仔猪胰腺干细胞的分离鉴定
发布时间：2018-09-14 19:56
【摘要】：近年来糖尿病的患病率日益增高,已成为继心血管疾病、肿瘤之后第三大高发慢性病。快速、有效、长期地补充机体胰岛素分泌不足是糖尿病治疗的根本方案。目前采用三种方法补充机体胰岛素分泌不足:传统皮下注射外源性胰岛素,虽然有效但很难阻止并发症的发生和发展,注射剂量经常与机体需要不相符,且每日每餐均需注射很繁琐;胰腺和胰岛器官移植,可解决胰岛素供需不符的问题,但移植器官难得、手术复杂、不良反应多;移植分泌胰岛素的干细胞,可以弥补上述两种方法不足,简易地注入人体成为自身胰腺细胞,一次给药长期有效,机体还可根据需要,调节产生胰岛素和其他辅助细胞因子等的多种细胞比例,避免并发症发生,干细胞增殖力强、数量充足、移植排斥反应低。但也是由于供体组织严重匮乏限制其应用发展,人们转向寻找异种动物胰腺组织作为供体组织来源时发现,猪繁殖性能强,组织来源充足,胰岛素结构和生物活性类似于人胰岛素,血糖调定点同人接近,猪胰腺细胞可在人血清中生存对人体无害,因此,仔猪胰腺干细胞可作为最佳的异种动物胰腺干细胞的供体细胞,对仔猪胰腺干细胞的研究是糖尿病治疗的新希望。本试验从仔猪胰腺干细胞分离培养、生物学性能测定及诱导分化多能性三个方面进行研究,为仔猪胰腺干细胞的临床应用提供基础资料。 1. 建立仔猪胰腺干细胞的分离方法和培养体系。 取新鲜的仔猪胰腺组织,冰上操作,0.25%胰酶消化,形成小细胞团和单个细胞,过滤,低速、多次离心,或以5%BSA作离心液离心,均可获得高活力的胰腺细胞,用层粘连蛋白或明胶铺被培养皿,以低糖DMEM 培养液添加10%血清及10 mmol/L 烟碱作为基础培养液,培养2d 后,除去未贴壁细胞,更换为去除烟碱、添加10 ng/mL EGF的基础培养液,长期培养以促进胰腺干细胞体外增殖生长。证实仔猪胰腺内分泌祖细胞呈多突起细胞形态。 2. 仔猪胰腺干细胞各项生物学特性。 原代细胞以细胞团和单个细胞贴壁,细胞团中不断有细胞脱离、悬浮,保留的贴壁细胞和单个细胞生长,约7d 出现多突起内分泌祖细胞,克隆样生长,很快连接成网状;小圆细胞数量增多;多角形导管上皮干细胞增殖慢。电镜观察多突起细胞具50~80μm长突起、有极性、核质比大、细胞器不发达;小圆细胞为直径10~12μm、有微绒毛、胰岛素阳性;多角形细胞呈圆形、核质比大、可见胞质内细胞器和细小分泌颗粒。成功地分离到三种形态的胰岛干细胞:多角形胰导管细胞、多突起内分泌祖细胞和小圆细胞。免疫组化鉴定,分离的细胞表达胚胎干细胞标志SSEA-1、SSEA-4、Oct4,也表达胰腺干细胞的标志PDX-1、CK7 等,证实分离的细胞具有胰腺干细胞特征。 测定仔猪胰腺干细胞的生长曲线。分离培养的胰腺干细胞体外生长12~15d 可传代,
[Abstract]:The prevalence of diabetes is increasing in recent years, and it has become the third most common chronic disease after cardiovascular disease and tumor. Rapid, effective and long-term supplementation of insulin deficiency is the fundamental treatment for diabetes mellitus. At present, three methods are used to supplement the body's insulin secretion: traditional subcutaneous injection of exogenous insulin, although effective but difficult to prevent the occurrence and development of complications, the injection dose often does not meet the needs of the body. The pancreas and islet organ transplantation can solve the problem that insulin supply and demand does not match, but the transplant organ is rare, the operation is complicated, the adverse reaction is many, transplant the stem cells that secrete insulin, It can make up for the deficiency of the above two methods. It can be easily injected into the human body to become its own pancreatic cells. It is effective for a long time. The body can also adjust the proportion of many kinds of cells producing insulin and other auxiliary cytokines as needed. To avoid complications, stem cell proliferation is strong, the number of stem cells is sufficient, and the rejection of transplantation is low. But it was also due to the severe lack of donor tissue that the development of its application was limited, and when people turned to look for xenogeneic animal pancreatic tissue as a source of donor tissue, they found that pigs had strong reproductive performance and sufficient tissue sources. The structure and biological activity of insulin are similar to that of human insulin. The pancreatic stem cells of piglets can be used as the best donor cells of pancreatic stem cells in xenogeneic animals. The study of pancreatic stem cells in piglets is a new hope for the treatment of diabetes mellitus. This experiment was conducted from three aspects: isolation and culture of pancreatic stem cells from piglets, determination of biological properties and pluripotency of inducing differentiation. To provide basic data for clinical application of pancreatic stem cells in piglets. 1. To establish the isolation method and culture system of pancreatic stem cells in piglets. High activity pancreatic cells were obtained from fresh porcine pancreatic tissue, digested with 0.25% trypsin on ice, formed small cell mass and single cell, filtered, centrifuged at low speed, repeatedly centrifuged, or centrifuged with 5%BSA as centrifuge solution. Laminin or gelatin was used to lay petri dish, 10% serum and 10 mmol/L nicotine were added to low glucose DMEM medium as the base culture medium. After 2 days of culture, the unadherent cells were removed and replaced with the basic culture medium of 10 ng/mL EGF. Long-term culture to promote the proliferation and growth of pancreatic stem cells in vitro. It was confirmed that the endocrine progenitor cells of piglet pancreas were polyprotuberous cells. 2. 2. Biological characteristics of pancreatic stem cells in piglets. The primary cells adhered to the wall by cell mass and single cell. The cells in the cell mass continued to be detached, suspended, retained adherent cells and single cells grew, about 7 days there appeared many protruded endocrine progenitor cells, clone like growth, quickly connected into a network; The number of small round cells increased and the proliferation of polygonal ductal epithelial stem cells was slow. Electron microscopic observation showed that the polygonal cells had a long protuberance of 50 ~ 80 渭 m, polarity, a large ratio of nucleus to cytoplasm, and an undeveloped organelle. The small round cells were 1012 渭 m in diameter, with microvilli and insulin positive. The polygonal cells were round, and the ratio of nucleus to cytoplasm was large. Cytoplasmic organelles and small secretory granules can be seen. Three types of islet stem cells were successfully isolated: polygonal pancreatic duct cells, polygonal endocrine progenitor cells and small round cells. The isolated cells expressed embryonic stem cells (SSEA-1,SSEA-4,Oct4,) and pancreatic stem cells (PDX-1,CK7), which confirmed that the isolated cells had the characteristics of pancreatic stem cells. The growth curve of pancreatic stem cells in piglets was measured. Pancreatic stem cells were isolated and cultured for 12 to 15 days in vitro.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2
本文编号：2243717
[Abstract]:The prevalence of diabetes is increasing in recent years, and it has become the third most common chronic disease after cardiovascular disease and tumor. Rapid, effective and long-term supplementation of insulin deficiency is the fundamental treatment for diabetes mellitus. At present, three methods are used to supplement the body's insulin secretion: traditional subcutaneous injection of exogenous insulin, although effective but difficult to prevent the occurrence and development of complications, the injection dose often does not meet the needs of the body. The pancreas and islet organ transplantation can solve the problem that insulin supply and demand does not match, but the transplant organ is rare, the operation is complicated, the adverse reaction is many, transplant the stem cells that secrete insulin, It can make up for the deficiency of the above two methods. It can be easily injected into the human body to become its own pancreatic cells. It is effective for a long time. The body can also adjust the proportion of many kinds of cells producing insulin and other auxiliary cytokines as needed. To avoid complications, stem cell proliferation is strong, the number of stem cells is sufficient, and the rejection of transplantation is low. But it was also due to the severe lack of donor tissue that the development of its application was limited, and when people turned to look for xenogeneic animal pancreatic tissue as a source of donor tissue, they found that pigs had strong reproductive performance and sufficient tissue sources. The structure and biological activity of insulin are similar to that of human insulin. The pancreatic stem cells of piglets can be used as the best donor cells of pancreatic stem cells in xenogeneic animals. The study of pancreatic stem cells in piglets is a new hope for the treatment of diabetes mellitus. This experiment was conducted from three aspects: isolation and culture of pancreatic stem cells from piglets, determination of biological properties and pluripotency of inducing differentiation. To provide basic data for clinical application of pancreatic stem cells in piglets. 1. To establish the isolation method and culture system of pancreatic stem cells in piglets. High activity pancreatic cells were obtained from fresh porcine pancreatic tissue, digested with 0.25% trypsin on ice, formed small cell mass and single cell, filtered, centrifuged at low speed, repeatedly centrifuged, or centrifuged with 5%BSA as centrifuge solution. Laminin or gelatin was used to lay petri dish, 10% serum and 10 mmol/L nicotine were added to low glucose DMEM medium as the base culture medium. After 2 days of culture, the unadherent cells were removed and replaced with the basic culture medium of 10 ng/mL EGF. Long-term culture to promote the proliferation and growth of pancreatic stem cells in vitro. It was confirmed that the endocrine progenitor cells of piglet pancreas were polyprotuberous cells. 2. 2. Biological characteristics of pancreatic stem cells in piglets. The primary cells adhered to the wall by cell mass and single cell. The cells in the cell mass continued to be detached, suspended, retained adherent cells and single cells grew, about 7 days there appeared many protruded endocrine progenitor cells, clone like growth, quickly connected into a network; The number of small round cells increased and the proliferation of polygonal ductal epithelial stem cells was slow. Electron microscopic observation showed that the polygonal cells had a long protuberance of 50 ~ 80 渭 m, polarity, a large ratio of nucleus to cytoplasm, and an undeveloped organelle. The small round cells were 1012 渭 m in diameter, with microvilli and insulin positive. The polygonal cells were round, and the ratio of nucleus to cytoplasm was large. Cytoplasmic organelles and small secretory granules can be seen. Three types of islet stem cells were successfully isolated: polygonal pancreatic duct cells, polygonal endocrine progenitor cells and small round cells. The isolated cells expressed embryonic stem cells (SSEA-1,SSEA-4,Oct4,) and pancreatic stem cells (PDX-1,CK7), which confirmed that the isolated cells had the characteristics of pancreatic stem cells. The growth curve of pancreatic stem cells in piglets was measured. Pancreatic stem cells were isolated and cultured for 12 to 15 days in vitro.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2
【引证文献】
相关期刊论文 前1条
1 张慧茹;冯若鹏;庞全海;刘珍;窦忠英;;仔猪胰腺干细胞的多向分化潜能[J];中国兽医科学;2011年01期
相关博士学位论文 前3条
1 楚元奎;胎猪胰岛间充质干细胞分离鉴定及向胰岛素分泌细胞分化的研究[D];西北农林科技大学;2011年
2 史明艳;山羊毛囊干细胞及组织工程皮肤构建研究[D];西北农林科技大学;2006年
3 王峗;猪胰腺干细胞分离鉴定[D];西北农林科技大学;2008年
相关硕士学位论文 前1条
1 冯若鹏;胎猪胰腺干细胞分离与克隆[D];西北农林科技大学;2006年
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