胰岛移植治疗糖尿病的临床前研究
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摘要
第一部分胰岛移植治疗糖尿病的临床前研究
糖尿病是对人类生命健康危害最为严重的疾病之一。胰岛移植是二十世纪七十年代建立的一种治疗糖尿病的技术方法,特别是本世纪采用Edmonton胰岛方案以来,胰岛移植已经发展成为一项成熟的技术,成为纠正糖代谢紊乱,防治并逆转糖尿病慢性并发症的有效方法。
目的建立人胰岛分离、纯化和功能评价的技术方案,并应用该方案对所分离和纯化的人胰岛进行体内、外功能的评价和安全性评估,为开展临床胰岛移植奠定基础,为并申报胰岛细胞治疗临床准入批文提供系统的数据资料。
方法获得成人尸体胰腺供体6例,采用胶原酶消化法及密度梯度离心法分离和纯化人尸体胰腺供体的胰岛,经双硫腙(DTZ)染色计数并分析胰岛细胞的纯度。通过AO/PI染色评价分离胰岛的活力;通过电子显微镜观察胰岛素分泌颗粒;采用免疫荧光染色分析分离胰岛的细胞组成和分布;通过RT-PCR检测分离胰岛主要激素和自身抗原mRNA的表达;通过静态葡萄糖刺激检测胰岛素分泌功能;将分离的人胰岛移植到糖尿病小鼠评价胰岛的代谢功能;并对分离胰岛的产品进行细菌污染,内毒素含量和凝血活性等安全性指标的检测。
结果成人尸体胰腺供体6例,冷缺血时间10-12个小时,胰腺重量65.61±19.31(41.30-94.87)g,未消化组织重量15.09±8.21(7.44-26.85)g,消化组织的重量为50.52±19.56(33.86-83.45)g;分离纯化后胰岛的数量为22.9±3.1万IEQs(4970±1620IEQs/g组织);经不连续密度梯度纯化后,胰岛的纯度为59.0%±8.9%(49%-70%);吖啶橙/碘化丙啶(AO/PI)染色显示,分离胰岛的活力为89%±3%(85%-94%);RT-PCR表明,分离胰岛的胰岛素、胰高糖素、IA2、IAR、ICAp69以及GAD65 mRNA表达阳性;静态葡萄糖孵育刺激胰岛素释放的刺激指数(SI)为8.1±4.0(3.8-10.2);透射电镜扫描可见分离的胰岛细胞中有大量的胰岛素分泌颗粒;冰冻切片的免疫荧光染色显示分离胰岛中C-肽(C-peptide)阳性细胞占大多数,主要分布于胰岛的中心部位,胰高糖素(glucagon)阳性细胞次之,主要分布在胰岛的周边部位,生长抑素(somatostatin)阳性细胞较少,主要散在分布于胰岛内,胰多肽(pancreaticpolytide)阳性细胞很少,主要分布于胰岛的周边;冻存复苏后的胰岛经AO/PI染色观察胰岛的活力为85%;冻存复苏后的胰岛经静态葡萄糖刺激后,胰岛素分泌的刺激指数为2.5;将分离纯化后的人胰岛移植于链脲菌素(STZ)制备糖尿病裸鼠的左肾包膜下,发现治疗组血糖3天后降至正常水平并长期保持;切除接受了胰岛移植的左侧肾脏后,糖尿病裸鼠的血糖明显升高;对分离胰岛的培养上清进行细菌培养的检测,结果为阴性,胰岛培养上清中内毒素含量＜0.5EU/ml,凝血活性＜300U/ml。这些结果和技术指标已经全面达到或超过美国迈阿密胰岛移植中心和宾夕法尼亚胰岛移植中心的技术指标。
结论采用我们制定的胰岛分离、纯化技术方案所分离的人胰岛在形态、结构、数量、纯度、活力、体内外功能以及安全性等方面的各项指标都达到实施临床胰岛移植的标准,为最终开展临床胰岛移植奠定了临床前基础.
第二部分胰腺干细胞诱导分化为胰岛素分泌细胞的实验研究
糖尿病是危害人类健康的慢性疾病,近年来发病率不断增加,糖尿病导致的各种严重并发症是患者致死、致残的主要原因。胰岛素治疗不能有效逆转糖尿病微血管并发症,胰岛移植是糖尿病的有效治疗手段。然而,胰腺供体的缺乏限制了胰岛移植的广泛开展。干细胞具有体外自我增殖的能力及分化成为各种组织细胞的潜能,将干细胞诱导分化成为胰岛的内分泌细胞有可能成为解决胰腺供体缺乏的途径。
目的分离纯化大鼠胰腺组织干细胞,体外诱导其向胰岛内分泌细胞分化并形成胰岛样结构,评价干细胞分化胰岛的体外功能并观察其在实验性糖尿病模型中的治疗作用。
方法应用胶原酶消化法和密度梯度离心法从大鼠胰腺分离出胰岛和胰腺导管细胞;应用nestin结合的免疫磁珠从大鼠胰腺导管细胞中分离纯化得到少量胰腺组织干细胞.经体外扩增,将大鼠胰腺干细胞在加入ITS、尼克酰胺、bFGF等诱导剂的条件培养基及悬浮培养于含有细胞外基质(extracellular matrix,ECM)成分的条件培养基将大鼠胰腺干细胞诱导分化为胰岛素分泌细胞并形成胰岛样结构;对干细胞诱导分化形成的胰岛进行体外和体内的功能评价。
结果从大鼠胰腺分离获得胰岛及胰腺导管细胞于体外培养:采用nestin抗体包被的免疫磁珠将其中的胰腺组织干细胞纯化出来;在体外扩增后,将大鼠胰腺干细胞在加入ITS、尼克酰胺、bFGF等诱导剂的条件培养基及悬浮培养于含有细胞外基质成分的条件培养基将大鼠胰腺干细胞诱导分化形成胰岛样结构;经RT-PCR检测显示干细胞诱导分化的胰岛表达胰岛素的mRNA;免疫荧光染色显示,胰岛结构内含有大量胰岛素阳性细胞和少量胰高糖素阳性细胞;在葡萄糖刺激下干细胞分化胰岛有胰岛素释放,其释放量约为正常胰岛的39.4%;将干细胞分化胰岛移植到免疫缺陷的糖尿病小鼠后可以明显改善糖代谢的紊乱。
结论可以应用大鼠胰腺组织中nestin阳性的干细胞可以在体外制备成为具有治疗糖尿病功能的胰岛细胞。
Pre-Clinical Research of Islet Transplantation For Diabetes therapy
Objective Diabetes mellitus is one of the diseases to harm seriously for human health. Islet transplantation has been demonstrated to be an effective therapy for insulin-dependent diabetes. This research is to establish the protocols for human islet isolation and purification and to evaluate the functions and safety of human islet products in vitro and in vivo as well as eventually to provide experimental basis for clinical application of islet transplantation.
Methods Six human pancreases were obtained from the 181st hospital of PLA. Human pancreases were digested using automatic method established by Ricordi and purificated by the Ficoll density gradient centrifugation in COBE 2991 blood cell processor. The islet counts and purities were calculated by a computer picture analysis program in a microscope (Nikon, SMZ1500) after DTZ staining. The mRNA expressions of insulin, glucagon and several islet relegated autoantigens were detected by RT-PCR. The glucose-induced insulin secretion was detected in vitro by chemiluminescence method. Insulin secretory granules of the isolated islets were observed by electron microscope. The cellular composition and distribution of the isolated islets were checked by a fluorescent microscope. The vitality of the isolated islets was analyzed by a computer picture analysis program after AO/PI staining. The insulin secretion function of the isolated islets was further evaluated in vivo by islet transplantation in diabetic nude mice. The medium of the islet products were cultured for detecting aerobic, anaerobic bacteria and fungus. The endotoxin content in the medium of the islet products was tested by TAL gel formation method. The procoagulant activity in medium of the islet products was determined by clotting time.
Results The cold ischemia time of the pancreas donors was 10 to 12 hours. The mean weight of these pancreas was 65.61g±19.31 g(41.30g-94.87g). The mean count of the islets was 229,000±31,000 IEQs (4970±1620 IEQs/g tissue) and the mean purity of the islets was 59.0%±8.9% (49%-70%) after purification. The vitality of the islets was 89%±3% (85%-94%) by AO/PI staining assay. By RT-PCR, the mRNA expressions of insulin, glucagons, IA2, IAR, ICAp69 and GAD65 were positive in the islets. The static insulin releasing test indicated that the mean insulin stimulation index was 8.1±4.0 (3.8-10.2) . By transmission electron microscope, a great quantity of insulin secretory granules was found in the cytoplasma of islet cells. The result of immunofluorescence staining showed that C-peptide positive cells were mainly distributed in the central of the islets, glucagons positive cells were mainly located the perimeter of the islets and polypeptide positive cells were distributed scatteringly in the islets. The vitalities of islets before freezing and after thawing were 90% and 85% respectively by AO/PI staining assay. The insulin stimulating index of the islets after thawing was 2.5. In order to evaluate function of the human islets, isolated human islets were transplanted under the capsule of left kidney in STZ induced diabetic nude mice. The result indicated that the glycemia of the diabetic nude mice decreased to normal levels on the third day after islet transplantation and maintained for a long-term, while the glycemia of the mice increased to diabetic state after the left kidneys with grafted islets were removed. The culture medium of islet products was collected to test bacteria and endotoxin. No aerobic or anaerobic bacteria and fungus was detected in the culture medium of islet products. The levels of endotoxin in the medium of islet products were below 0.5 EU/ml and procoagulant activities in that were below 300U/ml.
Conclusion We have established a practical protocol of human islets transplantation. According to this protocol, we have obtained adequate amount, high purity, functional and safe human pancreatic islets products for clinical islet transplantation. Our research works establish abundant experimental foundation for clinical islet transplantation in the future.
The experimental study of pancreatic stem cells differentiating into insulin producing cells
Diabetes mellitus is a chronic metabolic disorder disease characterized as hyperglycemia that results in several serious complications such as diabetic nephropathy, diabetic retinopathy, et al. Morbility of diabetes is increasing greatly worldwide these years. The diabetic complications threaten human health greatly and causing death and disability. Insulin injection therapy can not control metabolic disorder perfectly and can not reverse the diabetic complications. Pancreatic islet transplantation is an effective therapy to cure insulin-dependent diabetes mellitus (IDDM) . Howerver, the deficiency of pancreas donors limits the clinical application widespreadly. Stem cells own the potency of proliferation and differentiation into multi-kinds of tissue cells. Induction stem cells to differentiate into endocrine cells of pancreatic islet will possiblely solve the deficiency of pancreas donors.
Objective In the research, the pancreatic stem cells were isolated and purified from rat pancreas. Then the stem cells were induced to differentiate into pancreatic endocrinal cells and to form islet-like structures. To evaluate the potential application of stem cell for diabetes therapy, the function of these stem cell induced islets was further evaluated in vitro and in vivo.
Methods Firstly, rat islets and pancreatic duct cells were isolated from rat pancreas by collagenase digestion and density gradient centrifugation. Then, pancreatic stem cells were purified from pancreatic duct cells using nestin conjugated magnetic beads. The pancreatic stem cells were induced proliferation, differentiation and formation of islet-like structures by inductors such as insulin-transferrin-selenium, nicotinamide, basic fibroblast growth factor and following by suspension culture in additive extracellular matrix including fibronectin, laminin and collagen type IV. The function of these stem cell induced islets was further evaluated in vitro and in vivo.
Results Pancreatic islets and pancreatic duct cells were isolated from rat pancreas by collagenase digestion and density gradient centrifugation. Then, the pancreatic stem cells were purified from cultured pancreatic duct cells using nestin conjugated magnetic beads. After proliferation, the stem cells were induced to differentiate into insulin producing cells by inductors such as insulin-transferrin-selenium, nicotinamide and basic fibroblast growth factor and to form islet-like structure following by suspension culture in additive extracellular matrix including fibronectin, laminin and collagen type IV. The expression of insulin mRNA in the stem cells differentiated islets was significantly up-regulated by reverse transcription polymerase chain reaction (RT-PCR). Immunofluoresence staining indicated that lots of insulin positive cells and a few of glucagon positive cells were existed in the islet-like structures. The glucose-induced insulin releasing in stem cell differentiated islets was 39.4% of that in native rat pancreatic islets. In addition, the glycemia in diabetic nude mice was normalized after transplantation with the stem cell differentiated islets.
Conclusion: The nestin positive stem cells derived from rat pancreas could be induced differentiating into insulin producing cells, which exhibited the potential application for diabetes therapy.
糖尿病是对人类生命健康危害最为严重的疾病之一。胰岛移植是二十世纪七十年代建立的一种治疗糖尿病的技术方法,特别是本世纪采用Edmonton胰岛方案以来,胰岛移植已经发展成为一项成熟的技术,成为纠正糖代谢紊乱,防治并逆转糖尿病慢性并发症的有效方法。
目的建立人胰岛分离、纯化和功能评价的技术方案,并应用该方案对所分离和纯化的人胰岛进行体内、外功能的评价和安全性评估,为开展临床胰岛移植奠定基础,为并申报胰岛细胞治疗临床准入批文提供系统的数据资料。
方法获得成人尸体胰腺供体6例,采用胶原酶消化法及密度梯度离心法分离和纯化人尸体胰腺供体的胰岛,经双硫腙(DTZ)染色计数并分析胰岛细胞的纯度。通过AO/PI染色评价分离胰岛的活力;通过电子显微镜观察胰岛素分泌颗粒;采用免疫荧光染色分析分离胰岛的细胞组成和分布;通过RT-PCR检测分离胰岛主要激素和自身抗原mRNA的表达;通过静态葡萄糖刺激检测胰岛素分泌功能;将分离的人胰岛移植到糖尿病小鼠评价胰岛的代谢功能;并对分离胰岛的产品进行细菌污染,内毒素含量和凝血活性等安全性指标的检测。
结果成人尸体胰腺供体6例,冷缺血时间10-12个小时,胰腺重量65.61±19.31(41.30-94.87)g,未消化组织重量15.09±8.21(7.44-26.85)g,消化组织的重量为50.52±19.56(33.86-83.45)g;分离纯化后胰岛的数量为22.9±3.1万IEQs(4970±1620IEQs/g组织);经不连续密度梯度纯化后,胰岛的纯度为59.0%±8.9%(49%-70%);吖啶橙/碘化丙啶(AO/PI)染色显示,分离胰岛的活力为89%±3%(85%-94%);RT-PCR表明,分离胰岛的胰岛素、胰高糖素、IA2、IAR、ICAp69以及GAD65 mRNA表达阳性;静态葡萄糖孵育刺激胰岛素释放的刺激指数(SI)为8.1±4.0(3.8-10.2);透射电镜扫描可见分离的胰岛细胞中有大量的胰岛素分泌颗粒;冰冻切片的免疫荧光染色显示分离胰岛中C-肽(C-peptide)阳性细胞占大多数,主要分布于胰岛的中心部位,胰高糖素(glucagon)阳性细胞次之,主要分布在胰岛的周边部位,生长抑素(somatostatin)阳性细胞较少,主要散在分布于胰岛内,胰多肽(pancreaticpolytide)阳性细胞很少,主要分布于胰岛的周边;冻存复苏后的胰岛经AO/PI染色观察胰岛的活力为85%;冻存复苏后的胰岛经静态葡萄糖刺激后,胰岛素分泌的刺激指数为2.5;将分离纯化后的人胰岛移植于链脲菌素(STZ)制备糖尿病裸鼠的左肾包膜下,发现治疗组血糖3天后降至正常水平并长期保持;切除接受了胰岛移植的左侧肾脏后,糖尿病裸鼠的血糖明显升高;对分离胰岛的培养上清进行细菌培养的检测,结果为阴性,胰岛培养上清中内毒素含量＜0.5EU/ml,凝血活性＜300U/ml。这些结果和技术指标已经全面达到或超过美国迈阿密胰岛移植中心和宾夕法尼亚胰岛移植中心的技术指标。
结论采用我们制定的胰岛分离、纯化技术方案所分离的人胰岛在形态、结构、数量、纯度、活力、体内外功能以及安全性等方面的各项指标都达到实施临床胰岛移植的标准,为最终开展临床胰岛移植奠定了临床前基础.
第二部分胰腺干细胞诱导分化为胰岛素分泌细胞的实验研究
糖尿病是危害人类健康的慢性疾病,近年来发病率不断增加,糖尿病导致的各种严重并发症是患者致死、致残的主要原因。胰岛素治疗不能有效逆转糖尿病微血管并发症,胰岛移植是糖尿病的有效治疗手段。然而,胰腺供体的缺乏限制了胰岛移植的广泛开展。干细胞具有体外自我增殖的能力及分化成为各种组织细胞的潜能,将干细胞诱导分化成为胰岛的内分泌细胞有可能成为解决胰腺供体缺乏的途径。
目的分离纯化大鼠胰腺组织干细胞,体外诱导其向胰岛内分泌细胞分化并形成胰岛样结构,评价干细胞分化胰岛的体外功能并观察其在实验性糖尿病模型中的治疗作用。
方法应用胶原酶消化法和密度梯度离心法从大鼠胰腺分离出胰岛和胰腺导管细胞;应用nestin结合的免疫磁珠从大鼠胰腺导管细胞中分离纯化得到少量胰腺组织干细胞.经体外扩增,将大鼠胰腺干细胞在加入ITS、尼克酰胺、bFGF等诱导剂的条件培养基及悬浮培养于含有细胞外基质(extracellular matrix,ECM)成分的条件培养基将大鼠胰腺干细胞诱导分化为胰岛素分泌细胞并形成胰岛样结构;对干细胞诱导分化形成的胰岛进行体外和体内的功能评价。
结果从大鼠胰腺分离获得胰岛及胰腺导管细胞于体外培养:采用nestin抗体包被的免疫磁珠将其中的胰腺组织干细胞纯化出来;在体外扩增后,将大鼠胰腺干细胞在加入ITS、尼克酰胺、bFGF等诱导剂的条件培养基及悬浮培养于含有细胞外基质成分的条件培养基将大鼠胰腺干细胞诱导分化形成胰岛样结构;经RT-PCR检测显示干细胞诱导分化的胰岛表达胰岛素的mRNA;免疫荧光染色显示,胰岛结构内含有大量胰岛素阳性细胞和少量胰高糖素阳性细胞;在葡萄糖刺激下干细胞分化胰岛有胰岛素释放,其释放量约为正常胰岛的39.4%;将干细胞分化胰岛移植到免疫缺陷的糖尿病小鼠后可以明显改善糖代谢的紊乱。
结论可以应用大鼠胰腺组织中nestin阳性的干细胞可以在体外制备成为具有治疗糖尿病功能的胰岛细胞。
Pre-Clinical Research of Islet Transplantation For Diabetes therapy
Objective Diabetes mellitus is one of the diseases to harm seriously for human health. Islet transplantation has been demonstrated to be an effective therapy for insulin-dependent diabetes. This research is to establish the protocols for human islet isolation and purification and to evaluate the functions and safety of human islet products in vitro and in vivo as well as eventually to provide experimental basis for clinical application of islet transplantation.
Methods Six human pancreases were obtained from the 181st hospital of PLA. Human pancreases were digested using automatic method established by Ricordi and purificated by the Ficoll density gradient centrifugation in COBE 2991 blood cell processor. The islet counts and purities were calculated by a computer picture analysis program in a microscope (Nikon, SMZ1500) after DTZ staining. The mRNA expressions of insulin, glucagon and several islet relegated autoantigens were detected by RT-PCR. The glucose-induced insulin secretion was detected in vitro by chemiluminescence method. Insulin secretory granules of the isolated islets were observed by electron microscope. The cellular composition and distribution of the isolated islets were checked by a fluorescent microscope. The vitality of the isolated islets was analyzed by a computer picture analysis program after AO/PI staining. The insulin secretion function of the isolated islets was further evaluated in vivo by islet transplantation in diabetic nude mice. The medium of the islet products were cultured for detecting aerobic, anaerobic bacteria and fungus. The endotoxin content in the medium of the islet products was tested by TAL gel formation method. The procoagulant activity in medium of the islet products was determined by clotting time.
Results The cold ischemia time of the pancreas donors was 10 to 12 hours. The mean weight of these pancreas was 65.61g±19.31 g(41.30g-94.87g). The mean count of the islets was 229,000±31,000 IEQs (4970±1620 IEQs/g tissue) and the mean purity of the islets was 59.0%±8.9% (49%-70%) after purification. The vitality of the islets was 89%±3% (85%-94%) by AO/PI staining assay. By RT-PCR, the mRNA expressions of insulin, glucagons, IA2, IAR, ICAp69 and GAD65 were positive in the islets. The static insulin releasing test indicated that the mean insulin stimulation index was 8.1±4.0 (3.8-10.2) . By transmission electron microscope, a great quantity of insulin secretory granules was found in the cytoplasma of islet cells. The result of immunofluorescence staining showed that C-peptide positive cells were mainly distributed in the central of the islets, glucagons positive cells were mainly located the perimeter of the islets and polypeptide positive cells were distributed scatteringly in the islets. The vitalities of islets before freezing and after thawing were 90% and 85% respectively by AO/PI staining assay. The insulin stimulating index of the islets after thawing was 2.5. In order to evaluate function of the human islets, isolated human islets were transplanted under the capsule of left kidney in STZ induced diabetic nude mice. The result indicated that the glycemia of the diabetic nude mice decreased to normal levels on the third day after islet transplantation and maintained for a long-term, while the glycemia of the mice increased to diabetic state after the left kidneys with grafted islets were removed. The culture medium of islet products was collected to test bacteria and endotoxin. No aerobic or anaerobic bacteria and fungus was detected in the culture medium of islet products. The levels of endotoxin in the medium of islet products were below 0.5 EU/ml and procoagulant activities in that were below 300U/ml.
Conclusion We have established a practical protocol of human islets transplantation. According to this protocol, we have obtained adequate amount, high purity, functional and safe human pancreatic islets products for clinical islet transplantation. Our research works establish abundant experimental foundation for clinical islet transplantation in the future.
The experimental study of pancreatic stem cells differentiating into insulin producing cells
Diabetes mellitus is a chronic metabolic disorder disease characterized as hyperglycemia that results in several serious complications such as diabetic nephropathy, diabetic retinopathy, et al. Morbility of diabetes is increasing greatly worldwide these years. The diabetic complications threaten human health greatly and causing death and disability. Insulin injection therapy can not control metabolic disorder perfectly and can not reverse the diabetic complications. Pancreatic islet transplantation is an effective therapy to cure insulin-dependent diabetes mellitus (IDDM) . Howerver, the deficiency of pancreas donors limits the clinical application widespreadly. Stem cells own the potency of proliferation and differentiation into multi-kinds of tissue cells. Induction stem cells to differentiate into endocrine cells of pancreatic islet will possiblely solve the deficiency of pancreas donors.
Objective In the research, the pancreatic stem cells were isolated and purified from rat pancreas. Then the stem cells were induced to differentiate into pancreatic endocrinal cells and to form islet-like structures. To evaluate the potential application of stem cell for diabetes therapy, the function of these stem cell induced islets was further evaluated in vitro and in vivo.
Methods Firstly, rat islets and pancreatic duct cells were isolated from rat pancreas by collagenase digestion and density gradient centrifugation. Then, pancreatic stem cells were purified from pancreatic duct cells using nestin conjugated magnetic beads. The pancreatic stem cells were induced proliferation, differentiation and formation of islet-like structures by inductors such as insulin-transferrin-selenium, nicotinamide, basic fibroblast growth factor and following by suspension culture in additive extracellular matrix including fibronectin, laminin and collagen type IV. The function of these stem cell induced islets was further evaluated in vitro and in vivo.
Results Pancreatic islets and pancreatic duct cells were isolated from rat pancreas by collagenase digestion and density gradient centrifugation. Then, the pancreatic stem cells were purified from cultured pancreatic duct cells using nestin conjugated magnetic beads. After proliferation, the stem cells were induced to differentiate into insulin producing cells by inductors such as insulin-transferrin-selenium, nicotinamide and basic fibroblast growth factor and to form islet-like structure following by suspension culture in additive extracellular matrix including fibronectin, laminin and collagen type IV. The expression of insulin mRNA in the stem cells differentiated islets was significantly up-regulated by reverse transcription polymerase chain reaction (RT-PCR). Immunofluoresence staining indicated that lots of insulin positive cells and a few of glucagon positive cells were existed in the islet-like structures. The glucose-induced insulin releasing in stem cell differentiated islets was 39.4% of that in native rat pancreatic islets. In addition, the glycemia in diabetic nude mice was normalized after transplantation with the stem cell differentiated islets.
Conclusion: The nestin positive stem cells derived from rat pancreas could be induced differentiating into insulin producing cells, which exhibited the potential application for diabetes therapy.
引文
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