【作者】 曲惠廷；

【导师】 郑成云；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 研究目的：糖尿病是一组以高血糖为特征的代谢性疾病。导致高血糖的机制,一般认为来自两个方面,胰岛素分泌不足和胰岛素抵抗。糖尿病的自身症状和多种并发症,长期困扰着患者的身心健康。截止2014年,全国有1.14亿糖尿病病人,中国已经成为全世界第一糖尿病大国。糖尿病需要持续的医学治疗,目前常用的胰岛素增敏剂和外源性的胰岛素治疗,只能暂时性的缓解高血糖状况,增加靶器官对胰岛素的敏感性,但是都不能彻底治愈糖尿病。目前唯一有希望能治愈糖尿病的措施是胰岛移植,然而胰岛移植目前除了供体的来源的限制之外,还存在胰岛体外存活率有限,移植后发生宿主的免疫排斥等问题,因此胰岛移植尚不能理想的彻底治愈糖尿病。细胞治疗,特别是与胰岛移植类似的胰岛替代细胞的细胞治疗是近年来的研究热点,也是有希望治愈糖尿病的一种方式。近年的研究发现,胚胎干细胞(Embryonic stem cell, ESC)可以在体外经过多重细胞因子的诱导,或者基因修饰后,体外分化成为分泌胰岛素的细胞(Insulin producing cell, IPC),能在体外分泌胰岛素并且注射到糖尿病动物模型中可以起到一定的治疗作用。鉴于ESC在临床治疗中,存在伦理争议,并且有可能导致畸胎瘤等的发生,因此ESC相关的细胞治疗在大规模的临床应用上尚缺乏前途,亟待挖掘新的细胞来源。间充质干细胞(Mesenchymal stem cell, MSC),是近年来研究的热点,其多向分化和自我更新的特性,符合干细胞的一般特性,另外,MSC特有的免疫抑制作用,也有望用于抑制糖尿病的自身免疫。因此,MSC作为多能干细胞,很有希望作为细胞治疗糖尿病的主体。近年来,也有来自骨髓(bone marrow derived MSC, BM-MSC),脂肪组织(Adipose derived MSC, AD-MSC)等多种MSC向IPC分化的研究,但是研究发现,骨髓和脂肪组织来源的MSC存在年龄的差异和传代的老化等现象,脂肪组织来源的MSC的成骨分化能力较弱并且体外可以促进肿瘤的生长,而脐带组织来源的MSC就不在年龄的差异,另外脐带组织属于正常分娩的废弃物,也不存在伦理的问题,因此脐带组织来源的MSC应该是细胞治疗的良好来源。此外,在目前的技术手段下,IPC的产生效率较低,分化程度也低,体外分泌的胰岛素数量也不能满足将来大规模临床应用的需要。另外,现有的通过基因修饰的方法进行IPC诱导分化的方法,由于存在病毒载体,在临床应用过程中也有潜在的危险。因此,新的IPC诱导体系,或者旧的体系的改良以适应大规模的临床应用,都迫在眉睫。我们体外观察人脐带组织来源的原代间充质干细胞(Umbilical tissue Mesenchymal stem cell, UT-MSC or WJ-MSC)在适宜条件下诱导分化为胰岛素分泌样细胞(Insulin producing cell, IPC),研究细胞基底膜成分laminin调控IPC分化和成熟的作用及相关机制,对现有的方法和技术加以改良,力求得到体外胰岛素分泌量明显升高的IPC。并且在动物实验中,观察IPC的治疗作用。材料和方法：UC-MSC的培养和鉴定在获得山东大学第二医院伦理委员会的批准后,在取得捐献者签署知情同意书的前提下,我们收集健康剖宫产产妇的脐带组织,经过胶原酶和胰酶的消化处理,原代培养间充质干细胞(MSC),并且在体外大量扩增。经过形态学,流式细胞学免疫表型鉴定和多向分化潜能的鉴定之后,确认成功培育出可应用与临床治疗的符合GMP规范的脐带组织来源的MSC。IPC的诱导分化和改进参考现有的ESC和少量MSC分化的文献,在胰岛素转铁蛋白(ITS)、尼克酰胺等因子存在的适宜条件下,经过4个阶段的诱导分化,得到形态学相似的IPC细胞团,应用DTZ染色、RT-PCR、免疫荧光、Western blot等手段,确认IPC的成功分化,然后探求添加laminin进行进一步的分化改进,在体外实验部分继续应用RT-PCR, Western Blot,免疫荧光化学,流式细胞术等对IPC进行mRNA,蛋白水平的分析和鉴定。并利用多种检测手段,寻找laminin在IPC分化过程中的作用机制。动物实验在体内实验部分,在获取了山东大学第二医院伦理委员会的批准后,以STZ造模直接破坏大鼠p细胞的方法,制作1型糖尿病Wistar大鼠动物模型。在成功建立1型糖尿病动物模型后,分组输注细胞进行细胞治疗(生理盐水对照组、MSC输注治疗组、IPC输注治疗组和添加laminin分化的IPC治疗组等),观察经过不同处理的IPC对糖尿病大鼠的治疗作用,包括临床症状、动物的血糖水平、体重水平、生存分析等。临床试验我们同时进行了MSC直接治疗2型糖尿病的临床试验。从2009年7月到2012年10月,我们选取了在山东大学第二医院内分泌与代谢性疾病科治疗的2型糖尿病患者18例患者进行了MSC输注治疗。输注的平均剂量是1.8×106MSC/kg。输注的方法是静脉输注。随访的时间是6个月。结果：UC-MSC是贴壁生长的长梭形纤维样细胞,可以稳定传代,符合国际细胞治疗协会2006年的定义标准。UC-MSC在免疫表型上,高表达CD73,CD90,CD105,CD166,低表达HLA-DR, CD34, CD45。UC-MSC具有多向分化的潜能,可以在体外适宜的刺激条件下,分化为脂肪细胞、成骨细胞和软骨细胞。UC-MSC也可以在适宜的条件下,被诱导分化为IPC,经过多重手段得到验证,是具有胰岛素分泌功能的IPC,体外胰岛素分泌量可以达到189.8±93.05μIU/ml,并且在mRNA和蛋白水平上,高表达胰岛p细胞的相关标识(例如Pdxl,Insulin等)。在添加适宜浓度的人重组层黏连蛋白(laminin)后,可以显著的促进IPC的产生和成熟,与不添加组相比较,在mRNA和蛋白水平,添加laminin可以显著提高IPC细胞的基因Pdx1, Ngn3, Pax4和insulin等。在体外胰岛素释放试验中,在高糖刺激的环境下,可以分泌大量的胰岛素,其中laminin411添加后,体外胰岛素分泌可以达到612.1士40.78μIU/ml, laminin511添加后,体外胰岛素分泌量可以达到：561.6士31.81μIU/ml,均3倍以上高于不添加laminin组。在动物试验中,添加laminin组与纯MSC输注组相比较,在生存时间,血糖水平、糖化血红蛋白水平和体重水平等都有明显改善。其中添加laminin411的IPC输注组,可以更快的显著降低血糖的水平。添加laminin511的IPC输注治疗组,有效控制血糖的时间更长。添加laminin的IPC治疗组,总体显著改善糖化血红蛋白水平,有效缓解临床症状。此外,所有细胞治疗组,都是安全的。在临床试验中,经过长达6个月的随访,未发现明显的副反应,总体治疗有效率高达44.4%。我们的临床试验结果发现,经过MSC输注治疗,空腹血糖和餐后血糖水平可以有效缓解。结论：UC-MSC可以在适宜的条件下分化成为成熟的具有分泌胰岛素功能的细胞,在laminin存在的情况下,IPC的成熟度显著提升,体外可以分泌多达3倍以上的胰岛素,基本满足未来治疗的需要。MSC输注治疗,无论在动物实验还是在临床试验中,都是安全的。在动物实验中,添加laminin分化的IPC,显著控制动物血糖,缓解临床症状,显著改善生存。更多还原

【Abstract】 BackgroundDiabetes mellitus (DM) is one group of metabolic diseases characterized by high blood glucose level resulting mainly from hyposecretion of and tissue resistance to insulin. With improvement in economics and changes in life style, there will be approx.114million patients with diabetes mellitus in China by2014. DM requires constant and daily based medical treatment in order to control hyperglycaemia and related complications. At present, commonly used-insulin sensitization agent and exogenous insulin administration can only temporarily alleviate hyperglycemia condition, increase sensitivity of the target organs to insulin, but could not cure diabetes. Rencently, Islet transplantation has been tried in clinic and showed some clinical benefits. However, limited treatment efficacy due to short survival rate of transplanted islets and host immune rejection, and limited source of donors hinders developing an islet transplantation-based treatment approach for DM.Application of the insulin producing cells instead of islets seems to be an attractive approach for DM therapy in the future. In recent years, previous studies demonstrated that embryonic stem cells (ESC) could be in vitro induced to differentiate into insulin producing cells (IPC) in a culture system containing multiple cytokines and/or genetic modification, and transfusion of these IPCs reduced blood glucose level in diabetes animal models. However, to apply ESC-derived IPCs in clinic is still a long way to go because of its ethical issues and risk to develop a teratoma. Therefore to identify new sources of stem cells for IPC are urgently required.Mesenchymal stem cells (MSCs) are one type of adult stem cells having characteristics of self-renew and multi-directional differentiation capacity. Except from their differentiation activity, MSCs are endowed with potent immunosuppressive capacity, by which MSCs may excert therapeutic effects on auto-immune diseases and diabetes mellitus. Moreover, studies have demonstrated that bone marrow derived-MSC (BM-MSC) and adipose derived-MSC (AD-MSC) are able to differentiate into IPCs under suitable in vitro culture conditions. Howerve, their differentiation capacity are relative limited and also there were age-related issues in these kinds of MSCs. Furthermore, in vitro studies have also demonstrated that adiposed-derived MSCs support cancer cell growth while umbilical cord tissues derived MSCs not. In contrast, umbilical cord tissues are "wastes" of normal delivery, therefore ethical issues can be avoided. Moreover there was no risk of teratoma. Apparently, umbilical cord tissue is a good source of MSC for induction of IPC differentiation.Laminin is a heterotrimer glycoprotein that contains alpha, beta, and gamma chains. At least19laminin isoforms have been identified nowadays. Laminin is named according to its subchains (e.g., laminin411comprises the a4, β1, and γ1chains). Laminin is a key component of the basement membrane, and is involved in the structural scaffold, cell proliferation, and differentiation. Jiang, et al found that laminin111promoted the differentiation of fetal mouse pancreatic beta cells, whereas Leite et al reported that laminin could induce the expression of islet cell markers in the hepatic oval cells in vitro. Laminin was recently shown to promote the differentiation of hTERT-over-expressed human BM-MSCs into IPCs by activating Akt and Erk signals.In this study, we sought to induce human umbilical cord Wharton’s jelly derived MSCs (UC-MSC) into IPCs using laminin without any gene manipulation. In our culture system, insulin release increased significantly after a high concentration of glucose stimulation, and the yield of IPCs was much higher compared to that using published differentiation methods. In the animal tests, fasting blood glucose (FBG) levels declined rapidly following the administration of laminin-induced IPCs and glycosylated haemoglobin (HbAlc) level was significantly reduced70days after transfusion of IPC with laminin than IPC and MSC groups.Materials and MethodsThe culture of the UC-MSC and identificationAcquired the ethical approval from the Ethics Committee of Second Hospital of Shandong University, after signed informed consent in donors, we collect maternal health cesarean delivery of umbilical cord tissue, treated with collagenase and trypsin digestion, to obtain mesenchymal stem cells (MSC), and then made in a large number of amplification in vitro. MSCs were successfully confirmed by morphology, flow cytometry analysis and identification of multi-directional differentiation potential according to minima cretiaria of International Society of Cytotherapy in2006. MSCs can be applied to clinical treatment in accordance with GMP conditions.The IPC differentiation and improvement of published protocolsThen refer to the published literature, the differentiation of IPC was induced in presence of insulin transferrin (ITS), nicotinamide, with a four stages of differentiation. The IPC differentiation was confirmed by cell morphology, DTZ staining, RT-PCR, immunofluorescence, and Western blot. And then we explored adding laminin for further improvement of IPC differentiation. The mRNA and the protein level of IPC was confirmed again using RT-PCR, Western blot, immunofluorescence chemistry, and flow cytometry to determine the effect of adding laminin. And insulin release assay was also used to test the maturation and effecacy of adding laminin in the process of differentiation.Animal experimentIn the in vivo experimental section, after approved by the Ethics Committee of Second Hospital of Shandong University, we injected STZ intraperitoneal directly destroy the β cells of Wistar rats to make animal model of type1diabetes Wistar rats. After successfully established animal model of type1diabetes, infusion cells for cytotherapy group (normal saline control group, the group treated with infusion of MSC infusion treatment group, IPC and add laminin differentiation of IPC in treatment group, etc.), and observed the different processing of IPC effect on the treatment of diabetic rats, including clinical symptoms, animal weights, levels of fasting blood glucose level, glycosylated hemoglobin, survival analysis, etc.Clinical trialsTo evaluate safty and treatment effects of UC-MSC on type2DM, we have carried on one clinical trial (NCT01413035). From July2009to October2012,18patients with type2DM were enrolled the clinical trial from the Department of Endocrine and Metabolic diseases in the Second Hospital of Shandong University. Diagnoses were made based on the diagnostic criteria of World Health Organization (WHO). The study protocol was approved by the Ethical Committee of the Second Hospital of Shandong University. Informed consent according to the Declaration of Helsinki was obtained from every participated patient. The average dose of cell infusion was1.8X106MSC/kg for each patient.ResultsUC-MSCs are adherence to plastic flasks in accordance to the standards of The International Society for Cellular Therapy (ISTC). UC-MSCs were high expression CD73, CD90, CD105, CD166and low expression of HLA-DR, CD34, CD45. UC MSC has a multi-directional differentiation potential under the appropriate differentiation condition in vitro. MSCs could differentiate into adipocytes, osteoblasts and chondrocytes.UC-MSC can also be differentiated to IPCs under appropriate conditions, have been verified by multiple methods, it has insulin secretion function in vitro. Insulin secretion in vitro can reach189.8±93.05μIU/ml. And in the mRNA and protein levels, high expression of islet β cells associated genes (such as Pdxl, Insulin, etc.). Adding appropriate concentration of reconstituted human laminin, can significantly promote the generation and mature of IPCs, compared with no added group. In the mRNA and protein level, adding laminin could significantly improve the gene expression of Pdxl, Ngn3, Pax4and insulin. In the insulin releasing test in vitro, under the environment of high glucose stimulation, IPCs with laminin could secrete large amounts of insulin, insulin secretion in vitro can reach612.1±40.78μIU/ml in adding laminin411group, while laminin511adding group insulin secretion in vitro can achieve:561.6±31.81μIU/ml, add laminin group were more than three times higher than not.Laminin411and laminin511effectively induced IPC differentiation from MSCs, and upregulated both mRNA insulin expression and protein levels. The expression of genes known to govern insulin expression such as Pdxl and Ngn3were robustly induced by laminin411and laminin511, which suggested that Pdxl and Ngn3functioned as signaling pathways in the regulation of insulin expression and IPC differentiation induced by laminin. Administration of laminin induced-IPCs (laminin-IPCs) rapidly and significantly down-regulated fasting blood glucose level, and markedly improved the symptoms and survival of T1DM rats compared with controls.In animal experiments, adding laminin compared to MSC infusion group, the survival time, blood glucose levels and levels of weight are obviously improved. Add laminin411IPC infusion set, can significantly reduce the blood glucose level rapidly at0.5week after transfusion. Add laminin511IPC infusion treatment group, could effective control of blood glucose for longer periods of time.In the clinical trials, after six months of follow-up, found no obvious side effects, the overall treatment efficiency is as high as44.4%.Of clinical trial results, we found that after MSC infusion treatment, the fasting blood glucose and postprandial blood glucose levels can alleviate effectively.ConclusionsUC-MSC can be differentiated into mature insulin producing cells in vitro under the appropriate condition in the presence of laminin. IPC significantly induced IPC maturity and large amount of insulin secretion in vitro. In animal experiments, adding laminin differentiation of IPC, animals can successfully control blood glucose, ameliorate the clinical symptoms, and improve survival. Our results revealed that laminin411and511could induce Pdxl and Ngn3expression both at mRNA and protein levels, which provides a clue of how laminin increases the maturation of IPCs. Mature pancreatic (3-cells are also Pdxl positive. Our current protocols of IPCs differentiation are to form Pdxl+expressing pancreatic progenitors, indicating that laminin may play essential roles in affecting the yield of IPCs.Laminin acted as a potent differentiation inducer of IPCs from MSCs derived from umbilical cord tissue. Laminin induced differentiation through the Pdxl and Ngn3signaling pathways. Laminin-IPCs significantly reduced blood glucose level and improved the survival of T1DM rats. Our novel finding suggested the potential clinical use of laminin-IPCs in the treatment of T1DM. However, further studies on the biosafety of these cells in animal models are warranted before a clinical trial can be launched. In addition, the safety and efficacy of laminin-IPCs in human diabetes patients should be investigated.To conclude, laminin411and511significantly and efficiently induced differentiation of IPCs from MSCs derived from human cord tissues, and these IPC produced large amount of insulin through Pdxl and Ngn3signalling pathways. Moreover, IPC with laminin transfusion resulted in a long-term therapeutic effects on T1DM. Our findings here highlight a crucial role of laminin in the induction of IPCs differentiation from human MSC and a potential clinical use of IPCs with laminin in the treatment of T1DM as well as type2DM in the future.更多还原