the replication of existing beta-cells, reversible epithelial-to-mesenchymal transition and the reactivation of resident stem cells. EXPERT OPINION: Rather than a set mechanism, the pancreas appears to possess a wide range of facultative regeneration pathways. These are discussed in the context of the development of potential strategies aimed at restoring beta-cell function in insulin-dependent diabetes.     

Longitudinal monitoring of pancreatic islet damage in streptozotocin-treated mice with optical coherence microscopy

Pancreatic islets regulate glucose homeostasis in the body, and their dysfunction is closely related to diabetes. Islet transplantation into the anterior chamber of the eye (ACE) was recently developed for both in vivo islet study and diabetes treatment. Optical coherence microscopy (OCM) was previously used to monitor ACE transplanted islets in non-obese diabetic (NOD) mice for detecting autoimmune attack. In this study, OCM was applied to streptozotocin (STZ)-induced diabetic mouse models for the early detection of islet damage. A custom extended-focus OCM (xfOCM) was used to image islet grafts in the ACE longitudinally during STZ-induced beta cell destruction together with conventional bright-field (BF) imaging and invasive glucose level measurement. xfOCM detected local structural changes and vascular degradation during the islet damage which was confirmed by confocal imaging of extracted islet grafts. xfOCM detection of islet damage was more sensitive than BF imaging and glucose measurement. Longitudinal xfOCM images of islet grafts were quantitatively analyzed. All these results showed that xfOCM could be used as a non-invasive and sensitive monitoring method for the early detection of deficient islet grafts in the ACE with potential applications to human subjects.     

Increased islet apoptosis in Pdx1+/- mice

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.     

糖尿病小鼠理想的胰岛移植部位

背景：胰岛移植到糖尿病小鼠不同部位影响胰岛移植成功率。目的：比较糖尿病小鼠小网膜、肾被膜和腋窝3个部位胰岛移植效果,探索一个更理想的移植部位。方法：应用淋巴细胞分离液分离纯化BALB/c小鼠胰岛;将胰岛移植到造模成功的糖尿病C57BL/6小鼠,按照不同的移植部位小网膜、肾被膜和腋窝进行对比观察。结果与结论：分离纯化后获得高纯度胰岛,每只供体可获得胰岛（102±4）个,并具有良好生物活性;胰岛移植到糖尿病小鼠小网膜、肾被膜和腋窝3个部位后,均能降低血糖至正常范围,移植到小网膜与肾被膜的血糖值差异无显著性意义（P〉0.05）,但均低于腋窝血糖值（P〈0.05）。移植后第7天,苏木精-伊红染色见小网膜部位移植胰岛形态基本完整,肾被膜部位移植胰岛形态不规则,腋窝部位移植物胰岛完全被破坏,伴炎症细胞浸润,小网膜和肾被膜降血糖效果优于腋窝。说明小网膜血供充足容量大,能够接纳较大体积的移植物,可能作为胰岛移植的理想部位。     

Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats.

We have examined the effect of chronic (4 wk) hyperglycemia on insulin secretion in vivo in an awake, unstressed rat model. Three groups of animals were examined: control, partial (90%) pancreatectomy, and partial pancreatectomy plus phlorizin, in order to normalize plasma glucose levels. Insulin secretion in response to arginine (2 mM), hyperglycemia (+100 mg/dl), and arginine plus hyperglycemia was evaluated. In diabetic compared with control animals three specific alterations were observed: (a) a deficient insulin response, in both first and second phases, to hyperglycemia; (b) an augmented insulin response to the potentiating effect of arginine under basal glycemic conditions; and (c) an inability of hyperglycemia to augment the potentiating effect of arginine above that observed under basal glycemic conditions. Normalization of the plasma glucose profile by phlorizin treatment in diabetic rats completely corrected all three beta cell abnormalities. These results indicate that chronic hyperglycemia can lead to a defect in in vivo insulin secretion which is reversible when normoglycemia is restored.