【作者】 效梅；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 博士

【摘要】 糖尿病是危害人类健康的重大疾病之一,而传统的药物及胰岛素替代疗法却不能从根本上医治该病。近年来,随着免疫抑制剂的改进及细胞移植技术的不断成熟,移植胰岛细胞已成为治愈Ⅰ型和部分Ⅱ型糖尿病的有效途径。但是,由于每个临床胰岛移植受体通常需要2～4 个新鲜尸体胰腺,胰岛供体短缺的矛盾亦更为突出,制约了利用胰岛细胞治疗糖尿病技术的应用。胰腺干细胞(Pancreatic stem cells)是一类存在于胎儿和成年胰腺组织中的成体干细胞,能自我更新,具有多分化潜能,其特点是在自然分化过程中首先分化为胰腺组织的各种细胞。体外分离、克隆人类胰腺干细胞作为种子细胞,并定向诱导其分化为功能性胰岛细胞移植治疗糖尿病,是解决胰岛供体短缺的有效途径。目前,在世界范围内,分离克隆胰腺干细胞并将其定向诱导分化为功能性胰岛细胞移植治疗糖尿病的研究已成为细胞组织工程研究的又一热点。但是,由于胰腺干细胞的存在部位、标记物等还没有完全确定,世界范围内还没有建立公认的胰腺干细胞系。本研究以人流产胎儿胰腺组织为材料,对胰腺干细胞培养体系的建立,胰腺干细胞的形态和表达特征,胰腺干细胞的分化特性及胰腺干细胞体外诱导胰岛移植治疗大鼠糖尿病等方面进行了较为系统的研究,主要内容包括: 1 人胎儿胰腺干细胞培养体系的建立本研究建立1 例人胎儿胰腺干细胞系。以人胎儿胰腺组织为材料,采用0.1%Ⅳ型胶原酶消化分离人胎儿胰腺组织,获得胰腺单个细胞和细胞团。以低糖DMEM+10% NBS+0.08mg/mL 青霉素+0.1mg/mL 链霉素为基础培养液贴壁培养,获得原代上皮样人胎儿胰腺干细胞及少量成纤维细胞。0.25%胰酶+0.04%EDTA 消化传代,并在传代中逐渐消除成纤维样细胞及其它杂细胞,获得纯化的上皮样人胎儿胰腺干细胞。在基础培养液中添加10ng/mL EGF,扩增纯化人胎儿胰腺干细胞。继续消化传代,建立1 例人胎儿胰腺干细胞系,传50 代。人胎儿胰腺干细胞克隆性生长。当培养皿中细胞数较少时,可见多角形的上皮样胰腺干细胞呈典型的单克隆方式生长;干细胞生长至70%融合时,呈铺路石样。接种培养1～4 天,干细胞生长缓慢,5～6 天,进入倍增期。从干细胞生长曲线和1 年以上细胞扩增情况看,该人胎儿胰腺干细胞有无限增殖的趋势。不同浓度血清、EGF 和IGF-Ⅱ对人胎儿胰腺干细胞增殖有不同的影响。以低糖DMEM 为基础培养液,添加10%NBS,能促进人胎儿胰腺干细胞增殖;在低糖DMEM+10%NBS 的基础上,再添加15ng/mL EGF 或10ng/mL IGF-Ⅱ,均更有助于人胎更多还原

【Abstract】 Diabetes mellitus is a devastating disease suffered by about 150 million people in the world. At present, this disease could not cured completely by the therapy of traditional drugs or exogenous insulin injection. Along with the transplantation success of pancreatic islets and the improvement of immunosuppressive regimen, the possibilities of curing the type I and some of type II diabetes become more and more promising. However, the widespread application of this transplant technology has greatly been restricted by the deficient human pancreatic islets from the cadavers. Pancreatic stem cells are adult stem cells in pancreas, have the unlimited ability for self-renewal and to differentiate into many types of cell lineages in the appropriate local environment. They have the advantage of being already primed to specifically produce pancreatic cell types. An actual way to overcome the deficient human pancreatic islets is to isolate and clone pancreatic stem cells as “seed cells”and to induce their differentiation into functional pancreatic islets for an abundant transplant source. At present, attention has focused on the possible use of controlled differentiation of pancreatic stem cells to obtain islets useful in treating the diabetes mellitus. However, to date, no identifiable pancreatic stem cell line has been established in the world. The object of our research is to establish human fetal pancreatic stem cell line and to transplant the induced islets to cure rat diabetes mellitus. The main contents are as follows: 1. Establishment of cultural system of human fetal pancreatic stem cells A human fetal pancreatic stem cells line had been established in our lab. After treated with 0.1 % type Ⅳcallagenase, the human fetal pancreas was digested to many single cells and cell clusters. Culturing with modified glucose-low DMEM, these single cells and cell clusters adhered to the culture dish and primary epidermal-like cells and a few fibroblasts started to clone. After digestion with 0.25 % trypsin and 0.04 % EDTA, the purified epithelioid pancreatic stem cells were obtained gradually during passages. Supplemented with the EGF, the purified pancreatic stem cells proliferated quickly and formed a gravelstone-like monolayer. The growth curve showed that the purified human fetal pancreatic stem cells grew slowly in cultured 1-4 days, then they entered the logarithmic growth period during 5-6 days. Continuing culture,the human fetal pancreatic stem cells had been passed 50 passages. Comparing with the serum, EGF and IGF-Ⅱ, the proliferating speed of human fetal pancreatic stem cells was different. Supplemented with 10%NBS in basic DMEM culture media, the purified pancreatic stem cells proliferated quickly. The purified pancreatic stem cells proliferated more quickly with 15ng/mL EGF or 10ng/mL IGF-Ⅱin DMEM+10% NBS culture media, and that supplemented with 15ng/mL EGF>10ng/mL IGF-Ⅱ. The cryo-preserved system of human fetal pancreatic stem cells had been built. By the methods of reducing gradually temperature and preserving in liquid nitrogen, about 1×109 human fetal pancreatic stem cells in 80% DMEM+10% DMSO+10% NBS freezing media had been conserved. The thawing rate of the cryo-preserved human fetal pancreatic stem cells was≥93% by putting quickly the cryo-preserved cells into 36～38℃water. Chromosomes Karyotype analysis showed that the chromosome set of the human pancreatic stem cell was normal diploid (2n=46) after 47 passages. Also, we observed 5 pairs of metacentric chromosomes, 7 pairs of submetacentric chromosomes, 6 pairs of acrocentric chromosomes, 4 pairs of small chromosomes and 1 pair of xy sex chromosomes from the caryogram. 2. Morphology and expression features of human fetal pancreatic stem cells The Hematoxylin-eosin staining showed that the human fetal pancreatic stem cell had a polygonal epithelioid morphology with round caryon. Giemsa staining noted mono or double or polynucleolus. Ultrastructural analysis indicated a large ratio of caryon and cytoplasm, underdeveloped endoplasmic reticulum, mitochondria, and no excretory granule in cytoplasm. Human fetal pancreatic stem cells co-expressed the PCNA, Pdx1, glucagon, nestin and CK19 proteins. It indicated that the human fetal pancreatic stem cells line was a multi-potential stem cells line relative with pancreas, epithelium and nerve. Immunochemistry staining showed that the 18、25 and 48 passages human fetal pancreatic stem cells were 95% co-positive for the PCNA, Pdx1, glucagon, nestin and cytokeratin 19 protein expression and negative for CD34, CD44, CD45 protein expression and the control. Also, it is noted that the PCNA, Pdx1 and cytokeratin 19 were expressed in both nucleus and cytoplasm, the glucagon and nestin were only expressed in the nucleus, and a few cells expressed insulin in the nucleus. The RT-PCR further identified that human fetal pancreatic stem cells also expressed mRNA of the genes of the Pdx1, glucagon and nestin during in vitro culture. 3. Differentiation characteristics of human fetal pancreatic stem cells The human fetal pancreatic stem cells were induced directionally to differentiate into in vitro functional pancreatic islets clusters by the following method. Human fetal pancreatic stem cells were cultured on the gelatin in plate. After proliferated for 3 days, the human fetal pancreatic stem cells were induced directionally with modified DMEM supplemented with10mmol/L nicotinamide, 10ng/ml EGF, 10% NBS, 3.7g/L sodiumpyruvate, 100 U/mL penicillin and 100 ug/mL streptomycin at 37℃, 5 ℅CO2 for 25 days. Human fetal pancreatic stem cells were induced directionally to differentiate into functional pancreatic islets clusters. The induced pancreatic islets derived from human fetal pancreatic stem cells contained βcells. During the induced procession, human fetal pancreatic stem cells proliferated firstly into a monolayer like slabstones and then gradually epithelioid stem cells became round and aggregated islet clusters. The number and the size of islet clusters increased continuously throughout the induced course. Ultrastructural analysis showed a small ratio of caryon and cytoplasm, developed endoplasmic reticulum, mitochondria, and a few excretory granule in cytoplasm. On the induced day 28, most islet clusters became crimson with dithizone staining. The radioimmunoassay showed that after induced culture for 28 days, the induced pancreatic islets derived from 5.0×105 human fetal pancreatic stem cells secreted 7.299±1.166μIU/mL and 8.726±1.593μIU/mL insulin in 5.5mM and 25mM glucose media during 2 hours. It indicated that the induced islets derived from the human fetal pancreatic stem cells could secrete insulin and could secrete significantly more insulin with 25mM glucose media. The differentiated in vivo experiment indicated that human fetal pancreatic stem cells had pluripotentment to differentiate into the pancreas,epithelium and nerve . All three nude mice explanted the human fetal pancreatic stem cells displayed growing grafts that were white with rich blood vessel. One of the largest grafts was about ? 1 cm on the explanted day 30. Immunohistochemical analysis revealed that the cells of the grafts expressed the Pdx1, insulin, glucagons, CK19, MBP and NF proteins. 4. Reversal of rat diabetes after implantation in vitro differentiated islets After implanted induced islets derived from human fetal pancreatic stem cells into the subcapsular region of one kidney, the streptozotocin-induced diabetic rats showed prolonged life and a decrease in blood glucose. During 70d experiment, the blood glucose level of tow implanted rats decreased gradually from 26.56mmol/L to 9.6mmol/L. But the control diabetic rats kept the blood glucose in 18.47～26.81mmol/L.更多还原