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Application of Induced Pluripotent Stem Cells in Liver Diseases. 诱导多能干细胞在肝脏疾病中的应用。
Pub Date : 2014-11-05 DOI: 10.3727/215517914X680056
Yue Yu, Xuehao Wang, S. Nyberg
Tens of millions of patients are affected by liver disease worldwide. Many of these patients can benefit from therapy involving hepatocyte transplantation. Liver transplantation is presently the only proven treatment for many medically refractory liver diseases including end-stage liver failure and inherited metabolic liver disease. However, the shortage in transplantable livers prevents over 40% of listed patients per year from receiving a liver transplant; many of these patients die before receiving an organ offer or become too sick to transplant. Therefore, new therapies are needed to supplement whole-organ liver transplantation and reduce mortality on waiting lists worldwide. Furthermore, the remarkable regenerative capacity of hepatocytes in vivo is exemplified by the increasing number of innovative cell-based therapies and animal models of human liver disorders. Induced pluripotent stem cells (iPSCs) have similar properties to those of embryonic stem cells (ESCs) but bypass the ethical concerns of embryo destruction. Therefore, generation of hepatocyte-like cells (HLCs) using iPSC technology may be beneficial for the treatment of severe liver diseases, screening of drug toxicities, basic research of several hepatocytic disorders, and liver transplantation. Here we briefly summarize the growing number of potential applications of iPSCs for treatment of liver disease.
全世界有数千万患者受到肝脏疾病的影响。许多患者可以从肝细胞移植治疗中获益。肝移植是目前唯一被证实的治疗许多难治性肝病的方法,包括终末期肝衰竭和遗传性代谢性肝病。然而,可移植肝脏的短缺使每年超过40%的名单患者无法接受肝移植;许多这样的病人在接受器官移植之前就死了,或者病得太重而无法移植。因此,需要新的治疗方法来补充全器官肝移植,并减少全世界等待名单上的死亡率。此外,越来越多的基于细胞的创新疗法和人类肝脏疾病的动物模型证明了肝细胞在体内的显著再生能力。诱导多能干细胞(iPSCs)具有与胚胎干细胞(ESCs)相似的特性,但绕过了胚胎破坏的伦理问题。因此,利用iPSC技术生成肝细胞样细胞(hlc)可能有利于严重肝脏疾病的治疗、药物毒性筛选、几种肝细胞疾病的基础研究以及肝移植。在这里,我们简要总结了iPSCs在治疗肝脏疾病方面越来越多的潜在应用。
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引用次数: 15
Human Menstrual Blood-Derived Mesenchymal Cells as New Human Feeder Layer System for Human Embryonic Stem Cells. 人经血源性间充质细胞作为新的人胚胎干细胞喂养层体系。
Pub Date : 2014-11-05 DOI: 10.3727/215517914X679265
D. Silva Dos Santos, Vanessa Carvalho Coelho de Oliveira, K. Asensi, L. Vairo, A. B. Carvalho, A. C. Campos de Carvalho, R. Goldenberg
Human embryonic stem cells (hESCs) in general require coculture with feeder layers in order to remain undifferentiated. However, the use of animal-derived feeder layers is incompatible with the clinical setting. The objective of this work was to investigate whether human menstrual blood-derived mesenchymal cells (MBMCs) can substitute mouse embryonic fibroblasts (MEFs) as a feeder layer for H9-hESCs. Both feeder cell types were isolated and cultured in DMEM F-12 and high glucose DMEM, respectively. After three passages, they were inactivated with mitomycin C. To test MBMC feeder layer capacity, hESCs were grown over MBMCs and MEFs under standard conditions. hESC growth, proliferation, survival, and maintenance of the undifferentiated state were evaluated. hESCs grown over MBMCs preserved their undifferentiated state presenting standard morphology, expressing alkaline phosphatase, transcription factors OCT3/4, SOX2, and NANOG by RT-PCR and SSEA-4 and OCT3/4 by immunofluorescence assays. It is noteworthy that none of the feeder cells expressed these proteins. The average colony size of the hESCs on MBMCs was higher when compared to MEFs (p < 0.05; mean ± SD, n = 3). Growth factor analysis revealed amplification of the transcripts for FGF-2, BMP4, TGF-β, VEGF, and PEDF by RT-PCR in MBMCs and MEFs before and after inactivation. Furthermore, similar embryoid body formation, size, and morphology were observed in both feeder layers. In addition, EBs expressed marker genes for the three germ layers cultured on both feeder cells. In conclusion, MBMCs are able to maintain hESCs in an undifferentiated state with comparable efficiency to MEFs. Therefore, MBMCs are a suitable alternative to animal-derived feeder layers for growing hESCs.
人胚胎干细胞(hESCs)通常需要与饲养层共培养以保持未分化。然而,动物源性喂养层的使用与临床环境不相容。本研究的目的是探讨人经血源性间充质细胞(MBMCs)能否替代小鼠胚胎成纤维细胞(mef)作为H9-hESCs的喂养层。分别在DMEM F-12和高糖DMEM中分离培养两种饲养细胞。三次传代后,用丝裂霉素c灭活。为了测试MBMC饲养层容量,在标准条件下,hESCs在MBMC和mef上生长。评估hESC的生长、增殖、存活和维持未分化状态。在mbmc上生长的hESCs保持未分化状态,呈现标准形态,通过RT-PCR表达碱性磷酸酶、转录因子OCT3/4、SOX2和NANOG,通过免疫荧光检测表达SSEA-4和OCT3/4。值得注意的是,没有喂食细胞表达这些蛋白。mbmc上hESCs的平均菌落大小高于mef (p < 0.05;mean±SD, n = 3)。生长因子分析显示,在mbmc和mef失活前后,RT-PCR扩增了FGF-2、BMP4、TGF-β、VEGF和PEDF的转录本。此外,两饲喂层的胚状体形成、大小和形态相似。此外,EBs在两种饲养细胞上均表达了3种胚层的标记基因。综上所述,mbmc能够以与mef相当的效率将hESCs维持在未分化状态。因此,mbmc是替代动物源性饲养层生长hESCs的合适选择。
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引用次数: 7
Oligodendrocytes engineered with migratory proteins as effective graft source for cell transplantation in multiple sclerosis. 迁移蛋白工程少突胶质细胞作为多发性硬化症细胞移植的有效移植物来源。
Pub Date : 2014-04-10 DOI: 10.3727/215517913X674144
Ike de la Pena, Mibel Pabon, Sandra Acosta, Paul R Sanberg, Naoki Tajiri, Yuji Kaneko, Cesar V Borlongan

Multiple sclerosis (MS) is characterized by widespread immunomodulatory demyelination of the CNS resulting in nerve cell dysfunction. Accordingly, treatment strategies have been centered on immunodulation and remyelination, with the former primarily focused on reducing the pathology rather than enhancing myelin repair which the latter targets. While conceding to the emerging view of heterogeneity in the pathology of MS, which precludes variations in degree of immune response (i.e., inflammation) and demyelination, the concept of enhancing myelin repair is appealing since it is likely to provide both disease-reducing and disease-inhibiting therapeutic approach to MS. In this regard, we and several others, have proposed that cell replacement therapy is an effective strategy to repair the myelin in MS. Here, we hypothesize that transplantation of mouse bone marrow-derived oligodendrocytes (BMDOs) and BMDOs transfected with Ephrin proteins (BMDO+Ephrin), which are known to enhance cell and axonal migratory capacity, may produce therapeutic benefits in animal models of MS.

多发性硬化症(MS)的特点是中枢神经系统广泛的免疫调节性脱髓鞘,导致神经细胞功能障碍。因此,治疗策略集中于免疫调节和髓鞘再生,前者主要侧重于减少病理,而不是增强髓鞘修复,后者的目标。虽然承认多发性硬化症病理异质性的新观点,它排除了免疫反应(即炎症)和脱髓鞘程度的变化,但增强髓磷脂修复的概念很有吸引力,因为它可能为多发性硬化症提供减少疾病和抑制疾病的治疗方法。在这方面,我们和其他几个人提出细胞替代疗法是修复多发性硬化症髓磷脂的有效策略。我们假设移植小鼠骨髓源性少突胶质细胞(BMDO)和转染Ephrin蛋白(BMDO+Ephrin)的BMDO可以增强细胞和轴突的迁移能力,可能对MS动物模型产生治疗效果。
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引用次数: 9
Autologous Skeletal Myoblast Sheet Therapy for Porcine Myocardial Infarction Without Increasing Risk of Arrhythmia. 自体成骨肌细胞片治疗猪心肌梗死不增加心律失常风险。
Pub Date : 2014-04-10 DOI: 10.3727/215517913X672254
Y. Terajima, Tatsuya Shimizu, S. Tsuruyama, H. Sekine, Hikaru Ishii, K. Yamazaki, N. Hagiwara, T. Okano
Safety concerns of ventricular tachyarrhythmia have arisen from some clinical trials of autologous skeletal myoblast (SkM) injection therapy. This study examined the effect and safety of SkM sheet therapy in a pig model of chronic myocardial infarction. Minipigs underwent LAD occlusion using a balloon catheter for 2 h, followed by reperfusion. After 28 days, 12 SkM sheets were transplanted onto the infarcted myocardium (sheet group n = 8); the same number of cells was also injected into the myocardium (injection group n = 7), and sham operations were performed as a control (sham group n = 7). Implantable ECG loop recorders (ILR) were placed subcutaneously on the left thorax. At 28 days after transplantation, we assessed cardiac function with MDCT, interrogated ILR, and performed programmed ventricular stimulation (PVS), after which organs were harvested for histopathology. To assess the inflammatory and injury response, inflammation factors and high-sensitive CRP and troponin I were measured at 1, 3, 7, and 28 days after transplantation by the cytokine array method and ELISA, respectively. The sheet group showed an improvement in cardiac function compared with both the injection and sham groups (LVEF change: 5.8 ± 2.7%, -1.0 ± 2.6%, and -3.8 ± 1.8% in the sheet, injection, and sham groups, respectively, p < 0.05). VF was not detected in any group using ILR, while VT was detected in one pig from the injection group. VF was induced in 25.0%, 71.4%, and 28.6% of animals in the sheet, injection, and sham groups, respectively. In the injection group, anti-macrophage-positive cells were observed around the injected cells within the myocardium. Transmission electron microscopic images showed differentiated myofilaments, collagen layers, and a characteristic extracellular matrix surrounding the SkMs in the sheet group. Toroponin I and IL-6 levels were higher in the injection group compared with both the sheet and sham groups. SkM sheets transplanted onto infarcted myocardium improved cardiac function over SkM injection without increasing arrhythmogenicity.
一些自体骨骼肌母细胞(SkM)注射治疗的临床试验引起了室性心动过速的安全性问题。本研究考察了SkM薄片治疗猪慢性心肌梗死模型的效果和安全性。小猪采用球囊导管封堵LAD 2小时,然后再灌注。28天后,将12张SkM薄片移植到梗死心肌上(薄片组n = 8);将相同数量的细胞注射到心肌(注射组n = 7),假手术作为对照(假手术组n = 7)。在左胸皮下放置植入式心电环路记录仪(ILR)。在移植后28天,我们用MDCT评估心脏功能,询问ILR,并进行程序性心室刺激(PVS),之后摘取器官进行组织病理学检查。为了评估炎症和损伤反应,分别在移植后1、3、7和28天采用细胞因子阵列法和ELISA法检测炎症因子和高敏CRP和肌钙蛋白I。与注射组和假手术组比较,纸片组心功能均有改善(纸片组、注射组和假手术组LVEF变化分别为5.8±2.7%、-1.0±2.6%和-3.8±1.8%,p < 0.05)。ILR未检测到VF,而注射组有1头猪检测到VT。单片组、注射组和假手术组分别有25.0%、71.4%和28.6%的动物诱发VF。注射组心肌内注射细胞周围可见抗巨噬细胞阳性细胞。透射电子显微镜图像显示分化的肌丝、胶原层和特征性的细胞外基质围绕着薄板组的SkMs。注射组Toroponin I和IL-6水平均高于空白组和假手术组。与注射SkM相比,移植到梗死心肌上的SkM片改善了心功能,但不增加心律失常性。
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引用次数: 15
ESTROGEN REPLACEMENT THERAPY FOR STROKE. 雌激素替代疗法治疗中风。
Pub Date : 2014-04-10 DOI: 10.3727/215517913X672263
Mibel Pabon, Cyrus Tamboli, Sarosh Tamboli, Sandra Acosta, Ike De La Pena, Paul R Sanberg, Naoki Tajiri, Yuji Kaneko, Cesar V Borlongan

Stroke is the third most common cause of death and severe disability among Western populations. Overall, the incidence of stroke is uniformly higher in men than in women. Stroke is rare in women during the reproductive years, and rapidly increases after menopause, strongly suggesting that estrogen (E2) plays an important role in the prevention of stroke. Ongoing studies are currently evaluating both the benefits and risks associated with E2 replacement therapy and hormone replacement therapy in stroke. Equally important is the role of E2 receptor (ER), as studies indicate that ER populations in several tissue sites may significantly change during stress and aging. Such changes may affect the patient's susceptibility to neurological disorders including stroke, and greatly affect the response to selective E2 receptor modulators (SERMs). Replacement therapies may be inefficient with low ER levels. The goal of this review paper is to discuss an animal model that will allow investigations of the potential therapeutic effects of E2 and its derivatives in stroke. We hypothesize that E2 neuroprotection is, in part, receptor mediated. This hypothesis is a proof of principle approach to demonstrate a role for specific ER subtypes in E2 neuroprotection. To accomplish this, we use a retroviral mediated gene transfer strategy that express subtypes of the ER gene in regions of the rat brain most susceptible to neuronal damage, namely the striatum and cortex. The animal model is exposed to experimental stroke conditions involving middle cerebral artery occlusion (MCAo) method, and eventually the extent of neuronal damage will be evaluated. A reduction in neuronal damage is expected when E2 is administered with specific ER subtypes. From this animal model, an optimal E2 dose and treatment regimen can be determined. The animal model can help identify potential E2-like therapeutics in stroke, and screen for beneficial or toxic additives present in commercial E2 preparations that are currently available. Such studies will be informative in designing drug therapies for stroke.

中风是西方人口死亡和严重残疾的第三大常见原因。总的来说,男性中风的发病率普遍高于女性。中风在育龄期的女性中很少见,而在绝经后迅速增加,这强烈表明雌激素(E2)在预防中风中起着重要作用。目前正在进行的研究正在评估E2替代疗法和激素替代疗法对中风的益处和风险。同样重要的是E2受体(ER)的作用,因为研究表明,在压力和衰老过程中,几个组织部位的ER数量可能会发生显著变化。这种变化可能影响患者对包括中风在内的神经系统疾病的易感性,并极大地影响对选择性E2受体调节剂(SERMs)的反应。雌激素受体水平低时,替代疗法可能无效。这篇综述的目的是讨论一种动物模型,该模型将允许研究E2及其衍生物在中风中的潜在治疗作用。我们假设E2神经保护部分是由受体介导的。这一假设是证明特定ER亚型在E2神经保护中的作用的原理方法。为了实现这一目标,我们使用一种逆转录病毒介导的基因转移策略,在大鼠大脑中最容易受到神经元损伤的区域,即纹状体和皮层,表达内质网基因的亚型。采用大脑中动脉闭塞(MCAo)法对动物模型进行脑卒中实验,最终评估神经元损伤程度。当E2与特定ER亚型一起施用时,预计神经元损伤会减少。根据该动物模型,可以确定最佳E2剂量和治疗方案。该动物模型可以帮助确定潜在的类似E2的中风治疗方法,并筛选目前可用的商业E2制剂中存在的有益或有毒添加剂。这些研究将为中风药物治疗的设计提供信息。
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引用次数: 13
Disease and Stem Cell-Based Analysis of the 2013 ASNTR Meeting. 2013年ASNTR会议的疾病和干细胞分析。
Pub Date : 2014-04-10 DOI: 10.3727/215517913X674153
D. Eve
A wide diversity of subjects are presented at the annual American Society of Neural Therapy and Repair meeting every year, and 2013 was no exception. An insight into the current research trends in regenerative medicine was provided, including studies to elucidate disease mechanisms and the means to treat them. Different methods featured in 2013 included stem cell and tissue transplantation, gene therapy, dietary supplementation, and hydrogels as scaffold systems for the growth of stem cells. Diseases ranged from Parkinson's disease, spinal cord injury, and stroke to traumatic brain injury, pain, and epilepsy. Traumatic brain injury was an increasingly popular topic, highlighting the concerns of soldiers returning from duty overseas. A number of studies looked at ways to treat or elucidate mechanisms for more than one disorder. The studies including stem cells predominantly involved human-derived cells being transplanted, and the most common recipient of stem cells were rodents. Only one autologous transplant study, which featured mouse bone marrow cells being transplanted into mice for the treatment of stroke, was presented this year. The most popular stem cell studied was the neural stem cell, which in some instances was predifferentiated from induced pluripotent stem cells or embryonic stem cells. Other stem cells included the mesenchymal stem cell and adipose, amniotic fluid, and umbilical cord blood-derived cells. Many studies also looked at more than one stem cell type. Combinational studies, such as gene therapy and transplantation, were also commonly explored as well as studies using fetal ventral mesencephalon or spinal cord tissue rather than stem cells. Numerous studies also featured the use of "drugs"-some naturally derived or naturally occurring as well as drug cocktails. A number of possible treatments, including physical therapy and socialization, were explored for a number of different diseases, as well as reports on the current status of four gene therapy clinical trials for the treatment of Parkinson's disease. Other studies assessed possible causes of specific disorders. In this way, the ASNTR provides an important snapshot of developments in the field of regenerative medicine.
每年的美国神经治疗与修复学会年会都会提出各种各样的主题,2013年也不例外。对当前再生医学的研究趋势,包括阐明疾病机制和治疗方法的研究提供了见解。2013年的不同方法包括干细胞和组织移植、基因治疗、膳食补充和水凝胶作为干细胞生长的支架系统。疾病范围从帕金森氏症、脊髓损伤、中风到创伤性脑损伤、疼痛和癫痫。创伤性脑损伤是一个越来越受欢迎的话题,凸显了从海外服役归来的士兵的担忧。许多研究着眼于治疗或阐明一种以上疾病的机制。包括干细胞在内的研究主要涉及移植人类来源的细胞,最常见的干细胞受体是啮齿动物。今年只发表了一项自体移植研究,该研究将小鼠骨髓细胞移植到小鼠体内治疗中风。最受欢迎的干细胞研究是神经干细胞,在某些情况下,它是从诱导多能干细胞或胚胎干细胞中预分化出来的。其他干细胞包括间充质干细胞和脂肪、羊水和脐带血来源的细胞。许多研究也观察了不止一种干细胞类型。联合研究,如基因治疗和移植,以及使用胎儿腹侧中脑或脊髓组织而不是干细胞的研究也被广泛探索。许多研究也以“药物”的使用为特色——一些是天然衍生的或自然产生的,还有药物鸡尾酒。一些可能的治疗方法,包括物理治疗和社会化,对一些不同的疾病进行了探索,并报告了治疗帕金森病的四种基因治疗临床试验的现状。其他研究评估了特定疾病的可能原因。通过这种方式,ASNTR提供了再生医学领域发展的重要快照。
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引用次数: 0
Integration-Free Human Induced Pluripotent Stem Cells From Type 1 Diabetes Patient Skin Fibroblasts Show Increased Abundance of Pancreas-Specific microRNAs. 来自1型糖尿病患者皮肤成纤维细胞的无整合人诱导多能干细胞显示胰腺特异性microrna的丰度增加。
Pub Date : 2014-04-01 DOI: 10.3727/215517914X681785
Jun Liu, M. Joglekar, H. Sumer, A. Hardikar, H. Teede, P. Verma
Type 1 diabetes (T1D) is a disease that is typically associated with multigenetic changes as well as environmental triggers. Disease-specific induced pluripotent stem cells (iPSCs) are preferable cell sources to study T1D, as they are derived from patient cells and therefore capture the disease genotype in a stem cell line. The purpose of this study was to generate integration-free iPSCs from adult skin fibroblasts with T1D. iPSCs were generated by transfection of synthetic mRNAs encoding transcription factors OCT4, SOX2, KLF4, c-MYC, and LIN28. Phase-contrast microscopy, immunocytochemistry, karyotyping, bisulfite genomic sequencing, reverse transcription-polymerase chain reaction, and teratoma formation assay were used to determine reprogramming efficiency, pluripotency, and differentiation potential. Following 18 consecutive days of synthetic mRNA transfections, the T1D patient skin fibroblasts underwent morphological changes, and the aggregated clumps exhibited a human embryonic stem cell (ESC)-like morphology with a high nucleus/cytoplasm ratio. Highly efficient generation of iPSCs was achieved using the mRNA reprogramming approach. The disease-specific iPSCs expressed pluripotency markers, maintained a normal karyotype, and formed teratomas containing tissues representative of the three germ layers when injected into immune-deficient mice. Of interest, the iPSCs showed upregulations of pancreas-specific microRNAs, compared with parental fibroblasts. These data indicate that T1D patient skin fibroblasts can be reprogrammed to pluripotency using a synthetic mRNA approach. These cells can serve as a useful tool for the identification of genes that are involved in autoimmune reactions as well as generating patient-matched β-cells for cell-based therapy.
1型糖尿病(T1D)是一种通常与多基因变化和环境触发因素相关的疾病。疾病特异性诱导多能干细胞(iPSCs)是研究T1D的首选细胞来源,因为它们来源于患者细胞,因此可以捕获干细胞系中的疾病基因型。本研究的目的是从患有T1D的成人皮肤成纤维细胞中生成不整合的iPSCs。通过转染编码转录因子OCT4、SOX2、KLF4、c-MYC和LIN28的合成mrna,生成iPSCs。采用相对比显微镜、免疫细胞化学、核型、亚硫酸盐基因组测序、逆转录聚合酶链反应和畸胎瘤形成测定来确定重编程效率、多能性和分化潜力。在连续18天的合成mRNA转染后,T1D患者皮肤成纤维细胞发生了形态学变化,聚集的团块表现出高核/细胞质比的人胚胎干细胞(ESC)样形态。利用mRNA重编程方法实现了高效生成iPSCs。当注射到免疫缺陷小鼠体内时,疾病特异性iPSCs表达多能性标记物,保持正常核型,并形成含有代表三种胚层的组织的畸胎瘤。有趣的是,与亲代成纤维细胞相比,iPSCs表现出胰腺特异性microrna的上调。这些数据表明,T1D患者皮肤成纤维细胞可以使用合成mRNA方法重新编程为多能性。这些细胞可以作为一种有用的工具,用于鉴定参与自身免疫反应的基因,以及为细胞治疗产生患者匹配的β细胞。
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引用次数: 13
Preculturing Islets With Adipose-Derived Mesenchymal Stromal Cells Is an Effective Strategy for Improving Transplantation Efficiency at the Clinically Preferred Intraportal Site. 用脂肪来源的间充质间质细胞预培养胰岛是提高临床首选门静脉内移植效率的有效策略。
Pub Date : 2014-03-24 DOI: 10.3727/215517914X680047
C. Rackham, P. Dhadda, Aurélie Le Lay, A. King, P. Jones
We have recently shown that preculturing islets with kidney-derived mesenchymal stromal cells (MSCs) improves transplantation outcome in streptozotocin-diabetic mice implanted with a minimal mass of islets beneath the kidney capsule. In the present study, we have extended our previous observations to investigate whether preculturing islets with MSCs can also be used to enhance islet function at the clinically used intraportal site. We have used MSCs derived from adipose tissue, which are more readily accessible than alternative sources in human subjects and can be expanded to clinically efficacious numbers, to preculture islets throughout this study. The in vivo efficacy of grafts consisting of islets precultured alone or with MSCs was tested using a syngeneic streptozotocin-diabetic minimal islet mass model at the clinically relevant intraportal site. Blood glucose concentrations were monitored for 1 month. The vascularization of islets precultured alone or with MSCs was investigated both in vitro and in vivo, using immunohistochemistry. Islet insulin content was measured by radioimmunoassay. The effect of preculturing islets with MSCs on islet function in vitro was investigated using static incubation assays. There was no beneficial angiogenic influence of MSC preculture, as demonstrated by the comparable vascularization of islets precultured alone or with MSCs, both in vitro after 3 days and in vivo 1 month after islet transplantation. However, the in vitro insulin secretory capacity of MSC precultured islets was superior to that of islets precultured alone. In vivo, this was associated with improved glycemia at 7, 14, 21, and 28 days posttransplantation, in recipients of MSC precultured islets compared to islets precultured alone. The area of individual islets within the graft-bearing liver was significantly higher in recipients of MSC precultured islets compared to islets precultured alone. Our experimental studies suggest that preculturing islets with MSCs represents a favorable strategy for improving the efficiency of clinical islet transplantation.
我们最近的研究表明,用肾源间充质间质细胞(MSCs)预培养胰岛可以改善链脲佐菌素糖尿病小鼠在肾被膜下植入少量胰岛的移植结果。在本研究中,我们扩展了之前的观察结果,以研究用MSCs预培养胰岛是否也可以用于增强临床上使用的门静脉内部位的胰岛功能。在整个研究中,我们使用了来自脂肪组织的间充质干细胞来预培养胰岛,这比人类受试者的其他来源更容易获得,并且可以扩展到临床有效的数量。在临床相关的门静脉内部位,使用同基因链脲佐菌素-糖尿病最小胰岛块模型,测试由单独或与MSCs一起预培养的胰岛组成的移植物的体内疗效。监测血糖浓度1个月。采用免疫组织化学方法,在体外和体内研究了单独培养或与MSCs一起预培养的胰岛血管化情况。用放射免疫法测定胰岛胰岛素含量。采用静态孵育法研究体外培养MSCs对胰岛功能的影响。在体外3天和体内1个月的胰岛移植后,单独或与MSCs一起预培养的胰岛血管形成都证明了MSC预培养没有有益的血管生成影响。然而,MSC预培养的胰岛体外胰岛素分泌能力优于单独预培养的胰岛。在体内,与单独预培养的胰岛相比,MSC预培养的胰岛受体在移植后7、14、21和28天的血糖改善相关。移植肝脏内单个胰岛的面积在MSC预培养的胰岛受体中明显高于单独预培养的胰岛。我们的实验研究表明,用间充质干细胞预培养胰岛是提高临床胰岛移植效率的有利策略。
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引用次数: 37
Experimental Nonalcoholic Steatohepatitis Induced by Neonatal Streptozotocin Injection and a High-Fat Diet in Rats. 新生儿链脲佐菌素注射和高脂饮食诱导大鼠实验性非酒精性脂肪性肝炎。
Pub Date : 2013-12-30 DOI: 10.3727/215517913X674252
H. Hsu, M. Dozen, N. Matsuno, H. Obara, R. Tanaka, S. Enosawa
Nonalcoholic steatohepatitis (NASH) has become a major concern in clinical hepatology. To elucidate the disease mechanisms and to develop a treatment, the advent of an appropriate experimental model is crucial. Pregnant Sprague-Dawley rats were fed a high-fat diet from gestational day 16. Two days after birth, the neonates were injected subcutaneously with streptozotocin (STZ) (180, 200, or 256 mg/kg). The mothers were fed a high-fat diet during the nursing period. After being weaned (4 weeks of age), the juvenile rats were fed the same high-fat diet. The survival rates at the time of weaning were 25.6% (180 mg/kg STZ), 22.8% (200 mg/kg STZ), and 19.4% (256 mg/kg STZ). The mean body weight of NASH rats was approximately 20% less than that of normal rats. Serum levels of glucose, alanine aminotransferase, and hyaluronic acid increased in NASH rats. Histologically, typical features of steatohepatitis such as ballooning, inflammatory cell infiltration, and perivenular and pericellular fibrosis were observed. In an indocyanine green loading test, the blood half-life was significantly longer in NASH rats (5.04 ± 2.14 vs. 2.72 ± 0.72 min; p < 0.05), which was suggestive of an impaired hepatobiliary transportation function. Concomitantly, biliary ICG concentrations in NASH rats stabilized in a delayed fashion compared with normal rats. In addition, the amount of bile excreted in NASH rats was significantly lower than that in normal rats (4.32 ± 0.83 vs. 7.66 ± 1.05 mg/min; p < 0.01). The rat NASH model presented here mimics the clinical features of the disease and will be a helpful tool for medical and bioscience research.
非酒精性脂肪性肝炎(NASH)已成为临床肝病学关注的主要问题。为了阐明疾病机制并开发治疗方法,适当的实验模型的出现至关重要。从妊娠第16天开始,给怀孕的Sprague-Dawley大鼠喂食高脂肪饮食。出生2天后,分别皮下注射链脲佐菌素(STZ)(180、200、256 mg/kg)。在哺乳期间,母亲们被喂食高脂肪饮食。断奶后(4周龄),幼鼠喂食相同的高脂肪饲料。断奶时存活率分别为25.6% (180 mg/kg STZ)、22.8% (200 mg/kg STZ)和19.4% (256 mg/kg STZ)。NASH大鼠的平均体重比正常大鼠低约20%。NASH大鼠血清葡萄糖、丙氨酸转氨酶和透明质酸水平升高。组织学上,脂肪性肝炎的典型特征是水肿、炎症细胞浸润、静脉周围和细胞周围纤维化。在吲哚菁绿负荷试验中,NASH大鼠的血液半衰期明显更长(5.04±2.14∶2.72±0.72 min;P < 0.05),提示肝胆运输功能受损。与此同时,与正常大鼠相比,NASH大鼠胆道ICG浓度稳定的时间延迟。此外,NASH大鼠的胆汁排泄量显著低于正常大鼠(4.32±0.83 vs. 7.66±1.05 mg/min;P < 0.01)。这里提出的大鼠NASH模型模拟了该疾病的临床特征,将成为医学和生物科学研究的有用工具。
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引用次数: 4
Maintenance of Viability and Function of Rat Islets With the Use of ROCK Inhibitor Y-27632. ROCK抑制剂Y-27632对大鼠胰岛细胞活力和功能的维持
Pub Date : 2013-12-30 DOI: 10.3727/215517913X674199
Yasuhiro Kubota, H. Noguchi, M. Seita, Takeshi Yuasa, H. Sasamoto, S. Nakaji, T. Okitsu, T. Fujiwara, N. Kobayashi
The number of patients with diabetes is on an increasing trend, thus leading to the belief that diabetes will be the largest medical problem of the 21st century. Islet transplantation can improve glycometabolic control in patients with type 1 diabetes. We studied the viability of Rho-associated protein kinase (ROCK) inhibitor Y-27632 in a culture system in vitro on freshly isolated rat islets. Islet isolation was conducted on a Lewis rat, and studies of culture solutions were split into two groups, one group using ROCK inhibitor Y-27632, and another without. On the seventh day of culture, we evaluated the differences for the cell morphology, viability, and insulin secretion. The Y-27632 group maintained form better than the group without Y-27632. With strong expression of Bcl-2 observed with the Y-27632 group, and expression suppressed with Bax, inhibition of apoptosis by Y-27632 was confirmed. The Y-27632 group predominantly secreted insulin. For islet transplantation, Y-27632 inhibited cell apoptosis in a graft and was also effective in promoting insulin secretion. We were able to confirm effective morphological and functional culture maintenance by separating islets from a rat and adding ROCK inhibitor Y-27632 to the medium.
糖尿病患者的数量呈上升趋势,因此人们认为糖尿病将成为21世纪最大的医疗问题。胰岛移植可改善1型糖尿病患者的糖代谢控制。我们研究了rho相关蛋白激酶(ROCK)抑制剂Y-27632在新分离的大鼠胰岛体外培养系统中的生存能力。Lewis大鼠进行胰岛分离,培养液研究分为两组,一组使用ROCK抑制剂Y-27632,另一组不使用ROCK抑制剂Y-27632。在培养的第7天,我们评估了细胞形态、活力和胰岛素分泌的差异。Y-27632组比无Y-27632组保持形态更好。Y-27632组强表达Bcl-2, Bax抑制Bcl-2表达,证实Y-27632对细胞凋亡有抑制作用。Y-27632组主要分泌胰岛素。对于胰岛移植,Y-27632抑制移植细胞凋亡,并有效促进胰岛素分泌。通过分离大鼠胰岛并在培养基中添加ROCK抑制剂Y-27632,我们能够确认有效的形态和功能培养维持。
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引用次数: 0
期刊
Cell medicine
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