BMSCs向胰岛素细胞定向诱导分化及肠壁内自体移植治疗糖尿病的实验研究
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摘要
糖尿病(diabetes mellitus,DM)是目前危害人类特别是老年人健康的主要疾病之一,已成为导致全球人口死亡的第四大疾病。目前全球糖尿病患者已超过2.5亿人,在20年内就可能增至3.8亿人。全球每10秒钟就有2人被诊断为糖尿病,1人死于糖尿病相关性疾病。它是一种慢性非传染性疾病,由体内胰岛素缺乏或耐受所致。糖尿病可分为Ⅰ型和Ⅱ型糖尿病等,其中Ⅰ型糖尿病的病因是在遗传易感基因的基础上在外界环境因素的作用下,引发机体自身免疫功能紊乱,导致胰岛β细胞的损伤和破坏,最终使胰岛素分泌绝对不足。
目前临床上治疗糖尿病的主要方法是药物治疗和饮食疗法,虽然可以在一定程度上控制症状和避免并发症的发生,但只能治标,不能治本,于是胰岛细胞移植便成了治疗糖尿病最有希望的治本方法。供体胰岛细胞不足和免疫排斥限制了这种治疗方法的应用。近年来,随着干细胞理论和干细胞技术的发展,利用干细胞定向诱导分化和干细胞移植技术治疗糖尿病的研究成了糖尿病治疗研究中的一大热点。
干细胞(stem cell)是一类具有自我复制和多向分化潜能的的早期未分化细胞,在适宜的条件下或给予适宜的信号,可以分化为不同类型的具有特征性形态、特异分子标志和特殊功能的成熟细胞。根据干细胞分化潜力的不同,干细胞可以分为全能干细胞(totipotential stem cell)、多能干细胞(multipotentialstem cell)和单能干细胞(unipotential stem cell),单能干细胞又称为组织特异性干细胞(tissue-specific stem cell)或成体干细胞(adult stem cell,ASC)。成体干细胞存在于多种组织和器官中,具有自我复制能力,并能够分化为所在组织的细胞,补充失去生理功能的细胞或修复损伤,以维持组织内环境的稳定。现已明确,不仅胚胎干细胞可以纵向分化为不同组织的前体细胞,进而发育成相应组织的功能细胞,成体干细胞在特定的条件下也可以横向分化为其它组织细胞。由于成体干细胞取材相对容易,来源丰富,可实现个体化治疗、避免了免疫排斥反应及无伦理道德等问题而具有广阔的应用前景。
已有的研究发现胰腺导管上皮细胞、肝卵圆细胞等都有成体干细胞的特性。但因上述细胞来源和自我更新增殖能力有限,诱导分化得到的胰岛素分泌细胞量少,难以成为细胞治疗的种子细胞。骨髓中含有造血干细胞(hematopoieticstem cell)和间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)。研究发现,BMSCs在体内外不同的微环境中可以被诱导分化为多种细胞类型。由于BMSCs具有易于分离培养和体外扩增,并可自体移植无免疫排斥反应的特点,因此利用BMSCs的横向分化潜能,可以根据需要将其诱导分化为特定的组织细胞,用以修复受损的组织或器官。
体内可供胰岛素分泌细胞(insulin-producing cells,IPCs)移植的部位很多,实验中采取的大多数移植部位还没有应用于临床。以往肝脏被认为是最合适的移植部位。然而,研究者们越来越认识到肝脏的生理和解剖特性可以促使移植细胞的死亡;另外,手术方法也限制了足量的移植细胞进入肝门静脉循环。Bendayan和Park报道了十二指肠隐窝和肌层间的结缔组织中存在典型的胰岛结构。他们还发现,链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠的十二指肠壁中的胰岛主要包含胰高血糖素细胞,胰岛素分泌细胞已消失。研究发现,小肠黏膜下层能产生各种有利于细胞的生长因子,例如成纤维细胞生长因子-2(fibroblast growth factor-2)、转化生长因子(transforming growth factor)、血管内皮细胞生长因子(vascular endothelial growth factor)等。近来的研究也证实了体外培养于小肠黏膜下层的人胰岛可以保持较高的功能。体内的实验研究也表明,移植到黏膜下层的胰岛也可以保持较高的功能。
基于上述理念,我们设计了该项研究,目的是为了探讨干细胞自体移植治疗糖尿病的可行性,为此类疾病的治疗提供一条新途径。本研究由三部分组成,即骨髓间充质干细胞的分离培养和诱导分化;大鼠糖尿病动物模型的建立;胰岛素分泌细胞十二指肠壁内自体移植治疗糖尿病。论文发表于“The AnatomicalRecord”。第一部分骨髓间充质干细胞的分离培养和诱导分化
骨髓间充质干细胞的分离培养和体外扩增是进行干细胞诱导分化研究的前提。骨髓间充质干细胞在骨髓中的含量很少,10~6个骨髓单个核细胞中仅含有2～5个BMSCs,因此要获得足量细胞用于后续的诱导分化和自体移植实验,大量扩增纯化是十分必要的。本实验根据BMSCs密度比较低和容易贴壁生长的特点,采用密度梯度离心和贴壁培养相结合的方法。首先用淋巴细胞分离液经过梯度离心去除大部分的造血细胞,再通过贴壁培养、换液去除悬浮生长的造血细胞,用含10%胎牛血清的低糖DMEM培养扩增。结果显示,经密度梯度离心获得的细胞,原代培养4d时即可形成由BMSCs组成的细胞集落;约7～8d时,集落内细胞密集,细胞分裂相减少;用0.25%胰蛋白酶-0.02%EDTA消化细胞,含血清培养基中止消化,进行传代培养,传代细胞增殖较快,约5d可传一代,传至3代时经流式细胞术证实得到较纯的BMSCs。在成功分离培养BMSCs的基础了上,我们进行了干细胞体外定向诱导分化为胰岛素分泌细胞的研究。经过多次实验,我们成功地摸索出了一个BMSCs诱导分化为胰岛素分泌细胞的最佳方案:取生长良好的第3代骨髓间充质干细胞,按2×10~5/ml密度接种于6孔板中,每孔加2ml含10ng/ml碱性成纤维细胞生长因子(basic fibroblast growthfactor,bFGF)、10ng/ml表皮生长因子(epidermal growth factor,EGF)和2%的B_(27)的L—DMEM培养基;培养6天后,去除旧培养液,用D-Hank's液冲洗3遍,然后将细胞培养于含10ng/ml肝细胞生长因子(hepatocyte growth factor,HGF)、10ng/mlβ细胞调节素(β—cellulin)、10ng/ml活化素A(activinA)、10mmol/L尼克酰胺和2%B_(27)的H—DMEM培养基中,继续培养6天。诱导6天时细胞形态略有变化,多呈圆形,少数细胞呈多角形或长梭型,折光性明显增强,开始出现小的细胞簇;第12天时多数细胞已聚集成簇,结构类似胰岛。RT-PCR结果显示,分化细胞能表达胰岛β细胞分化和内分泌功能相关基因。进一步的Western blot和胰岛素免疫细胞化学染色结果表明,分化的细胞能合成和表达胰岛素。这一部分实验的结果说明,我们所设计的复合诱导剂可诱导大鼠骨髓间充质干细胞分化为功能性胰岛β细胞,从而为进一步的细胞自体移植治疗糖尿病提供了细胞来源。
第二部分大鼠糖尿病动物模型的建立
建立一稳定可重复的糖尿病动物模型是进行细胞移植治疗实验研究的基本条件。我们选用大鼠为实验动物,用腹腔注射链脲佐菌素的方法,选择性的破坏胰岛β细胞,成功地建立了该病的动物模型。给药后,大鼠出现明显多饮、多食、多尿、体重下降。大鼠空腹血糖测试结果显示,注射后3d模型组血糖明显升高,超过16.7mmol/L,与给药前血糖相比有显著性差异(P＜0.01),而对照组给药前后血糖无显著性差异(P＞0.05)。给药后1周、2周、3周、4周血糖检测结果显示,模型组大鼠血糖逐渐升高,始终保持在16.7mmol/L以上;而对照组大鼠血糖水平保持正常,两组相比有显著性差异(P＜0.01)。大鼠体重检测结果表明,给药后,模型组大鼠的体重持续下降;而对照组大鼠的体重保持增加,两组相比有显著性差异(P＜0.05)。给药后2周和4周处死模型鼠取胰腺,进行石蜡切片,常规HE染色和胰岛素免疫组化染色,光镜观察显示,胰岛素阳性表达的β细胞明显减少,HE染色可见胰腺外分泌细胞无异常,未发现炎性细胞浸润;在对照组中可观察到胰岛素阳性表达的胰岛β细胞无异常表现。该动物模型的成功建立,为干细胞移植治疗该病的实验研究提供了前提条件。
第三部分胰岛素分泌细胞十二指肠壁内自体移植治疗糖尿病
在成功建立动物模型的基础上,我们从模型鼠的股骨和胫骨中取骨髓间充质干细胞,定向诱导分化12天后,自体移植到供体鼠的十二指肠壁内,并设手术对照组。细胞移植1周后,血糖检测结果显示,胰岛素分泌细胞移植治疗组大鼠血糖恢复正常,而对照组大鼠血糖仍维持高值,两组间有显著性差异(P＜0.01);腹膜腔内糖耐量实验表明,细胞移植治疗组大鼠血糖反应曲线与正常对照组相近。移植后2、4和8周,组织学检测结果显示,移植细胞存活良好,主要存在于肠壁的黏膜下层,少部分分布于粘膜层;随着移植时间延长,细胞逐渐聚集形成细胞簇,胰岛素免疫组织化学染色呈阳性。这说明移植细胞能够在十二指肠壁内存活,并能分泌胰岛素,受体动物可恢复正常血糖水平。
结论
本项研究通过建立糖尿病的大鼠实验模型,成功地分离培养、体外扩增和鉴定了骨髓间充质干细胞,优化了体外诱导分化为胰岛素分泌细胞的条件,成功地将诱导分化后的胰岛素分泌细胞进行糖尿病大鼠十二指肠壁内自体移植并进行了多项移植后检测,结果显示:移植细胞在肠壁内存活,并呈现胰岛素免疫组化阳性着色,糖尿病症状明显改善,说明对糖尿病有明显治疗效果。
Diabetes mellitus (DM) is currently one of major diseases threatening the health of humanity, particularly the elderly people, and has become the fourth-largest disease led to the death of the world's population. The current global diabetes has exceed 250 million people, and could reach to 380 million people in 20 years. All over the world every 10 seconds 2 people were diagnosed with diabetes, and one person died of diabetes-related diseases.DM is a chronic non-communicable diseases caused by lack of insulin or insulin resistance in vivo. It can be divided into type I and type II diabetes. The causes of type I diabetes mellitus are that, at the basis of genetic susceptibility genes and on the role of environmental factors, the body's own immune dysfunction are triggered which resulted in pancreaticβ-cell damage and destruction and the absolute lack of insulin secretion.
Intensive insulin therapy is generally the best option for the treatment of diabetes mellitus. However, this therapy does not avoid serious microvascular complications. Therefore, pancreas and pancreatic islet transplantation are considered to be an effective approach for the treatment of diabetes. Currently, a limited supply of donor pancreatic islets and the risk of immunological rejection prevent widespread use of these approaches. In recent years, many researchers have tried to identify a substitute to pancreatic islet cell transplantation.
Recently more and more attention has been paid to the study on stem cells. The defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cell types. According to their capacity of differentiation, stem cells have been divided into three major groups: totipotential stem cells, multipotential stem cells and unipotential stem cells. Unipotential stem cells, that is adult stem cells, exist in a variety of tissues and organs, have the self-replication capacity and can differentiate into cells of the host organizations to supplement the loss of physiological activity of cells or to repair damage in order to maintain the stability of the organization environment. It is believed that not only embryonic stem cells can differentiate into different organizations precursor cells, which develop into fuctional cells of the corresponding tissue, but also adult stem cells in specific conditions can trans-differentiate into other tissues. Because they can be easily harvested and, when autologously transplanted, there is no immunological rejection and ethical issues have broad application prospects.
Researches have found that pancreatic ductal epithelial cells, hepatic oval cells and so on have characteristics of adult stem cells. However, the limited sources and self-renewal proliferation ability of these cells and a fewer of insulin-producing cells (IPCs) obtained after induction of these cells prevented it from becoming a seed cells for cell therapy of diabetes mellitus. Bone marrow contains hematopoietic stem cells and bone marrow-derived mesenchymal stem cells. The studies found that BMSCs in vitro and in vivo in different microenvironment could be induced to differentiate into multiple cells. Because of BMSCs with easily isolation, culture and in vitro amplification, and autologous transplantation without immune rejection characteristics, so the utilization of transdifferentiation potential of BMSCs, they can be induced to differentiate into specific tissue cells to repair damaged organization or organs.
Many sites have been evaluated for engraftment of pancreatic islet cell; however, most of the experimental sites have no clinical applicability. The liver has been considered as the most adequate site. However, it is increasingly recognized that the liver has physiologic and anatomic characteristics that may contribute to the death of transplanted islets and technical difficulties limit the introduction of sufficient numbers of islets into the portal circulation. Bendayan and Park reported that typical islets of Langerhans are located in the connective tissue between the duodenal crypts and the muscle layer. They also found that islets of streptozotocin (STZ)-induced diabetic rats, mainly containing glucagon cells, were present in the duodenal mucosa and the insulin cells in the islets had disappeared. Moreover, the intestinal submucosa produces various growth factors, such as fibroblast growth factor-2, transforming growth factor and vascular endothelial cell growth factor, which are beneficial for cell growth. A recent study demonstrated that human islets maintain a higher level of function in vitro when cultured in processed submucosa In another study, improved islet function was observed when islets were transplant into a submucosal site. Therefore, we designed this study and the purpose of the study is to explore the potential of stem cells autotransplantation as a therapeutic strategy for diabetes mellitus. This study is a series which consists of three parts as follows: isolation, culture and induced differentiation of BMSCs in vitro; establishment of the rat model for experimental diabetes; aiutotransplantation of insulin-producing cells in the duodenal wall for treating diabetes. The paper was publicated in "The Anatomical Record".
Part one: Isolation, culture and induced differentiation of BMSCs
Isolation, culture and expanding of BMSCs in vitro are the premise of the total experiment. There are a few of BMSCs in bone marrow ( about 2-5 BMSCs per 10~6 mononuclear cells), so it is important to expand and purify the BMSCs for the following experiments. BMSCs were isolated from adult rats using density centrifugation and anchoring culture, then cultured in low-glucose DMEM supplement with 10% fetal bovine serum (FBS) for expanding. Colonies of BMSCs were formed after 4 days at primary culture, after about 7 days, BMSCs got together and the number of division cells decreased. At a confluence of 90%, cells were resuspended with 0.05% trypsin-0.02%EDTA and seeded into fresh flasks. The BMSCs proliferated fast, and could be subcultured about every 5 days. The adherent, spindle-shaped BMSCs at passage 3 were harvested and analyzed by fluorescence-activated cell sorting. The result indicated that ralatively purified BMSCs were obtained after 3 generation.
On the basis of the successful isolation and culture of BMSCs, we carried out an experiment on transdifferentiation of BMSCs into insulin-producing cells in vitro. After repeated experiments, we have successfully explored a best proposal of differentiation of BMSCs into insulin-producing cells: At passage 3, rat BMSCs were cultured in medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 10 ng/ml epidermal growth factor (EGF) and 2% B_(27) at a concentration of 1×10~5 cells/ml. After 6 days, cells were cultured in a medium containing 10 ng/ml hepatocyte growth factor (HGF), 10 ng/mlβ-cellulin , 10 ng/ml activinA, 10 mmol/L nicotinamide and 2% B_(27) for another 6 days. The medium was replaced every 3 days. Cells cultured in medium without an inducer were used as controls. After 6 days, there was a slight change in cell shape which are mostly round , a small number of cells were polygonal or long shuttle type, the refractive index significantly increased, and began to appear in small clusters of cells; 12 days after induction, the structures of cell clusters were similar to those of isolated islets. RT-PCR analysis revealed that these IPCs could express Ins1, Ins2, glucagon, glucose transporter-2 and pancreatic duodenal homeobox-1 (Pdx-1). Insulin production by the IPCs was confirmed by immunocytochemistry and Western blot analysis. The BMSCs in vitro are differentiated into functional isletβcells by compound inducer involved in present experiment which provide a cell source for cell autologous transplantation in the treatment of diabetes.
Part two: Establishment of the rat model for experimental diabetes
According to the typical pathophysiology of diabetes mellitus, diabetes mellitus was introduced by a single intraperitoneal dose (60 mg/kg) of streptozotocin dissolved in citrate buffer (pH 4.4) into 12 h-fasted rats, whereas control rats received only citrate buffer. After administration, polydipsia, weight loss and polyuria were obseved in STZ treatment group. The results of fasting blood glucose test showed that 3d after injection, the blood glucose levels in STZ treatment group were significantly higher than that before administration (P<0.01), while blood glucose levels in the control group before and after administration have no significant difference (P>0.05). 1w , 2w, 3w, and 4w after STZ treatment, the blood glucose levels bagan to elevate and remained to exceed 16.7mmol/L, whereas blood glucose levels in control group remained normal and there are significant difference in two groups (P<0.01 ). In the STZ-treated group, body weight decreased, whereas body weight in control group kept the increase, and there are significant difference in two groups (P<0.05). The animals in each group were humanely killed at 2 weeks and 4 weeks after STZ treatment. Pancreas were harvested for histologic examination. HE staining showed that pancreatic exocrine cells without abnormality could be seen and inflammatory cell infiltration was not found. Immunohistochemical staining for insulin demonstrated that normal pancreaticβcells existed in section of the control group, whereas there was a fewer of insulin-positive cells in the STZ-treated group. This animal model will provide the basis for further studies on pathophysiology and new strategy for stem cell transplantation for the treatment of the disease.
Part three: Autotransplantation of insulin-producing cells in the duodenal wall for treating diabetes.
After BMSCs were isolated from femur and tibia in diabetes rat model, the donor rats supplying the BMSCs were made diabetic. Insulin-producing cells differentiated from BMSCs by the utilization of a compound medium were autologously engrafted into the duodenal wall. Control group rats received only medium without cells. The blood glucose levels in cell-implanted group that received the cellular implantation gradually decreased and reverted to the physiological range after 1 week. However, blood glucose levels in control group which underwent sham surgery without cellular implantation remained elevated (P<0.01). Intraperitoneal glucose tolerance test-induced responses were observed in the cell-implanted group similar to that in the normal rats. 2, 4, and 8 weeks after transplantation, immunohistochemical staining for insulin indicated that insulin-positive cells were frequently observed in the cell-implanted group, and most transplanted cells had migrated into the submucosa of the duodenum and some had migrated into the mucosa. With time transplanted cells gradually gathered to form cell clusters similar to isolated islets. These results indicated that insulin-producing cells differentiated from BMSCs could survive in the duodenal wall, and reversed the body's hyperglycemia.
Conclusion
In summary, we successfully set up an diabetes rat model, isolated, cultured, in vitro expanded and identified BMSCs, and induced BMSCs to differentiate into insulin-producing cells and then autotransplanted IPCs into the duodenal wall. It showed that the engrafted IPCs could survive and reversed hyperglycemia of rats. These results on autologous transplantation of IPCs derived from BMSCs into duodenal wall could offer a novel potential therapeutical protocol for diabetes mellitus.
目前临床上治疗糖尿病的主要方法是药物治疗和饮食疗法,虽然可以在一定程度上控制症状和避免并发症的发生,但只能治标,不能治本,于是胰岛细胞移植便成了治疗糖尿病最有希望的治本方法。供体胰岛细胞不足和免疫排斥限制了这种治疗方法的应用。近年来,随着干细胞理论和干细胞技术的发展,利用干细胞定向诱导分化和干细胞移植技术治疗糖尿病的研究成了糖尿病治疗研究中的一大热点。
干细胞(stem cell)是一类具有自我复制和多向分化潜能的的早期未分化细胞,在适宜的条件下或给予适宜的信号,可以分化为不同类型的具有特征性形态、特异分子标志和特殊功能的成熟细胞。根据干细胞分化潜力的不同,干细胞可以分为全能干细胞(totipotential stem cell)、多能干细胞(multipotentialstem cell)和单能干细胞(unipotential stem cell),单能干细胞又称为组织特异性干细胞(tissue-specific stem cell)或成体干细胞(adult stem cell,ASC)。成体干细胞存在于多种组织和器官中,具有自我复制能力,并能够分化为所在组织的细胞,补充失去生理功能的细胞或修复损伤,以维持组织内环境的稳定。现已明确,不仅胚胎干细胞可以纵向分化为不同组织的前体细胞,进而发育成相应组织的功能细胞,成体干细胞在特定的条件下也可以横向分化为其它组织细胞。由于成体干细胞取材相对容易,来源丰富,可实现个体化治疗、避免了免疫排斥反应及无伦理道德等问题而具有广阔的应用前景。
已有的研究发现胰腺导管上皮细胞、肝卵圆细胞等都有成体干细胞的特性。但因上述细胞来源和自我更新增殖能力有限,诱导分化得到的胰岛素分泌细胞量少,难以成为细胞治疗的种子细胞。骨髓中含有造血干细胞(hematopoieticstem cell)和间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)。研究发现,BMSCs在体内外不同的微环境中可以被诱导分化为多种细胞类型。由于BMSCs具有易于分离培养和体外扩增,并可自体移植无免疫排斥反应的特点,因此利用BMSCs的横向分化潜能,可以根据需要将其诱导分化为特定的组织细胞,用以修复受损的组织或器官。
体内可供胰岛素分泌细胞(insulin-producing cells,IPCs)移植的部位很多,实验中采取的大多数移植部位还没有应用于临床。以往肝脏被认为是最合适的移植部位。然而,研究者们越来越认识到肝脏的生理和解剖特性可以促使移植细胞的死亡;另外,手术方法也限制了足量的移植细胞进入肝门静脉循环。Bendayan和Park报道了十二指肠隐窝和肌层间的结缔组织中存在典型的胰岛结构。他们还发现,链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠的十二指肠壁中的胰岛主要包含胰高血糖素细胞,胰岛素分泌细胞已消失。研究发现,小肠黏膜下层能产生各种有利于细胞的生长因子,例如成纤维细胞生长因子-2(fibroblast growth factor-2)、转化生长因子(transforming growth factor)、血管内皮细胞生长因子(vascular endothelial growth factor)等。近来的研究也证实了体外培养于小肠黏膜下层的人胰岛可以保持较高的功能。体内的实验研究也表明,移植到黏膜下层的胰岛也可以保持较高的功能。
基于上述理念,我们设计了该项研究,目的是为了探讨干细胞自体移植治疗糖尿病的可行性,为此类疾病的治疗提供一条新途径。本研究由三部分组成,即骨髓间充质干细胞的分离培养和诱导分化;大鼠糖尿病动物模型的建立;胰岛素分泌细胞十二指肠壁内自体移植治疗糖尿病。论文发表于“The AnatomicalRecord”。第一部分骨髓间充质干细胞的分离培养和诱导分化
骨髓间充质干细胞的分离培养和体外扩增是进行干细胞诱导分化研究的前提。骨髓间充质干细胞在骨髓中的含量很少,10~6个骨髓单个核细胞中仅含有2～5个BMSCs,因此要获得足量细胞用于后续的诱导分化和自体移植实验,大量扩增纯化是十分必要的。本实验根据BMSCs密度比较低和容易贴壁生长的特点,采用密度梯度离心和贴壁培养相结合的方法。首先用淋巴细胞分离液经过梯度离心去除大部分的造血细胞,再通过贴壁培养、换液去除悬浮生长的造血细胞,用含10%胎牛血清的低糖DMEM培养扩增。结果显示,经密度梯度离心获得的细胞,原代培养4d时即可形成由BMSCs组成的细胞集落;约7～8d时,集落内细胞密集,细胞分裂相减少;用0.25%胰蛋白酶-0.02%EDTA消化细胞,含血清培养基中止消化,进行传代培养,传代细胞增殖较快,约5d可传一代,传至3代时经流式细胞术证实得到较纯的BMSCs。在成功分离培养BMSCs的基础了上,我们进行了干细胞体外定向诱导分化为胰岛素分泌细胞的研究。经过多次实验,我们成功地摸索出了一个BMSCs诱导分化为胰岛素分泌细胞的最佳方案:取生长良好的第3代骨髓间充质干细胞,按2×10~5/ml密度接种于6孔板中,每孔加2ml含10ng/ml碱性成纤维细胞生长因子(basic fibroblast growthfactor,bFGF)、10ng/ml表皮生长因子(epidermal growth factor,EGF)和2%的B_(27)的L—DMEM培养基;培养6天后,去除旧培养液,用D-Hank's液冲洗3遍,然后将细胞培养于含10ng/ml肝细胞生长因子(hepatocyte growth factor,HGF)、10ng/mlβ细胞调节素(β—cellulin)、10ng/ml活化素A(activinA)、10mmol/L尼克酰胺和2%B_(27)的H—DMEM培养基中,继续培养6天。诱导6天时细胞形态略有变化,多呈圆形,少数细胞呈多角形或长梭型,折光性明显增强,开始出现小的细胞簇;第12天时多数细胞已聚集成簇,结构类似胰岛。RT-PCR结果显示,分化细胞能表达胰岛β细胞分化和内分泌功能相关基因。进一步的Western blot和胰岛素免疫细胞化学染色结果表明,分化的细胞能合成和表达胰岛素。这一部分实验的结果说明,我们所设计的复合诱导剂可诱导大鼠骨髓间充质干细胞分化为功能性胰岛β细胞,从而为进一步的细胞自体移植治疗糖尿病提供了细胞来源。
第二部分大鼠糖尿病动物模型的建立
建立一稳定可重复的糖尿病动物模型是进行细胞移植治疗实验研究的基本条件。我们选用大鼠为实验动物,用腹腔注射链脲佐菌素的方法,选择性的破坏胰岛β细胞,成功地建立了该病的动物模型。给药后,大鼠出现明显多饮、多食、多尿、体重下降。大鼠空腹血糖测试结果显示,注射后3d模型组血糖明显升高,超过16.7mmol/L,与给药前血糖相比有显著性差异(P＜0.01),而对照组给药前后血糖无显著性差异(P＞0.05)。给药后1周、2周、3周、4周血糖检测结果显示,模型组大鼠血糖逐渐升高,始终保持在16.7mmol/L以上;而对照组大鼠血糖水平保持正常,两组相比有显著性差异(P＜0.01)。大鼠体重检测结果表明,给药后,模型组大鼠的体重持续下降;而对照组大鼠的体重保持增加,两组相比有显著性差异(P＜0.05)。给药后2周和4周处死模型鼠取胰腺,进行石蜡切片,常规HE染色和胰岛素免疫组化染色,光镜观察显示,胰岛素阳性表达的β细胞明显减少,HE染色可见胰腺外分泌细胞无异常,未发现炎性细胞浸润;在对照组中可观察到胰岛素阳性表达的胰岛β细胞无异常表现。该动物模型的成功建立,为干细胞移植治疗该病的实验研究提供了前提条件。
第三部分胰岛素分泌细胞十二指肠壁内自体移植治疗糖尿病
在成功建立动物模型的基础上,我们从模型鼠的股骨和胫骨中取骨髓间充质干细胞,定向诱导分化12天后,自体移植到供体鼠的十二指肠壁内,并设手术对照组。细胞移植1周后,血糖检测结果显示,胰岛素分泌细胞移植治疗组大鼠血糖恢复正常,而对照组大鼠血糖仍维持高值,两组间有显著性差异(P＜0.01);腹膜腔内糖耐量实验表明,细胞移植治疗组大鼠血糖反应曲线与正常对照组相近。移植后2、4和8周,组织学检测结果显示,移植细胞存活良好,主要存在于肠壁的黏膜下层,少部分分布于粘膜层;随着移植时间延长,细胞逐渐聚集形成细胞簇,胰岛素免疫组织化学染色呈阳性。这说明移植细胞能够在十二指肠壁内存活,并能分泌胰岛素,受体动物可恢复正常血糖水平。
结论
本项研究通过建立糖尿病的大鼠实验模型,成功地分离培养、体外扩增和鉴定了骨髓间充质干细胞,优化了体外诱导分化为胰岛素分泌细胞的条件,成功地将诱导分化后的胰岛素分泌细胞进行糖尿病大鼠十二指肠壁内自体移植并进行了多项移植后检测,结果显示:移植细胞在肠壁内存活,并呈现胰岛素免疫组化阳性着色,糖尿病症状明显改善,说明对糖尿病有明显治疗效果。
Diabetes mellitus (DM) is currently one of major diseases threatening the health of humanity, particularly the elderly people, and has become the fourth-largest disease led to the death of the world's population. The current global diabetes has exceed 250 million people, and could reach to 380 million people in 20 years. All over the world every 10 seconds 2 people were diagnosed with diabetes, and one person died of diabetes-related diseases.DM is a chronic non-communicable diseases caused by lack of insulin or insulin resistance in vivo. It can be divided into type I and type II diabetes. The causes of type I diabetes mellitus are that, at the basis of genetic susceptibility genes and on the role of environmental factors, the body's own immune dysfunction are triggered which resulted in pancreaticβ-cell damage and destruction and the absolute lack of insulin secretion.
Intensive insulin therapy is generally the best option for the treatment of diabetes mellitus. However, this therapy does not avoid serious microvascular complications. Therefore, pancreas and pancreatic islet transplantation are considered to be an effective approach for the treatment of diabetes. Currently, a limited supply of donor pancreatic islets and the risk of immunological rejection prevent widespread use of these approaches. In recent years, many researchers have tried to identify a substitute to pancreatic islet cell transplantation.
Recently more and more attention has been paid to the study on stem cells. The defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cell types. According to their capacity of differentiation, stem cells have been divided into three major groups: totipotential stem cells, multipotential stem cells and unipotential stem cells. Unipotential stem cells, that is adult stem cells, exist in a variety of tissues and organs, have the self-replication capacity and can differentiate into cells of the host organizations to supplement the loss of physiological activity of cells or to repair damage in order to maintain the stability of the organization environment. It is believed that not only embryonic stem cells can differentiate into different organizations precursor cells, which develop into fuctional cells of the corresponding tissue, but also adult stem cells in specific conditions can trans-differentiate into other tissues. Because they can be easily harvested and, when autologously transplanted, there is no immunological rejection and ethical issues have broad application prospects.
Researches have found that pancreatic ductal epithelial cells, hepatic oval cells and so on have characteristics of adult stem cells. However, the limited sources and self-renewal proliferation ability of these cells and a fewer of insulin-producing cells (IPCs) obtained after induction of these cells prevented it from becoming a seed cells for cell therapy of diabetes mellitus. Bone marrow contains hematopoietic stem cells and bone marrow-derived mesenchymal stem cells. The studies found that BMSCs in vitro and in vivo in different microenvironment could be induced to differentiate into multiple cells. Because of BMSCs with easily isolation, culture and in vitro amplification, and autologous transplantation without immune rejection characteristics, so the utilization of transdifferentiation potential of BMSCs, they can be induced to differentiate into specific tissue cells to repair damaged organization or organs.
Many sites have been evaluated for engraftment of pancreatic islet cell; however, most of the experimental sites have no clinical applicability. The liver has been considered as the most adequate site. However, it is increasingly recognized that the liver has physiologic and anatomic characteristics that may contribute to the death of transplanted islets and technical difficulties limit the introduction of sufficient numbers of islets into the portal circulation. Bendayan and Park reported that typical islets of Langerhans are located in the connective tissue between the duodenal crypts and the muscle layer. They also found that islets of streptozotocin (STZ)-induced diabetic rats, mainly containing glucagon cells, were present in the duodenal mucosa and the insulin cells in the islets had disappeared. Moreover, the intestinal submucosa produces various growth factors, such as fibroblast growth factor-2, transforming growth factor and vascular endothelial cell growth factor, which are beneficial for cell growth. A recent study demonstrated that human islets maintain a higher level of function in vitro when cultured in processed submucosa In another study, improved islet function was observed when islets were transplant into a submucosal site. Therefore, we designed this study and the purpose of the study is to explore the potential of stem cells autotransplantation as a therapeutic strategy for diabetes mellitus. This study is a series which consists of three parts as follows: isolation, culture and induced differentiation of BMSCs in vitro; establishment of the rat model for experimental diabetes; aiutotransplantation of insulin-producing cells in the duodenal wall for treating diabetes. The paper was publicated in "The Anatomical Record".
Part one: Isolation, culture and induced differentiation of BMSCs
Isolation, culture and expanding of BMSCs in vitro are the premise of the total experiment. There are a few of BMSCs in bone marrow ( about 2-5 BMSCs per 10~6 mononuclear cells), so it is important to expand and purify the BMSCs for the following experiments. BMSCs were isolated from adult rats using density centrifugation and anchoring culture, then cultured in low-glucose DMEM supplement with 10% fetal bovine serum (FBS) for expanding. Colonies of BMSCs were formed after 4 days at primary culture, after about 7 days, BMSCs got together and the number of division cells decreased. At a confluence of 90%, cells were resuspended with 0.05% trypsin-0.02%EDTA and seeded into fresh flasks. The BMSCs proliferated fast, and could be subcultured about every 5 days. The adherent, spindle-shaped BMSCs at passage 3 were harvested and analyzed by fluorescence-activated cell sorting. The result indicated that ralatively purified BMSCs were obtained after 3 generation.
On the basis of the successful isolation and culture of BMSCs, we carried out an experiment on transdifferentiation of BMSCs into insulin-producing cells in vitro. After repeated experiments, we have successfully explored a best proposal of differentiation of BMSCs into insulin-producing cells: At passage 3, rat BMSCs were cultured in medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 10 ng/ml epidermal growth factor (EGF) and 2% B_(27) at a concentration of 1×10~5 cells/ml. After 6 days, cells were cultured in a medium containing 10 ng/ml hepatocyte growth factor (HGF), 10 ng/mlβ-cellulin , 10 ng/ml activinA, 10 mmol/L nicotinamide and 2% B_(27) for another 6 days. The medium was replaced every 3 days. Cells cultured in medium without an inducer were used as controls. After 6 days, there was a slight change in cell shape which are mostly round , a small number of cells were polygonal or long shuttle type, the refractive index significantly increased, and began to appear in small clusters of cells; 12 days after induction, the structures of cell clusters were similar to those of isolated islets. RT-PCR analysis revealed that these IPCs could express Ins1, Ins2, glucagon, glucose transporter-2 and pancreatic duodenal homeobox-1 (Pdx-1). Insulin production by the IPCs was confirmed by immunocytochemistry and Western blot analysis. The BMSCs in vitro are differentiated into functional isletβcells by compound inducer involved in present experiment which provide a cell source for cell autologous transplantation in the treatment of diabetes.
Part two: Establishment of the rat model for experimental diabetes
According to the typical pathophysiology of diabetes mellitus, diabetes mellitus was introduced by a single intraperitoneal dose (60 mg/kg) of streptozotocin dissolved in citrate buffer (pH 4.4) into 12 h-fasted rats, whereas control rats received only citrate buffer. After administration, polydipsia, weight loss and polyuria were obseved in STZ treatment group. The results of fasting blood glucose test showed that 3d after injection, the blood glucose levels in STZ treatment group were significantly higher than that before administration (P<0.01), while blood glucose levels in the control group before and after administration have no significant difference (P>0.05). 1w , 2w, 3w, and 4w after STZ treatment, the blood glucose levels bagan to elevate and remained to exceed 16.7mmol/L, whereas blood glucose levels in control group remained normal and there are significant difference in two groups (P<0.01 ). In the STZ-treated group, body weight decreased, whereas body weight in control group kept the increase, and there are significant difference in two groups (P<0.05). The animals in each group were humanely killed at 2 weeks and 4 weeks after STZ treatment. Pancreas were harvested for histologic examination. HE staining showed that pancreatic exocrine cells without abnormality could be seen and inflammatory cell infiltration was not found. Immunohistochemical staining for insulin demonstrated that normal pancreaticβcells existed in section of the control group, whereas there was a fewer of insulin-positive cells in the STZ-treated group. This animal model will provide the basis for further studies on pathophysiology and new strategy for stem cell transplantation for the treatment of the disease.
Part three: Autotransplantation of insulin-producing cells in the duodenal wall for treating diabetes.
After BMSCs were isolated from femur and tibia in diabetes rat model, the donor rats supplying the BMSCs were made diabetic. Insulin-producing cells differentiated from BMSCs by the utilization of a compound medium were autologously engrafted into the duodenal wall. Control group rats received only medium without cells. The blood glucose levels in cell-implanted group that received the cellular implantation gradually decreased and reverted to the physiological range after 1 week. However, blood glucose levels in control group which underwent sham surgery without cellular implantation remained elevated (P<0.01). Intraperitoneal glucose tolerance test-induced responses were observed in the cell-implanted group similar to that in the normal rats. 2, 4, and 8 weeks after transplantation, immunohistochemical staining for insulin indicated that insulin-positive cells were frequently observed in the cell-implanted group, and most transplanted cells had migrated into the submucosa of the duodenum and some had migrated into the mucosa. With time transplanted cells gradually gathered to form cell clusters similar to isolated islets. These results indicated that insulin-producing cells differentiated from BMSCs could survive in the duodenal wall, and reversed the body's hyperglycemia.
Conclusion
In summary, we successfully set up an diabetes rat model, isolated, cultured, in vitro expanded and identified BMSCs, and induced BMSCs to differentiate into insulin-producing cells and then autotransplanted IPCs into the duodenal wall. It showed that the engrafted IPCs could survive and reversed hyperglycemia of rats. These results on autologous transplantation of IPCs derived from BMSCs into duodenal wall could offer a novel potential therapeutical protocol for diabetes mellitus.
引文
1. Bretzel RG, Brendel M, Eckhard M, Brandhorst D, Jaeger C, Hatziagelaki E, Federlin K: Islet transplantation: present clinical situation and future aspects. Exp Clin Endocrinol Diabetes 2001,109 Suppl 2:S384-399.
2. Gunnarsson R, Klintmalm G, Lundgren G, Wilczek H, Ostman J, Groth CG: Deterioration in glucose metabolism in pancreatic transplant recipients given cyclosporin. Lancet 1983,2(8349):571-572.
3. Inoue M, Maeno T, Hatchell DL: Survival of allografted pancreatic islets in the subretinal space in rats. Ophthalmic Res 2003, 35(1):48-53.
4. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000, 343(4):230-238.
5. Couzin J: Diabetes. Islet transplants face test of time. Science 2004, 306(5693):34-37.
6. Blyszczuk P, Czyz J, Kania G, Wagner M, Roll U, St-Onge L, Wobus AM: Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci U S A 2003,100(3):998-1003.
7. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R: Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001,292(5520):1389-1394.
8. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M: Insulin production by human embryonic stem cells. Diabetes 2001, 50(8):1691-1697.
9. Hori Y, Rulifson IC, Tsai BC, Heit JJ, Cahoy JD, Kim SK: Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci U S A 2002,99(25):16105-16110.
10. D'Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE: Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 2006,24(11):1392-1401.
11. Jiang W, Shi Y, Zhao D, Chen S, Yong J, Zhang J, Qing T, Sun X, Zhang P, Ding M et al: In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res 2007,17(4):333-344.
12. Shim JH, Kim SE, Woo DH, Kim SK, Oh CH, McKay R, Kim JH: Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia 2007, 50(6): 1228-1238.
13. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F: Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000,49(2): 157-162.
14. Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas MK, Habener JF: Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001, 50(3):521-533.
15. Yao ZX, Qin ML, Liu JJ, Chen XS, Zhou DS: In vitro cultivation of human fetal pancreatic ductal stem cells and their differentiation into insulin-producing cells. World J Gastroenterol 2004, 10(10):1452-1456.
16. Song KH, Ko SH, Ahn YB, Yoo SJ, Chin HM, Kaneto H, Yoon KH, Cha BY, Lee KW, Son HY: In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells. Biochem Biophys Res Commun 2004, 316(4):1094-1100.
17. Rosenberg L, Vinik AI, Pittenger GL, Rafaeloff R, Duguid WP: Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycaemia can be induced by a local growth factor(s). Diabetologia 1996, 39(3):256-262.
18. Friedenstein AJ, Piatetzkv S, II, Petrakova KV: Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol 1966, 16(3):381-390.
19. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M et al: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002,418(6893):41-49.
20. Evans MJ, Kaufman MH: Establishment in culture of pluripotential cells from mouse embryos. Nature 1981, 292(5819):154-156.
21. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science 1998,282(5391):1145-1147.
22. Keller GM: In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995,7(6):862-869.
23. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB: In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002,99(12):8078-8083.
24. Suzuki A, Nakauchi H, Taniguchi H: Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells into insulin-producing cells. Proc Natl Acad Sci U S A 2003, 100(9):5034-5039.
25. Timper K, Seboek D, Eberhardt M, Linscheid P, Christ-Crain M, Keller U, Muller B, Zulewski H: Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem Biophys Res Commun 2006, 341(4):1135-1140.
26. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstein V, Ansorge W et al: Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 2005,33(11):1402-1416.
27. Zalzman M, Gupta S, Giri RK, Berkovich I, Sappal BS, Karnieli O, Zern MA, Fleischer N, Efrat S: Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci U S A 2003,100(12):7253-7258.
28. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999,284(5411):143-147.
29. Conget PA, Minguell JJ: Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999, 181(1):67-73.
30. Lennon DP, Edmison JM, Caplan AI: Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. J Cell Physiol 2001,187(3):345-355.
31. Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM: Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001,98(8):2396-2402.
32. Barry F, Boynton R, Murphy M, Haynesworth S, Zaia J: The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem Biophys Res Commun 2001,289(2):519-524.
33. Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, Deliliers GL: Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002, 30(7):783-791.
34. Hung SC, Chen NJ, Hsieh SL, Li H, Ma HL, Lo WH: Isolation and characterization of size-sieved stem cells from human bone marrow. Stem Cells 2002,20(3):249-258.
35. Jahr H, Bretzel RG: Insulin-positive cells in vitro generated from rat bone marrow stromal cells. Transplant Proc 2003, 35(6):2140-2141.
36. Oh SH, Muzzonigro TM, Bae SH, LaPlante JM, Hatch HM, Petersen BE: Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab Invest 2004, 84(5):607-617.
37. Tang DQ, Cao LZ, Burkhardt BR, Xia CQ, Litherland SA, Atkinson MA, Yang LJ: In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 2004, 53(7):1721-1732.
38. Tayaramma T, Ma B, Rohde M, Mayer H: Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells. Stem Cells 2006, 24(12):2858-2867.
39. Roche E, Assimacopoulos-Jeannet F, Witters LA, Perruchoud B, Yaney G, Corkey B, Asfari M, Prentki M: Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1). J Biol Chem 1997, 272(5):3091-3098.
40. Leibiger B, Wahlander K, Berggren PO, Leibiger IB: Glucose-stimulated insulin biosynthesis depends on insulin-stimulated insulin gene transcription. J Biol Chem 2000,275(39):30153-30156.
41. Otonkoski T, Beattie GM, Mally MI, Ricordi C, Hayek A: Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. J Clin Invest 1993, 92(3): 1459-1466.
42. Mashima H, Ohnishi H, Wakabayashi K, Mine T, Miyagawa J, Hanafusa T, Seno M, Yamada H, Kojima I: Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells. J Clin Invest 1996, 97(7): 1647-1654.
43. Mngomezulu WT, Kramer B: Beneficial effect of nicotinamide on the proportion of insulin cells in developing chick pancreas. Dev Growth Differ 2000,42(2):187-193.
44. Reynolds BA, Weiss S: Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996,175(1):1-13.
45. Soria B: In-vitro differentiation of pancreatic beta-cells. Differentiation 2001, 68(4-5):205-219.
1.Rakieten N,Rakieten ML,Nadkarni MV:Studies on the diabetogenic action of streptozotocin(NSC-37917).Cancer Chemother Rep 1963,29:91-98.
2.Mendez JD,Ramos HG:Animal models in diabetes research.Arch Med Res 1994,25(4):367-375.
3.Bach JF:Immunotherapy of type 1 diabetes:lessons for other autoimmune diseases.Arthritis Res 2002,4 Suppl 3:S3-15.
4.Rees DA,Alcolado JC:Animal models of diabetes mellitus.Diabet Med 2005,22(4):359-370.
5.Mathews CE:Utility of murine models for the study of spontaneous autoimmune type 1 diabetes.Pediatr Diabetes 2005,6(3):165-177.
6.Junod A,Lambert AE,Orci L,Pictet R,Goner AE,Renold AE:Studies of the diabetogenic action of streptozotocin.Proc Soc Exp Biol Med 1967,126(1):201-205.
8.叶燕丽,王辉云,王莲桂,黄丽惜,冯启胜:四氧嘧啶制作大鼠糖尿病模型.实验动物科学与管理 2001,18(2):53,55.
9.徐叔云,卞如濂,陈修,等:药理实验方法学(第三版)[M].人民卫生出版社 2002:1066-1067.
10.Pettepher CC,LeDoux SP,Bohr VA,Wilson GL:Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin.J Biol Chem 1991,266(5):3113-3117.
11.Bennett RA,Pegg AE:Alkylation of DNA in rat tissues following administration of streptozotocin.Cancer Res 1981,41(7):2786-2790.
12.Lupi R,Dotta F,Marselli L,Del Guerra S,Masini M,Santangelo C,Patane G,Boggi U,Piro S,Anello Met al:Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets:evidence that beta-cell death is caspase mediated,partially dependent on ceramide pathway,and Bcl-2 regulated.Diabetes 2002,51(5):1437-1442.
13.沈亚非,徐焱成:链脲佐菌素诱导实验性搪尿病大鼠模型建立的研究.实用 诊断与治疗杂志 2005,19(2):79-80.
1. Limbert C, Path G, Jakob F, Seufert J: Beta-cell replacement and regeneration: Strategies of cell-based therapy for type 1 diabetes mellitus. Diabetes Res Clin Pract 2008, 79(3):389-399.
2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999,284(5411):143-147.
3. Oyagi S, Hirose M, Kojima M, Okuyama M, Kawase M, Nakamura T, Ohgushi H, Yagi K: Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CC14-injured rats. J Hepatol 2006, 44(4):742-748.
4. Hu X, Wang J, Chen J, Luo R, He A, Xie X, Li J: Optimal temporal delivery of bone marrow mesenchymal stem cells in rats with myocardial infarction. Eur J Cardiothorac Surg 2007, 31(3):438-443.
5. Tang DQ, Cao LZ, Burkhardt BR, Xia CQ, Litherland SA, Atkinson MA, Yang LJ: In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 2004, 53(7): 1721-1732.
6. Ianus A, Holz GG, Theise ND, Hussain MA: In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 2003, 111(6):843-850.
7. Oh SH, Muzzonigro TM, Bae SH, LaPlante JM, Hatch HM, Petersen BE: Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab Invest 2004, 84(5):607-617.
8. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T: Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001, 19(12):1134-1140.
9. Furuya H, Kimura T, Murakami M, Katayama K, Hirose K, Yamaguchi A: Revascularization and function of pancreatic islet isografts in diabetic rats following transplantation. Cell Transplant 2003,12(5):537-544.
10. Rayat GR, Korbutt GS, Elliott JF, Rajotte RV: Survival and function of syngeneic rat islet grafts placed within the thymus versus under the kidney capsule. Cell Transplant 1997,6(6):597-602.
11. Lee HC, Ahn KJ, Lim SK, Kim KR, Ahn YS, Lee KE, Huh KB: Allotransplantation of rat islets into the cisterna magna of streptozotocin-induced diabetic rats. Transplantation 1992,53(3):513-516.
12. Inoue M, Maeno T, Hatchell DL: Survival of allografted pancreatic islets in the subretinal space in rats. Ophthalmic Res 2003, 35(1):48-53.
13. Ferguson J, Scothorne RJ: Extended survival of pancreatic islet allografts in the testis of guinea-pigs. JAnat 1977,124(Pt 1):1-8.
14. Watt PC, Mullen Y, Nomura Y, Watanabe Y, Brunicardi FC, Anderson C, Zinner MJ, Passaro EP, Jr.: Successful engraftment of autologous and allogeneic islets into the porcine thymus. J Surg Res 1994, 56(4):367-371.
15. Robertson RP, Davis C, Larsen J, Stratta R, Sutherland DE: Pancreas and islet transplantation in type 1 diabetes. Diabetes Care 2006,29(4):935.
16. Strubbe JH, Steffens AB: Blood glucose levels in portal and peripheral circulation and their relation to food intake in the rat. Physiol Behav 1977, 19(2):303-307.
17. Al-Jazaeri A, Xu BY, Yang H, Macneil D, Leventhal JR, Wright JR, Jr.: Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice. Xenotransplantation 2005,12(3): 189-196.
18. Shapiro AM, Gallant H, Hao E, Wong J, Rajotte R, Yatscoff R, Kneteman N: Portal vein immunosuppressant levels and islet graft toxicity. Transplant Proc 1998, 30(2):641.
19. Guan J, Behme MT, Zucker P, Atkison P, Hramiak I, Zhong R, Dupre J: Glucose turnover and insulin sensitivity in rats with pancreatic islet transplants. Diabetes 1998,47(7): 1020-1026.
20. Kendall DM, Teuscher AU, Robertson RP: Defective glucagon secretion during sustained hypoglycemia following successful islet allo- and autotransplantation in humans. Diabetes 1997,46(1):23-27.
21. Gupta V, Wahoff DC, Rooney DP, Poitout V, Sutherland DE, Kendall DM, Robertson RP: The defective glucagon response from transplanted intrahepatic pancreatic islets during hypoglycemia is transplantation site-determined. Diabetes 1997,46(1 ):28-33.
22. Owen RJ, Ryan EA, O'Kelly K, Lakey JR, McCarthy MC, Paty BW, Bigam DL, Kneteman NM, Korbutt GS, Rajotte RV et al: Percutaneous transhepatic pancreatic islet cell transplantation in type 1 diabetes mellitus: radiologic aspects. Radiology 2003,229(1):165-170.
23. Villiger P, Ryan EA, Owen R, O'Kelly K, Oberholzer J, Al Saif F, Kin T, Wang H, Larsen I, Blitz SL et al: Prevention of bleeding after islet transplantation: lessons learned from a multivariate analysis of 132 cases at a single institution. Am J Transplant 2005, 5(12):2992-2998.
24. Casey JJ, Lakey JR, Ryan EA, Paty BW, Owen R, O'Kelly K, Nanji S, Rajotte RV, Korbutt GS, Bigam D et al: Portal venous pressure changes after sequential clinical islet transplantation. Transplantation 2002, 74(7):913-915.
25. Morrison CP, Wemyss-Holden SA, Dennison AR, Maddern GJ: Islet yield remains a problem in islet autotransplantation. Arch Surg 2002,137(1):80-83.
26. Halberstadt CR, Williams D, Emerich D, Goddard M, Vasconcellos AV, Curry W, Bhatia A, Gores PF: Subcutaneous transplantation of islets into streptozocin-induced diabetic rats. Cell Transplant 2005,14(8):595-605.
27. Figliuzzi M, Cornolti R, Plati T, Rajan N, Adobati F, Remuzzi G, Remuzzi A: Subcutaneous xenotransplantation of bovine pancreatic islets. Biomaterials 2005,26(28):5640-5647.
28. Bendayan M, Park IS: Presence of extrapancreatic islets of Langerhans in the duodenal wall of the rat. Diabetologia 1991, 34(8):604-606.
29. Bendayan M, Park IS: Extrapancreatic islets of Langerhans: ontogenesis and alterations in diabetic condition. J Endocrinol 1997,153(1):73-80.
30. Luo JC, Yang ZM: [Preparation and characteristics of small intestinal submucosa]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2003, 17(5):425-428.
31. Lakey JR, Woods EJ, Zieger MA, Avila JG, Geary WA, Voytik-Harbin SL, Critser JK: Improved islet survival and in vitro function using solubilized small intestinal submucosa. Cell Tissue Bank 2001,2(4):217-224.
32. Bharat A, Benshoff N, Olack B, Ramachandran S, Desai NM, Mohanakumar T: Novel in vivo murine model to study islet potency: engraftment and function. Transplantation 2005,79(11):1627-1630.
33. Caiazzo R, Gmyr V, Hubert T, Delalleau N, Lamberts R, Moerman E, Kerr-Conte J, Pattou F: Evaluation of alternative sites for islet transplantation in the minipig: interest and limits of the gastric submucosa. Transplant Proc 2007, 39(8):2620-2623.
1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. The New England journal of medicine 2000, 343(4):230-238.
2. Gunnarsson R, Klintmalm G, Lundgren G, Wilczek H, Ostman J, Groth CG: Deterioration in glucose metabolism in pancreatic transplant recipients given cyclosporin. Lancet 1983, 2(8349):571-572.
3. Guz Y, Nasir I, Teitelman G: Regeneration of pancreatic beta cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology 2001,142(11):4956-4968.
4. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R: Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science (New York, NY 2001, 292(5520):1389-1394.
5. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB: In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proceedings of the National Academy of Sciences of the United States of America 2002, 99(12):8078-8083.
6. Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patane G, Boggi U, Piro S, Anello M et al: Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 2002, 51(5):1437-1442.
7. Chang CF, Hsu KH, Chiou SH, Ho LL, Fu YS, Hung SC: Fibronectin and pellet suspension culture promote differentiation of human mesenchymal stem cells into insulin producing cells. Journal of biomedical materials research 2008,86(4):1097-1105.
8. Karnieli O, Izhar-Prato Y, Bulvik S, Efrat S: Generation of insulin-producing cells from human bone marrow mesenchymal stem cells by genetic manipulation. Stem cells (Dayton, Ohio) 2007, 25(11):2837-2844.
9. Sun Y, Chen L, Hou XG, Hou WK, Dong JJ, Sun L, Tang KX, Wang B, Song J, Li H et al: Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro. Chinese medical journal 2007,120(9):771-776.
10. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science (New York, NY 1999, 284(5411):143-147.
11. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ: Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001,105(3):369-377.
12. Pettepher CC, LeDoux SP, Bohr VA, Wilson GL: Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin. J Biol Chem 1991,266(5):3113-3117.
13. Dong QY, Chen L, Gao GQ, Wang L, Song J, Chen B, Xu YX, Sun L: Allogeneic diabetic mesenchymal stem cells transplantation in streptozotocin-induced diabetic rat. Clinical and investigative medicine 2008, 31(6):E328-337.
14. Choi KS, Shin JS, Lee JJ, Kim YS, Kim SB, Kim CW: In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract. Biochemical and biophysical research communications 2005,330(4): 1299-1305.
15. Basnet P, Kadota S, Shimizu M, Namba T: Bellidifolin: a potent hypoglycemic agent in streptozotocin (STZ)-induced diabetic rats from Swertia japonica. Planta medica 1994,60(6):507-511.
16. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F: Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000,49(2):157-162.
17. Otonkoski T, Gao R, Lundin K: Stem cells in the treatment of diabetes. Annals of medicine 2005, 37(7):513-520.
18. Burns CJ, Persaud SJ, Jones PM: Stem cell therapy for diabetes: do we need to make beta cells? The Journal of endocrinology 2004,183(3):437-443.
19. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M: Insulin production by human embryonic stem cells. Diabetes 2001, 50(8):1691-1697.
20. Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas MK, Habener JF: Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001, 50(3):521-533.
21. Bonner-Weir S, Sharma A: Pancreatic stem cells. The Journal of pathology 2002,197(4):519-526.
22. Baeyens L, De Breuck S, Lardon J, Mfopou JK, Rooman I, Bouwens L: In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells. Diabetologia 2005,48(1):49-57.
23. Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW: Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002,416(6880):542-545.
24. Ying QL, Nichols J, Evans EP, Smith AG: Changing potency by spontaneous fusion. Nature 2002,416(6880):545-548.
25. Ianus A, Holz GG, Theise ND, Hussain MA: In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. The Journal of clinical investigation 2003, 111(6):843-850.
26. Rayat GR, Korbutt GS, Elliott JF, Rajotte RV: Survival and function of syngeneic rat islet grafts placed within the thymus versus under the kidney capsule. Cell transplantation 1997, 6(6):597-602.
27. Lee HC, Ahn KJ, Lim SK, Kim KR, Ahn YS, Lee KE, Huh KB: Allotransplantation of rat islets into the cisterna magna of streptozotocin-induced diabetic rats. Transplantation 1992, 53(3):513-516.
28. Inoue M, Maeno T, Hatchell DL: Survival of allografted pancreatic islets in the subretinal space in rats. Ophthalmic research 2003, 35(1):48-53.
29. Ferguson J, Scothorne RJ: Extended survival of pancreatic islet allografts in the testis of guinea-pigs. Journal of anatomy 1977,124(Pt 1):1-8.
30. Watt PC, Mullen Y, Nomura Y, Watanabe Y, Brunicardi FC, Anderson C, Zinner MJ, Passaro EP, Jr.: Successful engraftment of autologous and allogeneic islets into the porcine thymus. The Journal of surgical research 1994, 56(4):367-371.
31. Robertson RP, Davis C, Larsen J, Stratta R, Sutherland DE: Pancreas and islet transplantation for patients with diabetes. Diabetes care 2000,23(1):112-116.
32. Al-Jazaeri A, Xu BY, Yang H, Macneil D, Leventhal JR, Wright JR, Jr.: Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice. Xenotransplantation 2005,12(3): 189-196.
33. Shapiro AM, Gallant H, Hao E, Wong J, Rajotte R, Yatscoff R, Kneteman N: Portal vein immunosuppressant levels and islet graft toxicity. Transplantation proceedings 1998, 30(2):641.
34. Guan J, Behme MT, Zucker P, Atkison P, Hramiak I, Zhong R, Dupre J: Glucose turnover and insulin sensitivity in rats with pancreatic islet transplants. Diabetes 1998,47(7):1020-1026.
35. Gupta V, Wahoff DC, Rooney DP, Poitout V, Sutherland DE, Kendall DM, Robertson RP: The defective glucagon response from transplanted intrahepatic pancreatic islets during hypoglycemia is transplantation site-determined. Diabetes 1997,46(1):28-33.
36. Bendayan M, Park IS: Presence of extrapancreatic islets of Langerhans in the duodenal wall of the rat. Diabetologia 1991,34(8):604-606.
37. Bendayan M, Park IS: Extrapancreatic islets of Langerhans: ontogenesis and alterations in diabetic condition. The Journal of endocrinology 1997, 153(1):73-80.
38. Luo JC, Yang ZM: [Preparation and characteristics of small intestinal submucosa]. Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 2003,17(5):425-428.
39. Lakey JR, Woods EJ, Zieger MA, Avila JG, Geary WA, Voytik-Harbin SL, Critser JK: Improved islet survival and in vitro function using solubilized small intestinal submucosa. Cell and tissue banking 2001, 2(4):217-224.
40. Caiazzo R, Gmyr V, Hubert T, Delalleau N, Lamberts R, Moerman E, Kerr-Conte J, Pattou F: Evaluation of alternative sites for islet transplantation in the minipig: interest and limits of the gastric submucosa. Transplantation proceedings 2007, 39(8):2620-2623.
2. Gunnarsson R, Klintmalm G, Lundgren G, Wilczek H, Ostman J, Groth CG: Deterioration in glucose metabolism in pancreatic transplant recipients given cyclosporin. Lancet 1983,2(8349):571-572.
3. Inoue M, Maeno T, Hatchell DL: Survival of allografted pancreatic islets in the subretinal space in rats. Ophthalmic Res 2003, 35(1):48-53.
4. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000, 343(4):230-238.
5. Couzin J: Diabetes. Islet transplants face test of time. Science 2004, 306(5693):34-37.
6. Blyszczuk P, Czyz J, Kania G, Wagner M, Roll U, St-Onge L, Wobus AM: Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci U S A 2003,100(3):998-1003.
7. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R: Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001,292(5520):1389-1394.
8. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M: Insulin production by human embryonic stem cells. Diabetes 2001, 50(8):1691-1697.
9. Hori Y, Rulifson IC, Tsai BC, Heit JJ, Cahoy JD, Kim SK: Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci U S A 2002,99(25):16105-16110.
10. D'Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE: Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 2006,24(11):1392-1401.
11. Jiang W, Shi Y, Zhao D, Chen S, Yong J, Zhang J, Qing T, Sun X, Zhang P, Ding M et al: In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res 2007,17(4):333-344.
12. Shim JH, Kim SE, Woo DH, Kim SK, Oh CH, McKay R, Kim JH: Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia 2007, 50(6): 1228-1238.
13. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F: Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000,49(2): 157-162.
14. Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas MK, Habener JF: Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001, 50(3):521-533.
15. Yao ZX, Qin ML, Liu JJ, Chen XS, Zhou DS: In vitro cultivation of human fetal pancreatic ductal stem cells and their differentiation into insulin-producing cells. World J Gastroenterol 2004, 10(10):1452-1456.
16. Song KH, Ko SH, Ahn YB, Yoo SJ, Chin HM, Kaneto H, Yoon KH, Cha BY, Lee KW, Son HY: In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells. Biochem Biophys Res Commun 2004, 316(4):1094-1100.
17. Rosenberg L, Vinik AI, Pittenger GL, Rafaeloff R, Duguid WP: Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycaemia can be induced by a local growth factor(s). Diabetologia 1996, 39(3):256-262.
18. Friedenstein AJ, Piatetzkv S, II, Petrakova KV: Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol 1966, 16(3):381-390.
19. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M et al: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002,418(6893):41-49.
20. Evans MJ, Kaufman MH: Establishment in culture of pluripotential cells from mouse embryos. Nature 1981, 292(5819):154-156.
21. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science 1998,282(5391):1145-1147.
22. Keller GM: In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995,7(6):862-869.
23. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB: In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002,99(12):8078-8083.
24. Suzuki A, Nakauchi H, Taniguchi H: Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells into insulin-producing cells. Proc Natl Acad Sci U S A 2003, 100(9):5034-5039.
25. Timper K, Seboek D, Eberhardt M, Linscheid P, Christ-Crain M, Keller U, Muller B, Zulewski H: Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem Biophys Res Commun 2006, 341(4):1135-1140.
26. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstein V, Ansorge W et al: Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 2005,33(11):1402-1416.
27. Zalzman M, Gupta S, Giri RK, Berkovich I, Sappal BS, Karnieli O, Zern MA, Fleischer N, Efrat S: Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci U S A 2003,100(12):7253-7258.
28. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999,284(5411):143-147.
29. Conget PA, Minguell JJ: Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999, 181(1):67-73.
30. Lennon DP, Edmison JM, Caplan AI: Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. J Cell Physiol 2001,187(3):345-355.
31. Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM: Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001,98(8):2396-2402.
32. Barry F, Boynton R, Murphy M, Haynesworth S, Zaia J: The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem Biophys Res Commun 2001,289(2):519-524.
33. Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, Deliliers GL: Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002, 30(7):783-791.
34. Hung SC, Chen NJ, Hsieh SL, Li H, Ma HL, Lo WH: Isolation and characterization of size-sieved stem cells from human bone marrow. Stem Cells 2002,20(3):249-258.
35. Jahr H, Bretzel RG: Insulin-positive cells in vitro generated from rat bone marrow stromal cells. Transplant Proc 2003, 35(6):2140-2141.
36. Oh SH, Muzzonigro TM, Bae SH, LaPlante JM, Hatch HM, Petersen BE: Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab Invest 2004, 84(5):607-617.
37. Tang DQ, Cao LZ, Burkhardt BR, Xia CQ, Litherland SA, Atkinson MA, Yang LJ: In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 2004, 53(7):1721-1732.
38. Tayaramma T, Ma B, Rohde M, Mayer H: Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells. Stem Cells 2006, 24(12):2858-2867.
39. Roche E, Assimacopoulos-Jeannet F, Witters LA, Perruchoud B, Yaney G, Corkey B, Asfari M, Prentki M: Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1). J Biol Chem 1997, 272(5):3091-3098.
40. Leibiger B, Wahlander K, Berggren PO, Leibiger IB: Glucose-stimulated insulin biosynthesis depends on insulin-stimulated insulin gene transcription. J Biol Chem 2000,275(39):30153-30156.
41. Otonkoski T, Beattie GM, Mally MI, Ricordi C, Hayek A: Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. J Clin Invest 1993, 92(3): 1459-1466.
42. Mashima H, Ohnishi H, Wakabayashi K, Mine T, Miyagawa J, Hanafusa T, Seno M, Yamada H, Kojima I: Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells. J Clin Invest 1996, 97(7): 1647-1654.
43. Mngomezulu WT, Kramer B: Beneficial effect of nicotinamide on the proportion of insulin cells in developing chick pancreas. Dev Growth Differ 2000,42(2):187-193.
44. Reynolds BA, Weiss S: Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996,175(1):1-13.
45. Soria B: In-vitro differentiation of pancreatic beta-cells. Differentiation 2001, 68(4-5):205-219.
1.Rakieten N,Rakieten ML,Nadkarni MV:Studies on the diabetogenic action of streptozotocin(NSC-37917).Cancer Chemother Rep 1963,29:91-98.
2.Mendez JD,Ramos HG:Animal models in diabetes research.Arch Med Res 1994,25(4):367-375.
3.Bach JF:Immunotherapy of type 1 diabetes:lessons for other autoimmune diseases.Arthritis Res 2002,4 Suppl 3:S3-15.
4.Rees DA,Alcolado JC:Animal models of diabetes mellitus.Diabet Med 2005,22(4):359-370.
5.Mathews CE:Utility of murine models for the study of spontaneous autoimmune type 1 diabetes.Pediatr Diabetes 2005,6(3):165-177.
6.Junod A,Lambert AE,Orci L,Pictet R,Goner AE,Renold AE:Studies of the diabetogenic action of streptozotocin.Proc Soc Exp Biol Med 1967,126(1):201-205.
8.叶燕丽,王辉云,王莲桂,黄丽惜,冯启胜:四氧嘧啶制作大鼠糖尿病模型.实验动物科学与管理 2001,18(2):53,55.
9.徐叔云,卞如濂,陈修,等:药理实验方法学(第三版)[M].人民卫生出版社 2002:1066-1067.
10.Pettepher CC,LeDoux SP,Bohr VA,Wilson GL:Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin.J Biol Chem 1991,266(5):3113-3117.
11.Bennett RA,Pegg AE:Alkylation of DNA in rat tissues following administration of streptozotocin.Cancer Res 1981,41(7):2786-2790.
12.Lupi R,Dotta F,Marselli L,Del Guerra S,Masini M,Santangelo C,Patane G,Boggi U,Piro S,Anello Met al:Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets:evidence that beta-cell death is caspase mediated,partially dependent on ceramide pathway,and Bcl-2 regulated.Diabetes 2002,51(5):1437-1442.
13.沈亚非,徐焱成:链脲佐菌素诱导实验性搪尿病大鼠模型建立的研究.实用 诊断与治疗杂志 2005,19(2):79-80.
1. Limbert C, Path G, Jakob F, Seufert J: Beta-cell replacement and regeneration: Strategies of cell-based therapy for type 1 diabetes mellitus. Diabetes Res Clin Pract 2008, 79(3):389-399.
2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999,284(5411):143-147.
3. Oyagi S, Hirose M, Kojima M, Okuyama M, Kawase M, Nakamura T, Ohgushi H, Yagi K: Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CC14-injured rats. J Hepatol 2006, 44(4):742-748.
4. Hu X, Wang J, Chen J, Luo R, He A, Xie X, Li J: Optimal temporal delivery of bone marrow mesenchymal stem cells in rats with myocardial infarction. Eur J Cardiothorac Surg 2007, 31(3):438-443.
5. Tang DQ, Cao LZ, Burkhardt BR, Xia CQ, Litherland SA, Atkinson MA, Yang LJ: In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 2004, 53(7): 1721-1732.
6. Ianus A, Holz GG, Theise ND, Hussain MA: In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 2003, 111(6):843-850.
7. Oh SH, Muzzonigro TM, Bae SH, LaPlante JM, Hatch HM, Petersen BE: Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab Invest 2004, 84(5):607-617.
8. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T: Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001, 19(12):1134-1140.
9. Furuya H, Kimura T, Murakami M, Katayama K, Hirose K, Yamaguchi A: Revascularization and function of pancreatic islet isografts in diabetic rats following transplantation. Cell Transplant 2003,12(5):537-544.
10. Rayat GR, Korbutt GS, Elliott JF, Rajotte RV: Survival and function of syngeneic rat islet grafts placed within the thymus versus under the kidney capsule. Cell Transplant 1997,6(6):597-602.
11. Lee HC, Ahn KJ, Lim SK, Kim KR, Ahn YS, Lee KE, Huh KB: Allotransplantation of rat islets into the cisterna magna of streptozotocin-induced diabetic rats. Transplantation 1992,53(3):513-516.
12. Inoue M, Maeno T, Hatchell DL: Survival of allografted pancreatic islets in the subretinal space in rats. Ophthalmic Res 2003, 35(1):48-53.
13. Ferguson J, Scothorne RJ: Extended survival of pancreatic islet allografts in the testis of guinea-pigs. JAnat 1977,124(Pt 1):1-8.
14. Watt PC, Mullen Y, Nomura Y, Watanabe Y, Brunicardi FC, Anderson C, Zinner MJ, Passaro EP, Jr.: Successful engraftment of autologous and allogeneic islets into the porcine thymus. J Surg Res 1994, 56(4):367-371.
15. Robertson RP, Davis C, Larsen J, Stratta R, Sutherland DE: Pancreas and islet transplantation in type 1 diabetes. Diabetes Care 2006,29(4):935.
16. Strubbe JH, Steffens AB: Blood glucose levels in portal and peripheral circulation and their relation to food intake in the rat. Physiol Behav 1977, 19(2):303-307.
17. Al-Jazaeri A, Xu BY, Yang H, Macneil D, Leventhal JR, Wright JR, Jr.: Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice. Xenotransplantation 2005,12(3): 189-196.
18. Shapiro AM, Gallant H, Hao E, Wong J, Rajotte R, Yatscoff R, Kneteman N: Portal vein immunosuppressant levels and islet graft toxicity. Transplant Proc 1998, 30(2):641.
19. Guan J, Behme MT, Zucker P, Atkison P, Hramiak I, Zhong R, Dupre J: Glucose turnover and insulin sensitivity in rats with pancreatic islet transplants. Diabetes 1998,47(7): 1020-1026.
20. Kendall DM, Teuscher AU, Robertson RP: Defective glucagon secretion during sustained hypoglycemia following successful islet allo- and autotransplantation in humans. Diabetes 1997,46(1):23-27.
21. Gupta V, Wahoff DC, Rooney DP, Poitout V, Sutherland DE, Kendall DM, Robertson RP: The defective glucagon response from transplanted intrahepatic pancreatic islets during hypoglycemia is transplantation site-determined. Diabetes 1997,46(1 ):28-33.
22. Owen RJ, Ryan EA, O'Kelly K, Lakey JR, McCarthy MC, Paty BW, Bigam DL, Kneteman NM, Korbutt GS, Rajotte RV et al: Percutaneous transhepatic pancreatic islet cell transplantation in type 1 diabetes mellitus: radiologic aspects. Radiology 2003,229(1):165-170.
23. Villiger P, Ryan EA, Owen R, O'Kelly K, Oberholzer J, Al Saif F, Kin T, Wang H, Larsen I, Blitz SL et al: Prevention of bleeding after islet transplantation: lessons learned from a multivariate analysis of 132 cases at a single institution. Am J Transplant 2005, 5(12):2992-2998.
24. Casey JJ, Lakey JR, Ryan EA, Paty BW, Owen R, O'Kelly K, Nanji S, Rajotte RV, Korbutt GS, Bigam D et al: Portal venous pressure changes after sequential clinical islet transplantation. Transplantation 2002, 74(7):913-915.
25. Morrison CP, Wemyss-Holden SA, Dennison AR, Maddern GJ: Islet yield remains a problem in islet autotransplantation. Arch Surg 2002,137(1):80-83.
26. Halberstadt CR, Williams D, Emerich D, Goddard M, Vasconcellos AV, Curry W, Bhatia A, Gores PF: Subcutaneous transplantation of islets into streptozocin-induced diabetic rats. Cell Transplant 2005,14(8):595-605.
27. Figliuzzi M, Cornolti R, Plati T, Rajan N, Adobati F, Remuzzi G, Remuzzi A: Subcutaneous xenotransplantation of bovine pancreatic islets. Biomaterials 2005,26(28):5640-5647.
28. Bendayan M, Park IS: Presence of extrapancreatic islets of Langerhans in the duodenal wall of the rat. Diabetologia 1991, 34(8):604-606.
29. Bendayan M, Park IS: Extrapancreatic islets of Langerhans: ontogenesis and alterations in diabetic condition. J Endocrinol 1997,153(1):73-80.
30. Luo JC, Yang ZM: [Preparation and characteristics of small intestinal submucosa]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2003, 17(5):425-428.
31. Lakey JR, Woods EJ, Zieger MA, Avila JG, Geary WA, Voytik-Harbin SL, Critser JK: Improved islet survival and in vitro function using solubilized small intestinal submucosa. Cell Tissue Bank 2001,2(4):217-224.
32. Bharat A, Benshoff N, Olack B, Ramachandran S, Desai NM, Mohanakumar T: Novel in vivo murine model to study islet potency: engraftment and function. Transplantation 2005,79(11):1627-1630.
33. Caiazzo R, Gmyr V, Hubert T, Delalleau N, Lamberts R, Moerman E, Kerr-Conte J, Pattou F: Evaluation of alternative sites for islet transplantation in the minipig: interest and limits of the gastric submucosa. Transplant Proc 2007, 39(8):2620-2623.
1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. The New England journal of medicine 2000, 343(4):230-238.
2. Gunnarsson R, Klintmalm G, Lundgren G, Wilczek H, Ostman J, Groth CG: Deterioration in glucose metabolism in pancreatic transplant recipients given cyclosporin. Lancet 1983, 2(8349):571-572.
3. Guz Y, Nasir I, Teitelman G: Regeneration of pancreatic beta cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology 2001,142(11):4956-4968.
4. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R: Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science (New York, NY 2001, 292(5520):1389-1394.
5. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB: In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proceedings of the National Academy of Sciences of the United States of America 2002, 99(12):8078-8083.
6. Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patane G, Boggi U, Piro S, Anello M et al: Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 2002, 51(5):1437-1442.
7. Chang CF, Hsu KH, Chiou SH, Ho LL, Fu YS, Hung SC: Fibronectin and pellet suspension culture promote differentiation of human mesenchymal stem cells into insulin producing cells. Journal of biomedical materials research 2008,86(4):1097-1105.
8. Karnieli O, Izhar-Prato Y, Bulvik S, Efrat S: Generation of insulin-producing cells from human bone marrow mesenchymal stem cells by genetic manipulation. Stem cells (Dayton, Ohio) 2007, 25(11):2837-2844.
9. Sun Y, Chen L, Hou XG, Hou WK, Dong JJ, Sun L, Tang KX, Wang B, Song J, Li H et al: Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro. Chinese medical journal 2007,120(9):771-776.
10. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science (New York, NY 1999, 284(5411):143-147.
11. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ: Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001,105(3):369-377.
12. Pettepher CC, LeDoux SP, Bohr VA, Wilson GL: Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin. J Biol Chem 1991,266(5):3113-3117.
13. Dong QY, Chen L, Gao GQ, Wang L, Song J, Chen B, Xu YX, Sun L: Allogeneic diabetic mesenchymal stem cells transplantation in streptozotocin-induced diabetic rat. Clinical and investigative medicine 2008, 31(6):E328-337.
14. Choi KS, Shin JS, Lee JJ, Kim YS, Kim SB, Kim CW: In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract. Biochemical and biophysical research communications 2005,330(4): 1299-1305.
15. Basnet P, Kadota S, Shimizu M, Namba T: Bellidifolin: a potent hypoglycemic agent in streptozotocin (STZ)-induced diabetic rats from Swertia japonica. Planta medica 1994,60(6):507-511.
16. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F: Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000,49(2):157-162.
17. Otonkoski T, Gao R, Lundin K: Stem cells in the treatment of diabetes. Annals of medicine 2005, 37(7):513-520.
18. Burns CJ, Persaud SJ, Jones PM: Stem cell therapy for diabetes: do we need to make beta cells? The Journal of endocrinology 2004,183(3):437-443.
19. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M: Insulin production by human embryonic stem cells. Diabetes 2001, 50(8):1691-1697.
20. Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas MK, Habener JF: Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001, 50(3):521-533.
21. Bonner-Weir S, Sharma A: Pancreatic stem cells. The Journal of pathology 2002,197(4):519-526.
22. Baeyens L, De Breuck S, Lardon J, Mfopou JK, Rooman I, Bouwens L: In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells. Diabetologia 2005,48(1):49-57.
23. Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW: Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002,416(6880):542-545.
24. Ying QL, Nichols J, Evans EP, Smith AG: Changing potency by spontaneous fusion. Nature 2002,416(6880):545-548.
25. Ianus A, Holz GG, Theise ND, Hussain MA: In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. The Journal of clinical investigation 2003, 111(6):843-850.
26. Rayat GR, Korbutt GS, Elliott JF, Rajotte RV: Survival and function of syngeneic rat islet grafts placed within the thymus versus under the kidney capsule. Cell transplantation 1997, 6(6):597-602.
27. Lee HC, Ahn KJ, Lim SK, Kim KR, Ahn YS, Lee KE, Huh KB: Allotransplantation of rat islets into the cisterna magna of streptozotocin-induced diabetic rats. Transplantation 1992, 53(3):513-516.
28. Inoue M, Maeno T, Hatchell DL: Survival of allografted pancreatic islets in the subretinal space in rats. Ophthalmic research 2003, 35(1):48-53.
29. Ferguson J, Scothorne RJ: Extended survival of pancreatic islet allografts in the testis of guinea-pigs. Journal of anatomy 1977,124(Pt 1):1-8.
30. Watt PC, Mullen Y, Nomura Y, Watanabe Y, Brunicardi FC, Anderson C, Zinner MJ, Passaro EP, Jr.: Successful engraftment of autologous and allogeneic islets into the porcine thymus. The Journal of surgical research 1994, 56(4):367-371.
31. Robertson RP, Davis C, Larsen J, Stratta R, Sutherland DE: Pancreas and islet transplantation for patients with diabetes. Diabetes care 2000,23(1):112-116.
32. Al-Jazaeri A, Xu BY, Yang H, Macneil D, Leventhal JR, Wright JR, Jr.: Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice. Xenotransplantation 2005,12(3): 189-196.
33. Shapiro AM, Gallant H, Hao E, Wong J, Rajotte R, Yatscoff R, Kneteman N: Portal vein immunosuppressant levels and islet graft toxicity. Transplantation proceedings 1998, 30(2):641.
34. Guan J, Behme MT, Zucker P, Atkison P, Hramiak I, Zhong R, Dupre J: Glucose turnover and insulin sensitivity in rats with pancreatic islet transplants. Diabetes 1998,47(7):1020-1026.
35. Gupta V, Wahoff DC, Rooney DP, Poitout V, Sutherland DE, Kendall DM, Robertson RP: The defective glucagon response from transplanted intrahepatic pancreatic islets during hypoglycemia is transplantation site-determined. Diabetes 1997,46(1):28-33.
36. Bendayan M, Park IS: Presence of extrapancreatic islets of Langerhans in the duodenal wall of the rat. Diabetologia 1991,34(8):604-606.
37. Bendayan M, Park IS: Extrapancreatic islets of Langerhans: ontogenesis and alterations in diabetic condition. The Journal of endocrinology 1997, 153(1):73-80.
38. Luo JC, Yang ZM: [Preparation and characteristics of small intestinal submucosa]. Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 2003,17(5):425-428.
39. Lakey JR, Woods EJ, Zieger MA, Avila JG, Geary WA, Voytik-Harbin SL, Critser JK: Improved islet survival and in vitro function using solubilized small intestinal submucosa. Cell and tissue banking 2001, 2(4):217-224.
40. Caiazzo R, Gmyr V, Hubert T, Delalleau N, Lamberts R, Moerman E, Kerr-Conte J, Pattou F: Evaluation of alternative sites for islet transplantation in the minipig: interest and limits of the gastric submucosa. Transplantation proceedings 2007, 39(8):2620-2623.