【作者】 孙宇；

【导师】 陈丽；

【作者基本信息】 山东大学 ， 内科学， 2007， 博士

【摘要】 研究背景胰岛β细胞功能的进行性衰竭是糖尿病(diabetes mellitus，DM)发身发展的根本原因，1型和2型DM都存在着这种不同程度的病理生理状态。目前临床治疗有多种途径，例如注射外源性胰岛素、口服降糖药物等，但这些治疗都只能减轻或延缓DM及其并发症的发生和发展，尚不能完全根治DM。以胰腺移植和胰岛细胞移植为主的再生治疗为DM的根治带了新的希望和思路，但胰腺移植和胰岛细胞移植仍然面临供体缺乏及免疫排斥反应这两大难题，使其效果和临床应用受到了极大的影响。随着近几年干细胞研究的进展以及认识的不断深入，人们逐渐认识到可以利用干细胞替代、修复或加强受损组织或器官的功能。间充质干细胞(Mesenchymal stem cells，MSCs)是存在于骨髓中的一种分泌多种细胞因子的较原始的骨髓基质细胞，具有基质细胞的特性，可以分化为造血实质，支持造血；同时具有多向分化潜能和自我更新能力，在适宜的培养条件下可增殖并被诱导分化成多种造血以外的组织细胞。自从1968年Friedenstein等人首先证实间充质干细胞在骨髓组织中大量存在以来，它的多分化潜能受到越来越多的广泛关注。国内外众多研究证实，在特定的诱导条件下，骨髓间充质干细胞(bode marrow-derived mesenchymal stem cells，BM-MSCs)可以分化为骨、软骨、脂肪、肌腱、肌肉和韧带等多种中胚层来源的细胞，不仅如此，还可以跨胚层分化为肝细胞、内皮细胞和神经细胞等内胚层和外胚层来源的各种细胞。所以近年来，BM-MSCs的功能作用和临床应用成为医学界普遍关注的研究热点，其已经成为一种理想的组织工程的种子细胞。BM-MSCs不仅具有取材方便、扩增迅速的特点，更重要的一点在于患者可以利用自身骨髓来源的干细胞治疗疾病，实现真正的自体移植，从而克服移植排斥反应。因此，MSCs作为一类具有自我更新和多向分化潜能的细胞，已经成为人们寻找胰岛β细胞替代物的新资源。在此基础之上，本研究选取DM患者的骨髓，建立适宜的BM-MSCs体外培养体系，并通过形态学特征、表面标志以及多向分化功能等方面进行鉴定，同时探讨在体外条件下自体BM-MSCs诱导分化为胰岛素分泌细胞(insulin-producing cells，IPCs)的可行性，证实病理状态下BM-MSCs的分化能力，从而为DM患者实现自体骨髓干细胞移植治疗糖尿病奠定理论基础。第一部分糖尿病患者骨髓间充质干细胞体外分离、培养、扩增及鉴定研究目的本研究旨在：1)建立DM患者BM-MSCs的体外培养体系；2)检测BM-MSCs的基本生物学特性；3)对BM-MSCs具有向骨、软骨、脂肪及神经等多方向的分化能力进行鉴定；从而为以MSCs为基础的组织细胞工程提供实验理论基础。研究方法随机选取10名行自体骨髓单个核细胞移植治疗DM的患者作为研究对象。无菌条件下，自髂后上嵴行多点穿刺取肝素化骨髓标本5ml，利用Ficoll-Hypaque分离液(相对密度1.077±0.001g／ml)密度梯度离心分离得到骨髓单个核细胞后，体外以DMEM-LG培养基(含有10％FCS)培养，常规胰蛋白酶-EDTA混合消化液进行传代扩增，通过H.E染色，Giemsa染色观察细胞形态，流式细胞仪检测BM-MSCs表面标志，并最后进行向成骨，软骨，脂肪，神经细胞方向的诱导分化，以期通过细胞形态学，表面标志，多向分化功能对BM-MSCs进行综合鉴定。研究结果1) DM患者的BM-MSCs为成纤维样细胞的细胞群体，以均一的、梭形的成纤维样细胞为主。H.E和Giemsa染色后，于光镜下可见细胞形态呈现梭形或多角形，核大，核浆比例高，居中，可见有3个核的细胞；2)原代培养周期大约为18～20天，胰蛋白酶-EDTA常规消化，经过3～5代后BM-MSCs基本达到形态均一。P10，P20的细胞仍可呈高度均质的梭形细胞群，平行或旋涡状方式生长。体外可以连续传至P20仍能保持原始未分化状态。3)流式细胞仪检测BM-MSCs表面抗原分子，结果显示BM-MSCs均一的表达CD29、CD44、CD106阳性，不表达CD14、CD34、CD45等造血细胞特异性表面分子；4) DM患者的BM-MSCs在体外适宜的条件下，成功向骨，软骨，脂肪，神经细胞方向进行分化，细胞组织化学染色，RT-PCR，免疫组化均支持上述结果；结论1)成功地进行了DM患者BM-MSCs的分离、培养及扩增。利用Ficoll-Hypaque分离液(相对密度1.077±0.001g／ml)密度梯度离心分离骨髓细胞，取骨髓MNCs以DMEM-LG培养基培养，可以获得纯度较高的BM-MSCs。并建立了适合其增殖的体外培养体系，证实BM-MSCs具有强大的体外增殖储备和扩增能力，并且可以在体外扩增达P20仍能够保持未分化状态，其可以用于进一步的组织工程、细胞和分子生物学研究；2)分离扩增的DM患者BM-MSCs不表达CD34、CD45、CD14这些造血干细胞的分子标记，高度表达CD29、CD44、CD106多种膜表面蛋白，与文献报道一致；3) DM患者BM-MSCs在体外具有多分化潜能，可以向中胚层3种细胞(骨、软骨和脂肪细胞)及中胚层(神经细胞)分化；4)结合细胞形态学、表面标记以及分化功能等多方面鉴定，表明所分离、培养及扩增的细胞群中确实存在具有多潜能ASCs特性的无造血细胞系污染的BM-MSCs；第二部分糖尿病患者骨髓间充质干细胞定向诱导分化为胰岛素分泌细胞研究目的探索诱导糖尿病患者BM-MSCs向IPCs分化的条件，证实在病理状态下糖尿病患者自体BM-MSCs仍然具备分化为IPCs的潜能，为获得大量胰岛β细胞用于细胞替代疗法治疗DM寻找新的出路。研究方法选取10名糖尿病患者的BM-MSCs，分离提纯后，选用三步诱导方案进行体外诱导分化为IPCs的实验。RT-PCR检测一系列与β细胞发育和功能相关的基因表达；免疫组化，双硫腙染色证实IPCs的生成；酶联免疫法检测胰岛素的分泌量。研究结果1)诱导分化18天后，镜下成功观察到胰岛样细胞团的生成，双硫腙及免疫组化染色胰岛素均呈现阳性反应；2)诱导分化的细胞在第二阶段(第10天)能够表达nestin，PDX-1，Ngn3，Pax4，insulin，glucagon，而第三阶段(第18天)则可以观察到PDX-1，insulin，glucagon的高表达；3)这些诱导分化的细胞接受葡萄糖刺激后能够分泌胰岛素，且胰岛素的分泌量随葡萄糖浓度的提高而增加(P＜0.05)；结论1)体外通过三阶段不同细胞因子组合法成功诱导DM患者BM-MSCs分化为IPCs，并且RT-PCR，免疫组化，细胞的双硫腙染色及酶联免疫分析等结果表明这些分化的细胞具有生理性胰岛β细胞的特性，证实DM患者自体BM-MSCs具有体外诱导分化为胰岛素分泌细胞的潜能；2)上述研究结果为DM患者实现自体骨髓干细胞移植治疗糖尿病奠定了坚实的理论基础，提示BM-MSCs作为临床细胞替代治疗DM的种子细胞具有可行性；更多还原

【Abstract】 BACKGROUND AND OBJECTIVEDiabetes mellitus (DM), results when there is the progressive failure of functional B-cells. The destruction of insulin-producing β-cells is the main cause of type 1 diabetes and also can be seen in type 2 diabetes at the later stages.At present, a lot of ways were used in treating DM, for axample, which include injection exogenous insulin, or using oral antidiabetic drug. While traditional recombinant insulin therapy can control blood glucose levels, the therapeutic efficacy reduces with time.The success of Edmonton Protocol for pancreatic islet transplantation and pancreas teansplantation have sparked new interests in treating DM. Unfortunately, they has historically been hampered by immune rejection as well as the scarcity of donor islets, which have greatly diminished the benefits to patients and limited its wider clinical applications. Stem cells, which have self-renewal and pluripotecy, appeal to people for developing new source of organ, tiusse replacement and regeneration in present years.To date, scientists have turned their focus on mesenchymal stem cells(MSCs), which exist in bone marrow mainly and belong to one of comparatively primary marrow stroma cells. MSCs can secret a lot of cytokines, and several reports revealed that MSCs have selt-renewal and pluripotecy differentiation capacity, since under suitable condition in vitro, MSCs can be differentiated into many different typeies tissue-derived cells. The multilineage differentiation potential of MSCs populations derived from a variety of different species has been extensively studied in vitro since their first discovery in 1968 by Friedenstein et al. Bone marrow contains bone marrow-derived mesenchymal stem cells (BM-MSCs), which carry the more significant implications for possible clinical development because of their pluripotecy differentiation capacity. More and more reports revealed that that populations of BM-MSCs from human, canine, rabbit, rat, and mouse have the capacity to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, fat, tendon, muscle and ligament et al. At the same time, hepatic, biliary duct epithelium, endotheliocyte and neuroectodermal cells of donor origin can be found inrecipient animals upon transplantation of BM-MSCs. BM-MSCs carry the more significant implications for possible clinical development, because they are easily accessible for an autograft and routinely collected from adults without ethical concern inherent to fetal embryonic tissues. Accordingly, BM-MSCs has spurred considerable interests in better understanding the biology of BM-MSCs and their potential clinical applications in the therapy of organ or system specific diseases in the future. Therefore, the utilization of BM-MSCs is becoming the most promising therapy of DM.Under these premises, in this present study, we isolated BM-MSCs from both typel and type 2 DM patients marrow by first taking advantage of their preferential adherence to the plastic surface of the culture dish and uncovered culture conditions to keep BM-MSCs from DM patients at undifferentiation state. We then went to characterize these BM-MSCs celllines and identify clones by testing morphology feature, surface marker and differentiation capacity for further investigation. Finally, we induced BM-MSCs to transdifferentiate into insulin-producing cells under culture conditions containing high concentrations of glucose and the additions of 8-cell stimulating growth factors. Taken together, our results indicated that the bone marrow-derived MSCs from diabetic patients themselves could differentiate into insulin-producing cells in vitro and hinted that using a diabetic patient’s own bone marrow-derived MSCs as a source of autologous insulin-producing cells for β-cell replacement would be feasible.Part 1 Isolation, culture-expanding and identification of bone marrow-derived mesenchymal stem cells from diabetic patients invitroOBJECTIVE1) Establish efficient methods for isolating, culture-expanding of BM-MSCs from diabetic patients;2) Investigate the chief biological properties of BM-MSCs from diabetic patients;3) Identify the differentiation capacity of BM-MSCs from diabetic patients into bone, cartilage, fat and neuroectodermal cells;MATERIALS AND METHODSUnder aseptic condition, 5 ml of heparinized bone marrow samples were obtained from the posterior superior iliac crest of DM patients. And then the diluted sample were isolated by Ficoll-Hypaque(1.077±0.001g/ml)density gradient centrifugation. Monuclear cells were culture-expanded in Dulbceco’s Modifed Eagle’s medium-low glucose (DMEM-LG) which contains 10% FCS. BM-MSCs from diabetic patients were characterized according to morphology by H.E and Giemsa staining. Representative cell surface antigen expression profiles of DM-MSCs analysed by flow cytometric analysis. Finally, the cells were differentiated into bone, cartilage, fat and neuroectodermal cells under certain conditionds in vitro.RESULTS1) BM-MSCs from diabetic patients exhibit a homonomous fibroblast-like morphology with spindle shape according to H.E and Giemsa staining;2) Primary cultures were maintained for 18～20 days. The cells were detached with a solution of trypsin-EDTA. BM-MSCs at passage 20 still can be kept in undifferentiation state; 3) Flow cytometric analysis of BM-MSCs showed that these cells expressed high levels of CD29, CD44 and CD106, and they negative for CD34, CD45 and CD14;4) BM-MSCs from diabetic patients have the multiple differentiation potentials, which can be differentiated into bone, cartilage, fat and neuroectodermal cells under certain conditionds in vitro;CONCLUSIONS1) BM-MSCs from diabetic patients can be successfully isolated, culture-expanded in vitro. By Ficoll-Hypaque(1.077±0.001g/mI)density gradient centrifugation, we can obtain relatively purified BM-MSCs in vitro.The results draw a conclusion that BM-MSCs posses powerful proliferation and amplification capacity in vitro, and BM-MSCs of passage 20 still can be kept in undifferentiation state, which will be more suitable for resesrch of organ or tiusse engineering, and cell/ molecular biology in the future;2) Flow cytometric analysis of the BM-MSCs from diabetic patients showed that these cells were negative for CD34, CD45 and CD14. They expressed high levels of CD29, CD44 and CD106. These results indicated that relatively purified BM-MSCs were isolated;3) BM-MSCs from diabetic patients were strong in the multiple differentiation potentials, which can be differentiated into bone, cartilage, fat and neuroectodermal cells under certain conditionds in vitro successfully;4) Culture-expandedh BM-MSCs were confirmed the presence of cells with adult multipotential stem cells-like characteristis and non-hematopoietic origin.Part 2 Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitroOBJECTIVE Study the differentiation capacity of the diabetic patient’s BM-MSCs and test the feasibility of using the BM-MSCs for autologous B-cell replacement in treating DM.MATERIALS AND METHODSBM-MSCs were obtained from 10 DM patients and induced to IPCs under a three-stage protocol with high concentrations of glucose and the additions of -cell stimulating growth factors. RT-PCR was performed to detect multiple genes related to pancreatic B-cell development and function. The identity of the IPCs was illustrated by the analysis of morphology, ditizone staining and immunocytochemistry. Release of insulin by these cells was confirmed by immunoradioassay.RESULTS1) Typical islet-like cell clusters were observed at the end of protocol (18 days). Ditizone staining and immunohistochemistry for insulin are both positive;2) These differentiated cells at stage 2 (10 days) expressed nestin, PDX-1, Ngn3, Pax4, insulin, glucagon, but at stage 3 (18 days) we observed the high expression of PDX-1, insulin, glucagons;3) Insulin was secreted by these cells in response to different concentration of glucose stimulation in a regulated manner (P<0.05);CONCLUSIONS1) BM-MSCs from DM patients can be differentiated into functional IPCs under certain conditions in vitro;2) Using diabetic patient’s own BM-MSCs as a source of autologous IPCs for β-cell replacement would be feasible;更多还原