仔豬胰腺干細(xì)胞的分離鑒定
發(fā)布時(shí)間：2018-09-14 19:56
【摘要】：近年來糖尿病的患病率日益增高,已成為繼心血管疾病、腫瘤之后第三大高發(fā)慢性病�？焖�、有效、長期地補(bǔ)充機(jī)體胰島素分泌不足是糖尿病治療的根本方案。目前采用三種方法補(bǔ)充機(jī)體胰島素分泌不足:傳統(tǒng)皮下注射外源性胰島素,雖然有效但很難阻止并發(fā)癥的發(fā)生和發(fā)展,注射劑量經(jīng)常與機(jī)體需要不相符,且每日每餐均需注射很繁瑣;胰腺和胰島器官移植,可解決胰島素供需不符的問題,但移植器官難得、手術(shù)復(fù)雜、不良反應(yīng)多;移植分泌胰島素的干細(xì)胞,可以彌補(bǔ)上述兩種方法不足,簡易地注入人體成為自身胰腺細(xì)胞,一次給藥長期有效,機(jī)體還可根據(jù)需要,調(diào)節(jié)產(chǎn)生胰島素和其他輔助細(xì)胞因子等的多種細(xì)胞比例,避免并發(fā)癥發(fā)生,干細(xì)胞增殖力強(qiáng)、數(shù)量充足、移植排斥反應(yīng)低。但也是由于供體組織嚴(yán)重匱乏限制其應(yīng)用發(fā)展,人們轉(zhuǎn)向?qū)ふ耶惙N動物胰腺組織作為供體組織來源時(shí)發(fā)現(xiàn),豬繁殖性能強(qiáng),組織來源充足,胰島素結(jié)構(gòu)和生物活性類似于人胰島素,血糖調(diào)定點(diǎn)同人接近,豬胰腺細(xì)胞可在人血清中生存對人體無害,因此,仔豬胰腺干細(xì)胞可作為最佳的異種動物胰腺干細(xì)胞的供體細(xì)胞,對仔豬胰腺干細(xì)胞的研究是糖尿病治療的新希望。本試驗(yàn)從仔豬胰腺干細(xì)胞分離培養(yǎng)、生物學(xué)性能測定及誘導(dǎo)分化多能性三個(gè)方面進(jìn)行研究,為仔豬胰腺干細(xì)胞的臨床應(yīng)用提供基礎(chǔ)資料。 1. 建立仔豬胰腺干細(xì)胞的分離方法和培養(yǎng)體系。 取新鮮的仔豬胰腺組織,冰上操作,0.25%胰酶消化,形成小細(xì)胞團(tuán)和單個(gè)細(xì)胞,過濾,低速、多次離心,或以5%BSA作離心液離心,均可獲得高活力的胰腺細(xì)胞,用層粘連蛋白或明膠鋪被培養(yǎng)皿,以低糖DMEM 培養(yǎng)液添加10%血清及10 mmol/L 煙堿作為基礎(chǔ)培養(yǎng)液,培養(yǎng)2d 后,除去未貼壁細(xì)胞,更換為去除煙堿、添加10 ng/mL EGF的基礎(chǔ)培養(yǎng)液,長期培養(yǎng)以促進(jìn)胰腺干細(xì)胞體外增殖生長。證實(shí)仔豬胰腺內(nèi)分泌祖細(xì)胞呈多突起細(xì)胞形態(tài)。 2. 仔豬胰腺干細(xì)胞各項(xiàng)生物學(xué)特性。 原代細(xì)胞以細(xì)胞團(tuán)和單個(gè)細(xì)胞貼壁,細(xì)胞團(tuán)中不斷有細(xì)胞脫離、懸浮,保留的貼壁細(xì)胞和單個(gè)細(xì)胞生長,約7d 出現(xiàn)多突起內(nèi)分泌祖細(xì)胞,克隆樣生長,很快連接成網(wǎng)狀;小圓細(xì)胞數(shù)量增多;多角形導(dǎo)管上皮干細(xì)胞增殖慢。電鏡觀察多突起細(xì)胞具50~80μm長突起、有極性、核質(zhì)比大、細(xì)胞器不發(fā)達(dá);小圓細(xì)胞為直徑10~12μm、有微絨毛、胰島素陽性;多角形細(xì)胞呈圓形、核質(zhì)比大、可見胞質(zhì)內(nèi)細(xì)胞器和細(xì)小分泌顆粒。成功地分離到三種形態(tài)的胰島干細(xì)胞:多角形胰導(dǎo)管細(xì)胞、多突起內(nèi)分泌祖細(xì)胞和小圓細(xì)胞。免疫組化鑒定,分離的細(xì)胞表達(dá)胚胎干細(xì)胞標(biāo)志SSEA-1、SSEA-4、Oct4,也表達(dá)胰腺干細(xì)胞的標(biāo)志PDX-1、CK7 等,證實(shí)分離的細(xì)胞具有胰腺干細(xì)胞特征。 測定仔豬胰腺干細(xì)胞的生長曲線。分離培養(yǎng)的胰腺干細(xì)胞體外生長12~15d 可傳代,
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R329.2
本文編號：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R329.2
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 張慧茹;馮若鵬;龐全海;劉珍;竇忠英;;仔豬胰腺干細(xì)胞的多向分化潛能[J];中國獸醫(yī)科學(xué);2011年01期
相關(guān)博士學(xué)位論文 前3條
1 楚元奎;胎豬胰島間充質(zhì)干細(xì)胞分離鑒定及向胰島素分泌細(xì)胞分化的研究[D];西北農(nóng)林科技大學(xué);2011年
2 史明艷;山羊毛囊干細(xì)胞及組織工程皮膚構(gòu)建研究[D];西北農(nóng)林科技大學(xué);2006年
3 王峗;豬胰腺干細(xì)胞分離鑒定[D];西北農(nóng)林科技大學(xué);2008年
相關(guān)碩士學(xué)位論文 前1條
1 馮若鵬;胎豬胰腺干細(xì)胞分離與克隆[D];西北農(nóng)林科技大學(xué);2006年
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