人胚胎干细胞诱导分化为胰岛素分泌细胞的研究
|
摘要
胰岛移植或β细胞替代足糖尿病最理想的治疗手段,但细胞来源严重匮乏及免疫排斥反应限制了临床的广泛应用。将人胚胎干细胞(human embryonicstem cell,hESCs)诱导为胰岛素分泌细胞,成为β细胞替代物的新资源,为解决细胞来源匮乏带来了希望。已有多种方案由ESCs获得胰岛素分泌细胞,但均存在各种限制,目前仍缺乏将ESCs诱导分化为有功能的胰岛素分泌细胞的最佳方案。探索诱导人hSCs分化为成熟且有功能的胰岛素分泌细胞,并制定出有效而高效的分化方案,成为本课题的主要研究目的。
第一,本研究摸索了在体外培养、扩增、冻存、复苏hESCs细胞株(PKU1)的条件,并对其特异性标志物进行了检测。结果显示,hESCs经多次传代后仍然表达OCT-4和Nanog基因,免疫荧光法显示呈OCT-4、SSEA-4阳性,流式细胞术分析显示,OCT-4、SSEA-4阳性细胞比例分别达89.38%、83.44%;染色体核型分析表明为正常女性染色体核型(46,XX)。
第二,采用酶消化法分离出胎龄14-18W的胎儿胰岛,经悬浮培养、双硫腙(DTZ)染色后种植到组织培养皿中,再以免疫荧光法检测一些细胞标志物的表达。结果显示,悬浮培养条件下,胎龄14-18W的胎儿胰岛细胞聚集成球形细胞簇,呈DTZ阳性,培养液中能检测到一定量的胰岛素和C-肽,贴壁培养后还能检测到胰岛细胞标志物insulin和glucagons及前体细胞标志物PDX-1、CK-19和Nestin的表达,证实14-17W胎儿胰岛已具备一定的胰岛特性和前体细胞的特征。
第三,模拟胰腺发育分化的生理过程,研究设计出五阶段诱导方案,具体如下:(1).撤除分化抑制因素使hESCs形成拟胚体(EBs),悬浮培养48h;(2).将EBs转移至明胶包被的组织培养皿后,加入含Activin A和bFGF及LY294002的诱导培养基Ⅰ诱导5天,向定形内胚层诱导分化。RT-PCR法鉴定SOX17和FOXA2的基因表达,与同期自发分化的EBs进行比较,免疫荧光法检测SOX17的表达。(3).将细胞转移至laminin包被的培养皿,加入含bFGF、KGF、EGF、GLP-1、尼克酰胺的低血清诱导培养基Ⅱ作用14d,向胰腺前体细胞诱导。RT-PCR法和免疫荧光化学法分别检测前体细胞标志物PDX-1及Ngn3表达。(4).添加含IGF-1、betacellulin、尼克酰胺、GLP-1、laminin的低血清诱导培养基Ⅲ作用14d,促进胰岛细胞分化,RT-PCR法检测细胞胰岛素基因的表达,免疫荧光化学法检测分化细胞胰岛素和C-肽的表达。(5).将细胞转移至低黏附性培养皿中,撤除IGF-I继续培养3天,促进细胞成熟。应用双硫腙(DTZ)染色液对获得的细胞团进行染色,应用RT-PCR法检测了胰岛素和胰高血糖素基因表达,应用RIA法分别测定了获得的细胞团培养液上清和胞浆中的胰岛素、C-肽含量,并与胎儿胰岛培液上清进行比较,静态培养胰岛素释放试验测定胰岛素与C-肽分泌是否呈葡萄糖反应性。结果(1).hESCs悬浮培养2d后,形成类球形EBs;(2).经第二阶段诱导后,细胞表达SOX17和FOXA2基因,且表达量高于同期自发分化的EBs,基本不表达外胚层标志物SOX1,随诱导时间延长,FOXA2表达逐渐增强;免疫荧光化学法显示,诱导5天的细胞胞核呈SOX17。(3).经第三阶段的诱导,细胞呈上皮样集落,RT-PCR法检测到细胞表达PDX-1、Ngn3及微弱的Pax4基因,免疫荧光法显示细胞呈PDX-1阳性。(4)经第四阶段的诱导,集落中一部分细胞聚集成多层,RT-PCR显示,不同诱导时期均有胰岛素基因的表达,且随诱导时间延长表达水平逐渐升高;免疫荧光显示,部分细胞呈胰岛素和C-肽阳性。(5).经第五阶段诱导后,hESCs来源的细胞呈类似胎儿胰岛的胰岛样细胞簇(ICCs),呈DTZ阳性,RT-PCR检测到Insulin和Glucagon基因表达,RIA法检测到ICCs培液上清中含胰岛素与C-肽,水平与15-17W胎儿胰岛培液上清相近(P＞0.05),胞浆内的胰岛素和C-肽水平分别为1089.2±217.06μIU/ml和181.33±30.12ng/ml,静态培养胰岛素释放试验验证了ICCs分泌的胰岛素呈糖反应性。
应用五阶段分化方案,hESCs能够有效的分化为具有部分β细胞特性和糖反应性胰岛素分泌功能的细胞,为DM细胞替代治疗提供了有潜力的细胞来源和良好的临床应用前景。
Islet transplantation orβcells replacement therapy are taken as a promising approach for diabetes treatment,but widespread application of this treatment is restricted by absence of islet source and immunological rejection.Progresses made in human embryonic stem cells(hESCs)field brought hope for settling the shortage of islet source.Many reports have obtained insulin-producing cells from ESCs using different strategies,however,these all have various limitations and there is lack of an optimal protocol producing functionalβcells from ESCs.To explore a high-performance differentiation protocol that can obtaine mature and functional insulin-producing cells from hESCs is the major problem that will be settled in this study.
At first,this study explored the condition to culture,amplificate,freeze and reanimate the hESCs line PKUl and the expression of several specific markers of hESCs were detected.Results showed,after high passage,hESCs expressed OCT-4 and Nanog gene,remained positive for OCT-4 and SSEA-4,and the percentage of OCT-4- and SSEA-4-positive cells was respectively 89.38%and 83.44%.Chromatosome karyotype analysis showed the 114 generation hESCs karyotype is normal female(46,XX).Thus,the study successfully established the hESCs culture system that could maintain their stem cell features.
In order to realize the morphology and character of islets during embryo development and provide reference for identification of the insulin-producing cells derived from hESCs,the fetal islets gestation age between 14-18 weeks were isolated by enzymatic digestion and cultured in suspension for 3 days.After DTZ staining,fetal islets were changed to tissue culture dish,then some islet markers and precursor cells markers were identified by immunofluorescence.The insulin and C-peptide content in culture supernatant was measured by radioimmunoassay (RIA).Results showed,fetal islets were spherical clusters and were positive for DTZ,insulin and C-peptide were detected in the culture supernatant,and some of the cells in the epithelioid cell colonies were positive for insulin and glucagon, and several precursor cell markers such as PDX-1、CK19 and Nestin.From this, fetal islets of gestation age between 14-17W have some features of islet and some precursor cells.
This study designed a proposal of five stages minic embryo pancreas development.Firstly,withdraw the inhibitor of differentiation and hESCs formed embryonic bodies(EBs),after 2 days the EBs were planted to 0.1%gelatin-coated culture dishes and cultured in DF12 medium containing serum replacement(SR) supplemented with bFGF,Activin A and LY294002 for 5 days(stage 2),then the cells were dissociated with trypsin and planted to lminin-coated culture dishes in DF12 medium containing low fetal calf serum(FBS)supplemented with bFGF, KGF,EGF,GLP-1 and nicotinamide(stage 3).After 14 days,bFGF,EGF and KGF were withdrew and replaced by IGF-1 and BTC and cultured for another 14 days(stage 4).The cells were dissociated with collagenaseⅣand transferred into low-attachment plates and cultured in RPMI-1640 medium supplemented with GLP-1 and nicotinamide for 3-5 days(stage 5).Ihe induction effects were identified by RT-PCR,immunofluorescence,DTZ staining and RIA.The indexes markers included definitive endoderm(DE)markers such as SOX17 and FOXA2, pancreatic markers PDX-1 and Ngn3 and islet markers insulin,glucagons and C-peptide,and the insulin and C-peptide content in the last stage culture supernatant and glucose-stimulated insulin and C-peptide release analysis were carried out by RIA.
The results showed,the cells induced in the stage 2 for 5d expressed SOX17 and FOXA2 genes,their expression levels were higher than that of the same days EBs,and the FOXA2 level increased with the induction time.The immunofluorescence displayed the cells were positive for SOX17 in nuclei.After stage 3,the cells were positive for PDX-1 and expressed pancreatic precursor marker PDX-1 and islet precursor marker Nng3 gene,even a very low expression of Pax4.After stage 4,the induced cells expressed Insulin gene and expression level raised with induction days,part of them were positive for insulin and C-peptide.At last stage,the hESC- derived cell clusters were spherical just like fetal islets,and were positive for DTZ.RT-PCR showed,the cells expressed Insulin and Glucagon gene.By RIA,insulin and C-peptide content in culture supernatant were similar with that were detected in fetal islets culture supernatant. Intracellular insulin and C-peptide content was 1089.2±217.06μIU/ml and 181.33±30.12ng/ml,and insulin release from the ICCs was regulated by glucose.
Thus,results presented in this study provide credence that the five stages protocol differentiated hESCs into mature insulin-producing cells and can offers a promising source of cells for diabetes cell transplantation therapy.
第一,本研究摸索了在体外培养、扩增、冻存、复苏hESCs细胞株(PKU1)的条件,并对其特异性标志物进行了检测。结果显示,hESCs经多次传代后仍然表达OCT-4和Nanog基因,免疫荧光法显示呈OCT-4、SSEA-4阳性,流式细胞术分析显示,OCT-4、SSEA-4阳性细胞比例分别达89.38%、83.44%;染色体核型分析表明为正常女性染色体核型(46,XX)。
第二,采用酶消化法分离出胎龄14-18W的胎儿胰岛,经悬浮培养、双硫腙(DTZ)染色后种植到组织培养皿中,再以免疫荧光法检测一些细胞标志物的表达。结果显示,悬浮培养条件下,胎龄14-18W的胎儿胰岛细胞聚集成球形细胞簇,呈DTZ阳性,培养液中能检测到一定量的胰岛素和C-肽,贴壁培养后还能检测到胰岛细胞标志物insulin和glucagons及前体细胞标志物PDX-1、CK-19和Nestin的表达,证实14-17W胎儿胰岛已具备一定的胰岛特性和前体细胞的特征。
第三,模拟胰腺发育分化的生理过程,研究设计出五阶段诱导方案,具体如下:(1).撤除分化抑制因素使hESCs形成拟胚体(EBs),悬浮培养48h;(2).将EBs转移至明胶包被的组织培养皿后,加入含Activin A和bFGF及LY294002的诱导培养基Ⅰ诱导5天,向定形内胚层诱导分化。RT-PCR法鉴定SOX17和FOXA2的基因表达,与同期自发分化的EBs进行比较,免疫荧光法检测SOX17的表达。(3).将细胞转移至laminin包被的培养皿,加入含bFGF、KGF、EGF、GLP-1、尼克酰胺的低血清诱导培养基Ⅱ作用14d,向胰腺前体细胞诱导。RT-PCR法和免疫荧光化学法分别检测前体细胞标志物PDX-1及Ngn3表达。(4).添加含IGF-1、betacellulin、尼克酰胺、GLP-1、laminin的低血清诱导培养基Ⅲ作用14d,促进胰岛细胞分化,RT-PCR法检测细胞胰岛素基因的表达,免疫荧光化学法检测分化细胞胰岛素和C-肽的表达。(5).将细胞转移至低黏附性培养皿中,撤除IGF-I继续培养3天,促进细胞成熟。应用双硫腙(DTZ)染色液对获得的细胞团进行染色,应用RT-PCR法检测了胰岛素和胰高血糖素基因表达,应用RIA法分别测定了获得的细胞团培养液上清和胞浆中的胰岛素、C-肽含量,并与胎儿胰岛培液上清进行比较,静态培养胰岛素释放试验测定胰岛素与C-肽分泌是否呈葡萄糖反应性。结果(1).hESCs悬浮培养2d后,形成类球形EBs;(2).经第二阶段诱导后,细胞表达SOX17和FOXA2基因,且表达量高于同期自发分化的EBs,基本不表达外胚层标志物SOX1,随诱导时间延长,FOXA2表达逐渐增强;免疫荧光化学法显示,诱导5天的细胞胞核呈SOX17。(3).经第三阶段的诱导,细胞呈上皮样集落,RT-PCR法检测到细胞表达PDX-1、Ngn3及微弱的Pax4基因,免疫荧光法显示细胞呈PDX-1阳性。(4)经第四阶段的诱导,集落中一部分细胞聚集成多层,RT-PCR显示,不同诱导时期均有胰岛素基因的表达,且随诱导时间延长表达水平逐渐升高;免疫荧光显示,部分细胞呈胰岛素和C-肽阳性。(5).经第五阶段诱导后,hESCs来源的细胞呈类似胎儿胰岛的胰岛样细胞簇(ICCs),呈DTZ阳性,RT-PCR检测到Insulin和Glucagon基因表达,RIA法检测到ICCs培液上清中含胰岛素与C-肽,水平与15-17W胎儿胰岛培液上清相近(P＞0.05),胞浆内的胰岛素和C-肽水平分别为1089.2±217.06μIU/ml和181.33±30.12ng/ml,静态培养胰岛素释放试验验证了ICCs分泌的胰岛素呈糖反应性。
应用五阶段分化方案,hESCs能够有效的分化为具有部分β细胞特性和糖反应性胰岛素分泌功能的细胞,为DM细胞替代治疗提供了有潜力的细胞来源和良好的临床应用前景。
Islet transplantation orβcells replacement therapy are taken as a promising approach for diabetes treatment,but widespread application of this treatment is restricted by absence of islet source and immunological rejection.Progresses made in human embryonic stem cells(hESCs)field brought hope for settling the shortage of islet source.Many reports have obtained insulin-producing cells from ESCs using different strategies,however,these all have various limitations and there is lack of an optimal protocol producing functionalβcells from ESCs.To explore a high-performance differentiation protocol that can obtaine mature and functional insulin-producing cells from hESCs is the major problem that will be settled in this study.
At first,this study explored the condition to culture,amplificate,freeze and reanimate the hESCs line PKUl and the expression of several specific markers of hESCs were detected.Results showed,after high passage,hESCs expressed OCT-4 and Nanog gene,remained positive for OCT-4 and SSEA-4,and the percentage of OCT-4- and SSEA-4-positive cells was respectively 89.38%and 83.44%.Chromatosome karyotype analysis showed the 114 generation hESCs karyotype is normal female(46,XX).Thus,the study successfully established the hESCs culture system that could maintain their stem cell features.
In order to realize the morphology and character of islets during embryo development and provide reference for identification of the insulin-producing cells derived from hESCs,the fetal islets gestation age between 14-18 weeks were isolated by enzymatic digestion and cultured in suspension for 3 days.After DTZ staining,fetal islets were changed to tissue culture dish,then some islet markers and precursor cells markers were identified by immunofluorescence.The insulin and C-peptide content in culture supernatant was measured by radioimmunoassay (RIA).Results showed,fetal islets were spherical clusters and were positive for DTZ,insulin and C-peptide were detected in the culture supernatant,and some of the cells in the epithelioid cell colonies were positive for insulin and glucagon, and several precursor cell markers such as PDX-1、CK19 and Nestin.From this, fetal islets of gestation age between 14-17W have some features of islet and some precursor cells.
This study designed a proposal of five stages minic embryo pancreas development.Firstly,withdraw the inhibitor of differentiation and hESCs formed embryonic bodies(EBs),after 2 days the EBs were planted to 0.1%gelatin-coated culture dishes and cultured in DF12 medium containing serum replacement(SR) supplemented with bFGF,Activin A and LY294002 for 5 days(stage 2),then the cells were dissociated with trypsin and planted to lminin-coated culture dishes in DF12 medium containing low fetal calf serum(FBS)supplemented with bFGF, KGF,EGF,GLP-1 and nicotinamide(stage 3).After 14 days,bFGF,EGF and KGF were withdrew and replaced by IGF-1 and BTC and cultured for another 14 days(stage 4).The cells were dissociated with collagenaseⅣand transferred into low-attachment plates and cultured in RPMI-1640 medium supplemented with GLP-1 and nicotinamide for 3-5 days(stage 5).Ihe induction effects were identified by RT-PCR,immunofluorescence,DTZ staining and RIA.The indexes markers included definitive endoderm(DE)markers such as SOX17 and FOXA2, pancreatic markers PDX-1 and Ngn3 and islet markers insulin,glucagons and C-peptide,and the insulin and C-peptide content in the last stage culture supernatant and glucose-stimulated insulin and C-peptide release analysis were carried out by RIA.
The results showed,the cells induced in the stage 2 for 5d expressed SOX17 and FOXA2 genes,their expression levels were higher than that of the same days EBs,and the FOXA2 level increased with the induction time.The immunofluorescence displayed the cells were positive for SOX17 in nuclei.After stage 3,the cells were positive for PDX-1 and expressed pancreatic precursor marker PDX-1 and islet precursor marker Nng3 gene,even a very low expression of Pax4.After stage 4,the induced cells expressed Insulin gene and expression level raised with induction days,part of them were positive for insulin and C-peptide.At last stage,the hESC- derived cell clusters were spherical just like fetal islets,and were positive for DTZ.RT-PCR showed,the cells expressed Insulin and Glucagon gene.By RIA,insulin and C-peptide content in culture supernatant were similar with that were detected in fetal islets culture supernatant. Intracellular insulin and C-peptide content was 1089.2±217.06μIU/ml and 181.33±30.12ng/ml,and insulin release from the ICCs was regulated by glucose.
Thus,results presented in this study provide credence that the five stages protocol differentiated hESCs into mature insulin-producing cells and can offers a promising source of cells for diabetes cell transplantation therapy.
引文
[1].Ramiya V K,Maraist M,Arfors K E,et al.Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.Nat Med,2000,6(3):278-282.
[2].Bonnet-Weir S,Taneja M,Weir G C,et al.In vitro cultivation of human islets from expanded ductal tissue.PNAS,2000,97(14):7999-8004.
[3].Kim SY,Lee SH,Kim BM,et al.Activation of nestin-positive duct stem (NPDS)cells in pancreas upon neogenic motivation and possible cytodifferentiation into insulin-secreting cells from NPDS cells.Dev Dyn,2004,230(1):1-11.
[4].Huang H,Tang X.Phenotypic determination and characterizationof nestin-positive precursors derived from human fetal pancreas.Lab Inve,2003,83(4):539-547.
[5].Yang L J,Li S W,Hatch H,et al.In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells.PNAS,2002,99(12):8078-8083.
[6].Zalzman M,Gupta S,Giri R K,et al.Reversal of hyperglycemia in mice by using human expandable insulin-producing cell sdifferentiated from fetal liver progenitor cells.PNAS,2003,100(12):7253-7258.
[7].Tamar S,Keren S,Irit M L,et al.Cell-replacement therapy for diabetes:Generating functional insulin-producing tissue from adult human liver cells.PNAS,2005,102(22):7964-7969.
[8].Oh SH,Muzzonigro TM,Bae S H,et al.Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type 1diabetes.Lab Invest,2004,84:607-617.
[9].Tang DQ,Cao LZ,Burkhardt BR,et al.In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow.Diabetes,2004,53(7):1721-1732.
[10].李艳华,白慈贤,谢超,等成人骨髓间充质干细胞体外定向诱导分化为胰岛样细胞团的研究.自然科学进展,2003,13(6):593-597.
[11].Bums CJ,Minger SL,Hall S,et al.The in vitro differentiation of rat neural stem cells into an insulin-expressing phenotype.Biochem Biophys Res Commun,2005,326(3):570-577.
[12].Hori Y,Gu X,Xie X,et al.Differentiation of insulin-producing cells from human neural progenitor cells.PLOS Medicine,2005,2(4):347-356.
[13].Kojima H,Nakamura T,Fujita Y,et al.Combined expression of pancreatic duodenal homeobox-1 and islet factor 1 induces immature entereeytes to produce insulin.Diabetes,2002,51(5):1398-1408.
[14].Sofia B,Roche E,Berna G,et al.Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000,49:157-162.
[15].Suheir A,Gila M,Michal A,et al.Insulin Production by Human Embryonic Stem Cells. Diabetes, 2001, 50:1691-1697.
[16]. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R: Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science, 2001, 292: 1389-1394.
[17]. Blyszczuk P and Wobus AM. Stem cells and pancreatic differentiation in vitro. J. Biotechnol, 2004, 113:3-13.
[18]. Hanna S, Bettina F, Anna Z, et al. Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells, 2004, 22:265-274.
[19]. Hori Y, Rulifson I C, Tsai B C, et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. PNAS 2002,99: 16105-16110.
[20]. Bai L, Meredith G and Tuch BE. Glucagon-like peptide-1 enhances production of insulin in insulin- producing cells derived from mouse embryonic stem cells. Journal of Endocrinology, 2005, 186: 343-352 .
[21].Yue FM, Cui L, Johkura K, et al. Glucagon-like peptide-1 differentiation of primate embryonic stem cells into insulin-producing cells. Tissue Engineering, 2006, 12(8): 2105-2116.
[22]. Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. PNAS, 2003, 100:998-1003.
[23]. Satsuki M, Eiji Y, and Jun-ichi M. Regulated Expression of pdx-l Promotes In Vitro Differentiation of Insulin- producing Cells From Embryonic Stem Cells. Diabetes 2004, 53:1030-1037.
[24]. Leon-Quinto T, Jones J, Skoudy A, et al. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia, 2004, 47:1442-1451.
[25]. Akira S, Shigehiko U, Yukiteru O, et al. Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer. World J Gastroenterol, 2005, 11(27):4161-4166.
[26]. Brolen KC, Nico H, Josefina E, et al. Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing β-cell-like cells. Diabetes, 2005, 54:2867-2874.
[27]. Vaca P, Martin F, Vegara-Meseguer JM, et al. Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors. Stem Cells, 2006, 24(2):258-265.
[28]. Rajagopal J, Anderson, WJ, Kume, S, et al. Insulin staining of ES cell progeny from insulin uptake. Science, 2003, 299: 363.
[29]. Sipione S, Eshpeter A, Lyon JG. Insulin-expressing cells from differentiated embryonic stem cells are not beta cells. Diabetologia, 2004, 47:499-508.
[30]. Hansson M, Tonning A, Frandsen U. et al. Artifactual insulin release from differentiated embryonic stem cells. Diabetes, 2004, 53:2603-2609.
[31]. Enrique R, Pilar S, Juan AR, et al. Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells. FASEB.J, 2005, 19(10)1341-1343.
[32]. Xu X, Kahan B, Forgianni A, et al. Endoderm and pancreatic islet lineage differentiation from human embryonic stem cells. Cloning Stem Cells, 2006, 8(2): 96-107.
[33]. Slack JMW. Developmental biology of the pancreas. Development, 1995, 121:1569-1580.
[34]. Thomson JA, Itskovitz - Eldor J, Shapiro SS, et al . Embryonic stem cell lines derived from human blastocysts. Science, 1998, 282: 1145-1147.
[35]. Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci USA, 1998,95: 13726-13731.
[36]．薄爱华,夏苓,白雪梅,等．人胚胎期胰腺的形态学研究．解剖学报,1990,21(3)：333—336．
[37]. Shiroi A, Yoshikawa M, Yokota H, et al. Identification of Insulin-Producing Cells Derived from Embryonic Stem Cells by Zinc-Chelating Dithizone. Stem Cells, 2002, 20:284-292.
[38]. Palle S, Thomas M P. Islet and stem cell transplantation for reating diabetes. BMJ, 2001,22:29-32.
[39]. Ahlgren U, Jonsson J, Edlund H. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/ PDX-1 deficient mice. Development, 1996, 122: 1409-1416.
[40]. Offield MF, Jetton TL, Labosky PA, et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development, 1996, 122:983-995.
[41]. Bouwens L, Lu WG, De Krijer R. Proliferation and differentation in the human fetal endocrine panereas. Diabetologia, 1997, 40: 398-404.
[42]. Bonner-Weir S, Taneja M, Weir GC, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA, 2000, 97 (14): 7999-8004.
[43]. Hunziker E, Stein M. Nestin-expressing cells in the pancreatic islets of langerhans. Biochem Biophys Res Commun, 2000, 271(1): 116-119.
[44]. Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-postive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, excrine, and hepaticphenotypes. Diabetes, 2001, 50:521-533.
[45]. Huang H, Tang X. Phenotypic determination and characterization of nestin-positive precursors derived from human fetal pancreas. Lab Inve, 2003, 83 (4): 539-547.
[46]. Silvya SM, Maria LC, Leticia L, et al. Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures. Journal of Endocrinology, 2004, 183, 455-467.
[47]. Burns CJ, Minger SL, Hall S, et al. The in vitro differentiation of rat neural stem cells into an insulin-expressing phenotype. Biochem Biophys Res Commun, 2005, 326(3): 570-577.
[48]. Selander L, Edlund H. Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas. Mech Dev, 2002, 113 (2): 189-192.
[49]. Lardon J, Rooman I, Bouwens L, et al. Nestin expression in pancreatic stellate cells and angiogenic endothelial cells. Histochem Cell Biol, 2002, 117:535-540.
[51]. Klein T, Ling ZD, Heimberg H, et al. Nestin Is Expressed in Vascular Endothelial Cells in the Adult Human Pancreas. J Histochem Cytochem, 2003, 51:697-706.
[52]. Hopt UT, Drognitz O. Pancreas organ transplantation. Short and long-term results in terms of diabetes control. Langenbecks Arch Surg, 2000, 385(6):379-389.
[53]. Shapiro AM , Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid -free immuno-suppressive regimen. N Engl J Med, 2000, 343:230-238.
[54]. Kim SK, Hebrok M. Intercellular signals regulating pancreas development and function. Genes Dev, 2001, 15:111-127.
[55]. Li H, Arber S, Jessell TM, et al. Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9. Nat Genet, 1999, 23:67-70.
[56]. Schwitzgebel VM, Scheel DW, Conners JR, et al. Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development, 2000, 127:3533-3542.
[57]. Edlund H. Developmental biology of the pancreas. Diabetes, 2001, 50 (Suppl.1): S5-S9.
[58]. Moritoh Y, Yamato E, Yasui Y, et al. Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells. Diabetes, 2003,52:1163-1168.
[59]. Kim D, Gu Y, Ishii M,et al. In vivo functioning and transplantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cell. Pancreas, 2003, 27: E34-E41.
[60]. Edlund H. Transcribing pancreas. Diabetes, 1998, 47: 1817-1823.
[61]. Sander M, German MS. The beta cell transcription factors and development of the pancreas. J Mol Med, 1997, 75:327-340.
[62]. Habener JF, Kemp DM, Thomas MK. Minireview: Transcription al regulation in pancreatic development. Endocrinology, 2005, 146:1025-1034.
[63]. Stoffers DA, Thomas MK, Habener J F. Homeodomain protein IDX-1: a master regulator of pancreas development and insulin gene expression. Trends Endocrinol Metab, 1997, 8: 145-151.
[64]. Wang H, Maechler P, Ritz-Laser B, et al. Pdx1 level defines pancreatic gene expression pattern and cell lineage differentiation. J Biol Chem, 2001, 276 (27):25279-25286.
[65]. Gradwohl G, Dierich A, Lemur M, et al. Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. PNAS, 2000, 97:1607-1611.
[66]. Gu G, Dubauskaite J, Melton MA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development, 2002, 129:2447-2457.
[67]. Sosa-Pineda B, Chowdhury K, Torres M, et al. The Pax4 gene is essential for differentiation of insulin-producing β cells in the mammalian pancreas. Nature, 1997,386:399-402.
[68]. Dohrmann C, Gruss P, Lemaire L, et al. Pax genes and the differentiation of hormone-producing endocrine cells in the pancreas. Mech Dev, 2000, 92: 47-54.
[69]. Soria, B. In-vitro differentiation of pancreatic β cells. Differentiation, 2001, 68: 205-219.
[70]. Sussel L, Kalamaras J, Hartigan-O'Connor DJ, et al. Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic β-cells. Development, 1998, 125:2213-2221.
[71]. Wang JF, Elghazi L, Parker SE, et al. The concerted activities of Pax4 and Nkx2.2 are essential to initiate pancreatic β-cell differentiation. Developmental Biology, 2004, 266:178-189.
[72]. Rodaway A and Patient R. Mesendoderm: an ancient germ layer? Cell, 2001, 105: 169-172.
[73]. Shook D and Keller R. Mechanisms, mechanics and function of epithelial- mesenchymal transitions in early development. Mech Dev, 2003, 120:1351-1383.
[74]. Schier AF and Shen MM. Nodal signalling in vertebrate development. Nature, 2000,403:385-389.
[75]. Stainier DYR. A glimpse into the molecular entrails of endoderm formation. Genes Dev, 2002, 16:893-907.
[76]. Ninomiya H, Takahashi S, Tanegashima K, et al. Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus. Dev Growth Differ, 1999,41:391-400.
[77]. Tremblay KD, Hoodless PA, Bikoff EK et al. Formation of the definitive endoderm in mouse is a Smad-dependent process. Development, 2000, 127:3079-3090.
[78]. Kubo A, Shinozaki K, Shannon JM., et al. Development of definitive endoderm from embryonic stem cells in culture. Development, 2004, 131(7): 1651-1662.
[79]. D'Amour KA, Agulnick AD, Eliazer S et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol, 2005, 23:1534-1541.
[80]. Shiraki N, Lai CJ, Hishikari Y, et al. TGF-β signaling potentiates differentiation of embryonic stem cells to Pdx-1 expressing endodermal cells. Genes to Cells, 2005, 10:503-516.
[81]. Mclean AB, D'Amour KA, Jones KL. et al. Activin A efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. STEM CELLS, 2007, 25:29-38.
[82]. Kim SK, Hebrok M, Melton DA. Notochord to endoderm signaling is required for pancreas development. Development, 1997, 124:4243-4252.
[83]. Deutsch G, Jung J, Zheng M, et al. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development, 2001, 128: 871-881.
[84]. Kim SK and Macdonald RJ. Signaling and transcriptional control of pancreatic organogenesis. Curr Opin Genet Dev, 2002, 12: 540-547.
[85]. Ang SL, Wierda A, Wong D, et al. The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development, 1993, 119:1301-1315.
[86]. Kanai-Azuma M, Kanai Y, Gad JM, et al. Depletion of definitive gut endoderm in Soxl 7-null mutant mice. Developmen, 2002, 129: 2367-2379.
[87]. Wu KL, Gannon M, Peshavaria M, et al. Hepatocyte nuclear factor 3 beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene. Mol Cell Biol, 1997, 17:6002-6013.
[88]. Lee CS, Sund NJ, Vatamaniuk MZ, et al. Foxa2 controls Pdxl gene expression in pancreatic beta-cells in vivo. Diabetes, 2002, 51:2546-2551.
[89]. Itkin-Ansari P, Geron I, Hao E, et al . Cell-based therapies for diabetes : progress towards a transplantable human beta-cell line. Ann N Y Acad Sci, 2003, 1005: 138-147.
[90]. Jensen J. Gene regulatory factors in pancreatic development. Dev Dyn, 2004, 229: 176-200.
[91]. Dunbar AJ, Goddard C, Structure-function and biological role of betacellulin. Int J Biochem Cell Biol, 2000, 32:805-815.
[92]. Soria B. In vitro differentiation of pancreatic β cell. Differentiation, 2001, 68(2):205-219.
[93]. Yi ES, Yin S, Harclerode DL et al. Keratinocyte growth factor induces pancreatic ductal epithelial proliferation. Am J Pathol, 1994, 145:80-85.
[94]. Movassat J, Beattie GM, Lopez AD, et al. Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells. Diabetologia, 2003, 46:822-829.
[95]. Lam NT and Kieffer TJ. The multifaceted potential of glucagon-like peptide-1 as a therapeutic agent. Minerva Endocrinol, 2002, 27:79-93.
[96]. Hui HX, Wright C and Perfetti R. Glucagon-Like Peptide 1 Induces Differentiation of Islet Duodenal Homeobox-1 -Positive Pancreatic Ductal Cells Into Insulin-Secreting Cells. Diabetes, 2001, 50:785-796.
[97]. Abraham EJ, Leech CA, Lin JC, et al. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulinproducing cells. Endocrinology, 2002, 143:3152-3161.
[98]. Hardikar AA, Wang XY, Williams L, et al. Functional maturation of fetal porcine beta cells by glucagon-like peptide 1 and cholecystokinin. Endocrinology, 2002, 143: 3505-3514.
[99]. Huotari MA, Palgi J, Otonkoski T, et al. Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel beta-cell mitogen. Endocrinology, 1998, 139:1494-1499.
[100]. Li L, Seno M, Yamada H, et al. Promotion of beta-cell regeneration by betacellulin in ninety percent-pancreatectomized rats. Endocrinology, 2001, 142: 5379-5385.
[101]. Sugiyama K, Yonemura Y, Okamoto H, Effects of poly (ADP-ribose) synthetase inhibitor on B-cells of a canine pancreas after massive pancreatectomy. Int J Pancreatol, 1991,8:85-95.
[102]. Otonkoski T, Beattie GM, Mally MI, et al. Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. J Clin Invest, 1993,92:1459-1466.
[103]. Cho YM, Lim JM, Yoo DH, et al. Betacellulin and nicotinamide sustain PDX1 expression and induce pancreatic β-cell differentiation in human embryonic stem cells. Biochemical and Biophysical Research Communications, 2008, 366:129-134.
[1].Tyden G,Bolinder J,Solders G,et al.Improved survival in patients with insulin-dependedt diabetes mellitus and end-stage diabetic nephropathy 10years after combined pancreas and kidney transplantation.Transplantation,1999,67:645-648.
[2].Shapiro AM,Lakey JR,Ryan EA,et al.Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.N Engl J Med,2000,343:230-238.
[3].Ryan EA,Lakey JRT,Rajotte RV,et al.Clinical outcomes and insulin secretion after islet transplantation with the Endmonton protocol.Diabetes,2001,50:710-719.
[4].Ryan EA,Lakey J R,Paty BW,et al.Successful islet transplantation:continued insulin reserve provides long-term glycemic control.Diabetes,2002,51(7):2148-2157.
[5].Ryan EA,Paty BW,Senior PA,et al.Five-year follow-up after clinical islet transplantation.Diabetes,2005,54(7):2060-2069.
[6].Matsumoto S and Tanaka K.Pancreatic islet cell transplantation using non-heart-beating donors(NHBDs).J Hepa Pancreat Surg,2005,12:227-230.
[7].Voros P,Farkas G,Lengyel Z,et al.Albuminuria after fetal pancreatic islet transplantation:a 10-year follow-up.Nephrol Dial Transplant,1998,13(11):2899-2904.
[8].王海慧,陈兵,张平,等.人胎胰岛细胞的分离培养及其意义.重庆医学, 2006, 35(9): 783-784.
[9]. Yoon KH, Quikel RR, Tatarkieuicz K, et al. Differentiation and expansion of beta cell mass in porcine neonatal pancreatic cell clusters transplanted into nude mice. Cell Transplant, 1999, 8:673-677.
[10].Van Der Laan LJ, Lockey C, Griffeth BC, et al. Infection by procine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature, 2000,407:29-31.
[11]. Ramiya VK, Maraist M, Arfors K E, et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med, 2000, 6(3):278-282.
[12]. Bonner-Weir S, Taneja M, Weir G C, et al. In vitro cultivation of human islets from expanded ductal tissue. PNAS, 2000, 97 (14): 7999-8004.
[13]. Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-postive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, excrine, and hepaticphenotypes. Diabetes, 2001, 50:521-533.
[14]. Kim SY, Lee SH, Kim BM, et al. Activation of nestin-positive duct stem (NPDS) cells in pancreas upon neogenic motivation and possible cytodifferentiation into insulin-secreting cells from NPDS cells. Dev Dyn, 2004, 230(1): 1-11.
[15]. Huang H, Tang X. Phenotypic determination and characterizationof nestin-positive precursors derived from human fetal pancreas. Lab Invest, 2003, 83 (4):539-547.
[16]. Yang L J, Li S W, Hatch H, et al. In vitro transdifferentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. PNAS, 2002, 99(12):8078-8083.
[17]. Zalzman M, Gupta S, Giri R K, et al. Reversal of hyperglycemia in mice by using human expandable insulin-producing cell sdifferentiated from fetal liver progenitor cells. PNAS, 2003, 100 (12):7253-7258.
[18]. Tamar S, Keren S, Irit M L, et al. Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells. PNAS, 2005, 102(22):7964-7969.
[19]. Imai J, Katagiri H, Yamada T, Ishigaki Y, Ogihara T, Uno K, et al. Constitutively active PDX1 induced efficient insulin production in adult murine liver. Biochem Biophys Res Commun, 2005, 326:402-409.
[20]. Oh SH, Muzzonigro TM, Bae S H, et al. Adult bone marrow-derived cells trans- differentiating into insulin-producing cells for the treatment of type 1 diabetes. Lab Invest, 2004, 84: 607-617.
[21]. Tang DQ, Cao LZ, Burkhardt BR, et al. In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes, 2004, 53(7): 1721-1732.
[22]. Jahr H, Bretzel RG. Insulin-positive cells in vitro generated from rat bone marrow stromal cells. Transplant Proc, 2003, 35: 2140-2141.
[23].贾延劼,钟乐,宋建辉,等。体外诱导大鼠骨髓间充质干细胞分化为胰岛素分泌细胞.中国当代儿科杂志,2003,5(5):393-397。
[24].李艳华,白慈贤,谢超,等。成人骨髓间充质干细胞体外定向诱导分化为胰岛样细胞团的研究.自然科学进展,2003,13(6):593-597.
[25].陆琰,张洹,迟作华.体外诱导人骨髓间充质干细胞向胰岛β样细胞分化的研究.暨南大学学报(医学版),2005,26(6):729-733.
[26].Burns CJ,Minger SL,Hall S,et al.The in vitro differentiation of rat neural stem cells into an insulin-expressing phenotype.Biochem Biophys Res Commun,2005,326(3):570-577.
[27].Hori Y,Gu X,Xie X,et al.Differentiation of insulin-producing cells from human neural progenitor cells.PLOS Medicine,2005,2(4):347-356.
[28].Kodama S,Kuhtreiher W,Fujimura S,et al.Islet regeneration during the reversal of autoimmune diabetes in NOD mice.Science,2003,302:1223-1226.
[29].Kojima H,Nakamura T,Fujita Y,et al.Combined expression of pancreatic duodenal homeobox-1 and islet factor 1 induces immature entereeytes to produce insulin.Diabetes,2002,51(5):1398-1408.
[30].Ende N,Chen R F,Reddi A S.Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice.Biochem Biophys Res Commun,2004,325(3):665-669.
[31].Ende N,Chen R F and Reddi A S.Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice.Biochem Biophys Res Commun,2004,321(1):168-171.
[32].Soria B,Roche E,Berna G,et al.Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000,49:157-162.
[33].Suheir A,Gila M,Michal At,et al.Insulin Production by Human Embryonic Stem Cells.Diabetes,2001,50:1691-1697.
[34].Silva DG,Petrovsky N,Socha L,et al.Mechanisms of accelerated immune-mediated diabetes resulting from islet beta cell expression of a Fas ligand transgene.J Immunol,2003,170:4996-5002.
[35].Ichim TE,Zhong R,MinWP.Prevention of allograft rejection by in vitro generated tolerogenic dendritic cells.Transplant Immunology,2003,11:295-306.
[1].Shapiro AMJ,Lakey JRT,and Ryan EA.Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immuno-suppressive regimen.N Engl J Med,2000,343:230-238.
[2].Soria B,Skoudy A,and Martin F.From stem cells to beta cells:new strategies in cell therapy of diabetes mellitus.Diabetologia,2001,44:407-415.
[3].Soria B,Roche E,Berna G,et al.Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000,49:157-162.
[4].Suheir A,Gila M,Michal A,et al.Insulin Production by Human Embryonic Stem Cells.Diabetes,2001,50:1691-1697.
[5].Blyszczuk P,Czyz J,Kania G,et al.Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulinproducing cells.PNAS,2003,100:998-1003.
[6].Satsuki M,Eiji Y,and Jun-ichi M Regulated Expression of pdx-1 Promotes In Vitro Differentiation of Insulin- producing Cells From Embryonic Stem Cells.Diabetes,2004,53:1030-1037.
[7].Akira S,Shigehiko U,Yukiteru O,et al.Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer.World J Gastroenterol,2005,11(27):4161-4166.
[8].Vaca P,Berna' G,Marti'n F,and Sofia B.Nicotinamide Induces Both Proliferation and Differentiation of Embryonic Stem Cells Into Insulin-Producing Cells.Transplantation Proceedings,2003,35:2021-2023.
[9].张妍,郭希民,王常勇,等.硒促进小鼠胚胎干细胞分化为胰岛细胞的实验研究.军事医学科学院院刊,2004,28(2):126-129.
[11].Brole'n KC,Nico H,Josefina E,et al.Signals From the Embryonic Mouse Pancreas Induce Differentiation of Human Embryonic Stem Cells Into Insulin-Producing β-Cell-Like Cells.Diabetes,2005,54:2867-2874.
[12].Vaca P,Martin F,Vegara-Meseguer JM,et al.Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors.Stem Cells,2006,24(2):258-265.
[13].Moritoh Y,Yamato E,Yasui Y,et al.Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells.Diabetes,2003,52:1163-1168.
[14].Leon-Quinto T,Jones J,Skoudy A,et al.In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells.Diabetologia,2004,47:1442-1451.
[15].Lumelsky N,Blondel O,Laeng P,et al.Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets.Science,2001:292:1389-1394.
[16].周光纪,屈纪富,徐海伟,等.无血清培养法诱导小鼠胚胎干细胞分化为胰岛素分泌细胞的实验条件优化选择.中国临床康复,2005,9(34):23-26.
[17].Linda B.L,Hung-Chih K,Laura A,et al.Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes.Reproductive Biology and Endocrinology,2004,2:42-46.
[18].Masahide Y,Hiroshi Y,Hiroshi F,et al.Identification of Insulin-Producing Cells Derived from Embryonic Stem Cells by Zinc-Chelating Dithizone.Stem Cells,2002,20:284-292.
[19].Rajagopal J,Anderson,WJ,Kume S,et al.Insulin staining of ES cell progeny from insulin uptake.Science,2003,299:363.
[20].Hansson M,Tonning,A,Frandsen,U.et al.Artifactual insulin release from differentiated embryonic stem cells.Diabetes,2004,53:2603-2609.
[21].Enrique R,Pilar S,Juan AR,et al.Ectodermal commitment of insulinproducing cells derived from mouse embryonic stem cells.FASEB,2005,19(10):1341-1343.
[22].Xu X,Kahan B,Forgianni A,et al.Endoderm and pancreatic islet lineage differentiation from human embryonic stem cells.Cloning Stem Cells,2006,8(2):96-107.
[2].Bonnet-Weir S,Taneja M,Weir G C,et al.In vitro cultivation of human islets from expanded ductal tissue.PNAS,2000,97(14):7999-8004.
[3].Kim SY,Lee SH,Kim BM,et al.Activation of nestin-positive duct stem (NPDS)cells in pancreas upon neogenic motivation and possible cytodifferentiation into insulin-secreting cells from NPDS cells.Dev Dyn,2004,230(1):1-11.
[4].Huang H,Tang X.Phenotypic determination and characterizationof nestin-positive precursors derived from human fetal pancreas.Lab Inve,2003,83(4):539-547.
[5].Yang L J,Li S W,Hatch H,et al.In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells.PNAS,2002,99(12):8078-8083.
[6].Zalzman M,Gupta S,Giri R K,et al.Reversal of hyperglycemia in mice by using human expandable insulin-producing cell sdifferentiated from fetal liver progenitor cells.PNAS,2003,100(12):7253-7258.
[7].Tamar S,Keren S,Irit M L,et al.Cell-replacement therapy for diabetes:Generating functional insulin-producing tissue from adult human liver cells.PNAS,2005,102(22):7964-7969.
[8].Oh SH,Muzzonigro TM,Bae S H,et al.Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type 1diabetes.Lab Invest,2004,84:607-617.
[9].Tang DQ,Cao LZ,Burkhardt BR,et al.In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow.Diabetes,2004,53(7):1721-1732.
[10].李艳华,白慈贤,谢超,等成人骨髓间充质干细胞体外定向诱导分化为胰岛样细胞团的研究.自然科学进展,2003,13(6):593-597.
[11].Bums CJ,Minger SL,Hall S,et al.The in vitro differentiation of rat neural stem cells into an insulin-expressing phenotype.Biochem Biophys Res Commun,2005,326(3):570-577.
[12].Hori Y,Gu X,Xie X,et al.Differentiation of insulin-producing cells from human neural progenitor cells.PLOS Medicine,2005,2(4):347-356.
[13].Kojima H,Nakamura T,Fujita Y,et al.Combined expression of pancreatic duodenal homeobox-1 and islet factor 1 induces immature entereeytes to produce insulin.Diabetes,2002,51(5):1398-1408.
[14].Sofia B,Roche E,Berna G,et al.Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000,49:157-162.
[15].Suheir A,Gila M,Michal A,et al.Insulin Production by Human Embryonic Stem Cells. Diabetes, 2001, 50:1691-1697.
[16]. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R: Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science, 2001, 292: 1389-1394.
[17]. Blyszczuk P and Wobus AM. Stem cells and pancreatic differentiation in vitro. J. Biotechnol, 2004, 113:3-13.
[18]. Hanna S, Bettina F, Anna Z, et al. Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells, 2004, 22:265-274.
[19]. Hori Y, Rulifson I C, Tsai B C, et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. PNAS 2002,99: 16105-16110.
[20]. Bai L, Meredith G and Tuch BE. Glucagon-like peptide-1 enhances production of insulin in insulin- producing cells derived from mouse embryonic stem cells. Journal of Endocrinology, 2005, 186: 343-352 .
[21].Yue FM, Cui L, Johkura K, et al. Glucagon-like peptide-1 differentiation of primate embryonic stem cells into insulin-producing cells. Tissue Engineering, 2006, 12(8): 2105-2116.
[22]. Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. PNAS, 2003, 100:998-1003.
[23]. Satsuki M, Eiji Y, and Jun-ichi M. Regulated Expression of pdx-l Promotes In Vitro Differentiation of Insulin- producing Cells From Embryonic Stem Cells. Diabetes 2004, 53:1030-1037.
[24]. Leon-Quinto T, Jones J, Skoudy A, et al. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia, 2004, 47:1442-1451.
[25]. Akira S, Shigehiko U, Yukiteru O, et al. Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer. World J Gastroenterol, 2005, 11(27):4161-4166.
[26]. Brolen KC, Nico H, Josefina E, et al. Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing β-cell-like cells. Diabetes, 2005, 54:2867-2874.
[27]. Vaca P, Martin F, Vegara-Meseguer JM, et al. Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors. Stem Cells, 2006, 24(2):258-265.
[28]. Rajagopal J, Anderson, WJ, Kume, S, et al. Insulin staining of ES cell progeny from insulin uptake. Science, 2003, 299: 363.
[29]. Sipione S, Eshpeter A, Lyon JG. Insulin-expressing cells from differentiated embryonic stem cells are not beta cells. Diabetologia, 2004, 47:499-508.
[30]. Hansson M, Tonning A, Frandsen U. et al. Artifactual insulin release from differentiated embryonic stem cells. Diabetes, 2004, 53:2603-2609.
[31]. Enrique R, Pilar S, Juan AR, et al. Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells. FASEB.J, 2005, 19(10)1341-1343.
[32]. Xu X, Kahan B, Forgianni A, et al. Endoderm and pancreatic islet lineage differentiation from human embryonic stem cells. Cloning Stem Cells, 2006, 8(2): 96-107.
[33]. Slack JMW. Developmental biology of the pancreas. Development, 1995, 121:1569-1580.
[34]. Thomson JA, Itskovitz - Eldor J, Shapiro SS, et al . Embryonic stem cell lines derived from human blastocysts. Science, 1998, 282: 1145-1147.
[35]. Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci USA, 1998,95: 13726-13731.
[36]．薄爱华,夏苓,白雪梅,等．人胚胎期胰腺的形态学研究．解剖学报,1990,21(3)：333—336．
[37]. Shiroi A, Yoshikawa M, Yokota H, et al. Identification of Insulin-Producing Cells Derived from Embryonic Stem Cells by Zinc-Chelating Dithizone. Stem Cells, 2002, 20:284-292.
[38]. Palle S, Thomas M P. Islet and stem cell transplantation for reating diabetes. BMJ, 2001,22:29-32.
[39]. Ahlgren U, Jonsson J, Edlund H. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/ PDX-1 deficient mice. Development, 1996, 122: 1409-1416.
[40]. Offield MF, Jetton TL, Labosky PA, et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development, 1996, 122:983-995.
[41]. Bouwens L, Lu WG, De Krijer R. Proliferation and differentation in the human fetal endocrine panereas. Diabetologia, 1997, 40: 398-404.
[42]. Bonner-Weir S, Taneja M, Weir GC, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA, 2000, 97 (14): 7999-8004.
[43]. Hunziker E, Stein M. Nestin-expressing cells in the pancreatic islets of langerhans. Biochem Biophys Res Commun, 2000, 271(1): 116-119.
[44]. Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-postive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, excrine, and hepaticphenotypes. Diabetes, 2001, 50:521-533.
[45]. Huang H, Tang X. Phenotypic determination and characterization of nestin-positive precursors derived from human fetal pancreas. Lab Inve, 2003, 83 (4): 539-547.
[46]. Silvya SM, Maria LC, Leticia L, et al. Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures. Journal of Endocrinology, 2004, 183, 455-467.
[47]. Burns CJ, Minger SL, Hall S, et al. The in vitro differentiation of rat neural stem cells into an insulin-expressing phenotype. Biochem Biophys Res Commun, 2005, 326(3): 570-577.
[48]. Selander L, Edlund H. Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas. Mech Dev, 2002, 113 (2): 189-192.
[49]. Lardon J, Rooman I, Bouwens L, et al. Nestin expression in pancreatic stellate cells and angiogenic endothelial cells. Histochem Cell Biol, 2002, 117:535-540.
[51]. Klein T, Ling ZD, Heimberg H, et al. Nestin Is Expressed in Vascular Endothelial Cells in the Adult Human Pancreas. J Histochem Cytochem, 2003, 51:697-706.
[52]. Hopt UT, Drognitz O. Pancreas organ transplantation. Short and long-term results in terms of diabetes control. Langenbecks Arch Surg, 2000, 385(6):379-389.
[53]. Shapiro AM , Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid -free immuno-suppressive regimen. N Engl J Med, 2000, 343:230-238.
[54]. Kim SK, Hebrok M. Intercellular signals regulating pancreas development and function. Genes Dev, 2001, 15:111-127.
[55]. Li H, Arber S, Jessell TM, et al. Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9. Nat Genet, 1999, 23:67-70.
[56]. Schwitzgebel VM, Scheel DW, Conners JR, et al. Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development, 2000, 127:3533-3542.
[57]. Edlund H. Developmental biology of the pancreas. Diabetes, 2001, 50 (Suppl.1): S5-S9.
[58]. Moritoh Y, Yamato E, Yasui Y, et al. Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells. Diabetes, 2003,52:1163-1168.
[59]. Kim D, Gu Y, Ishii M,et al. In vivo functioning and transplantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cell. Pancreas, 2003, 27: E34-E41.
[60]. Edlund H. Transcribing pancreas. Diabetes, 1998, 47: 1817-1823.
[61]. Sander M, German MS. The beta cell transcription factors and development of the pancreas. J Mol Med, 1997, 75:327-340.
[62]. Habener JF, Kemp DM, Thomas MK. Minireview: Transcription al regulation in pancreatic development. Endocrinology, 2005, 146:1025-1034.
[63]. Stoffers DA, Thomas MK, Habener J F. Homeodomain protein IDX-1: a master regulator of pancreas development and insulin gene expression. Trends Endocrinol Metab, 1997, 8: 145-151.
[64]. Wang H, Maechler P, Ritz-Laser B, et al. Pdx1 level defines pancreatic gene expression pattern and cell lineage differentiation. J Biol Chem, 2001, 276 (27):25279-25286.
[65]. Gradwohl G, Dierich A, Lemur M, et al. Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. PNAS, 2000, 97:1607-1611.
[66]. Gu G, Dubauskaite J, Melton MA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development, 2002, 129:2447-2457.
[67]. Sosa-Pineda B, Chowdhury K, Torres M, et al. The Pax4 gene is essential for differentiation of insulin-producing β cells in the mammalian pancreas. Nature, 1997,386:399-402.
[68]. Dohrmann C, Gruss P, Lemaire L, et al. Pax genes and the differentiation of hormone-producing endocrine cells in the pancreas. Mech Dev, 2000, 92: 47-54.
[69]. Soria, B. In-vitro differentiation of pancreatic β cells. Differentiation, 2001, 68: 205-219.
[70]. Sussel L, Kalamaras J, Hartigan-O'Connor DJ, et al. Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic β-cells. Development, 1998, 125:2213-2221.
[71]. Wang JF, Elghazi L, Parker SE, et al. The concerted activities of Pax4 and Nkx2.2 are essential to initiate pancreatic β-cell differentiation. Developmental Biology, 2004, 266:178-189.
[72]. Rodaway A and Patient R. Mesendoderm: an ancient germ layer? Cell, 2001, 105: 169-172.
[73]. Shook D and Keller R. Mechanisms, mechanics and function of epithelial- mesenchymal transitions in early development. Mech Dev, 2003, 120:1351-1383.
[74]. Schier AF and Shen MM. Nodal signalling in vertebrate development. Nature, 2000,403:385-389.
[75]. Stainier DYR. A glimpse into the molecular entrails of endoderm formation. Genes Dev, 2002, 16:893-907.
[76]. Ninomiya H, Takahashi S, Tanegashima K, et al. Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus. Dev Growth Differ, 1999,41:391-400.
[77]. Tremblay KD, Hoodless PA, Bikoff EK et al. Formation of the definitive endoderm in mouse is a Smad-dependent process. Development, 2000, 127:3079-3090.
[78]. Kubo A, Shinozaki K, Shannon JM., et al. Development of definitive endoderm from embryonic stem cells in culture. Development, 2004, 131(7): 1651-1662.
[79]. D'Amour KA, Agulnick AD, Eliazer S et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol, 2005, 23:1534-1541.
[80]. Shiraki N, Lai CJ, Hishikari Y, et al. TGF-β signaling potentiates differentiation of embryonic stem cells to Pdx-1 expressing endodermal cells. Genes to Cells, 2005, 10:503-516.
[81]. Mclean AB, D'Amour KA, Jones KL. et al. Activin A efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. STEM CELLS, 2007, 25:29-38.
[82]. Kim SK, Hebrok M, Melton DA. Notochord to endoderm signaling is required for pancreas development. Development, 1997, 124:4243-4252.
[83]. Deutsch G, Jung J, Zheng M, et al. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development, 2001, 128: 871-881.
[84]. Kim SK and Macdonald RJ. Signaling and transcriptional control of pancreatic organogenesis. Curr Opin Genet Dev, 2002, 12: 540-547.
[85]. Ang SL, Wierda A, Wong D, et al. The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development, 1993, 119:1301-1315.
[86]. Kanai-Azuma M, Kanai Y, Gad JM, et al. Depletion of definitive gut endoderm in Soxl 7-null mutant mice. Developmen, 2002, 129: 2367-2379.
[87]. Wu KL, Gannon M, Peshavaria M, et al. Hepatocyte nuclear factor 3 beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene. Mol Cell Biol, 1997, 17:6002-6013.
[88]. Lee CS, Sund NJ, Vatamaniuk MZ, et al. Foxa2 controls Pdxl gene expression in pancreatic beta-cells in vivo. Diabetes, 2002, 51:2546-2551.
[89]. Itkin-Ansari P, Geron I, Hao E, et al . Cell-based therapies for diabetes : progress towards a transplantable human beta-cell line. Ann N Y Acad Sci, 2003, 1005: 138-147.
[90]. Jensen J. Gene regulatory factors in pancreatic development. Dev Dyn, 2004, 229: 176-200.
[91]. Dunbar AJ, Goddard C, Structure-function and biological role of betacellulin. Int J Biochem Cell Biol, 2000, 32:805-815.
[92]. Soria B. In vitro differentiation of pancreatic β cell. Differentiation, 2001, 68(2):205-219.
[93]. Yi ES, Yin S, Harclerode DL et al. Keratinocyte growth factor induces pancreatic ductal epithelial proliferation. Am J Pathol, 1994, 145:80-85.
[94]. Movassat J, Beattie GM, Lopez AD, et al. Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells. Diabetologia, 2003, 46:822-829.
[95]. Lam NT and Kieffer TJ. The multifaceted potential of glucagon-like peptide-1 as a therapeutic agent. Minerva Endocrinol, 2002, 27:79-93.
[96]. Hui HX, Wright C and Perfetti R. Glucagon-Like Peptide 1 Induces Differentiation of Islet Duodenal Homeobox-1 -Positive Pancreatic Ductal Cells Into Insulin-Secreting Cells. Diabetes, 2001, 50:785-796.
[97]. Abraham EJ, Leech CA, Lin JC, et al. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulinproducing cells. Endocrinology, 2002, 143:3152-3161.
[98]. Hardikar AA, Wang XY, Williams L, et al. Functional maturation of fetal porcine beta cells by glucagon-like peptide 1 and cholecystokinin. Endocrinology, 2002, 143: 3505-3514.
[99]. Huotari MA, Palgi J, Otonkoski T, et al. Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel beta-cell mitogen. Endocrinology, 1998, 139:1494-1499.
[100]. Li L, Seno M, Yamada H, et al. Promotion of beta-cell regeneration by betacellulin in ninety percent-pancreatectomized rats. Endocrinology, 2001, 142: 5379-5385.
[101]. Sugiyama K, Yonemura Y, Okamoto H, Effects of poly (ADP-ribose) synthetase inhibitor on B-cells of a canine pancreas after massive pancreatectomy. Int J Pancreatol, 1991,8:85-95.
[102]. Otonkoski T, Beattie GM, Mally MI, et al. Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. J Clin Invest, 1993,92:1459-1466.
[103]. Cho YM, Lim JM, Yoo DH, et al. Betacellulin and nicotinamide sustain PDX1 expression and induce pancreatic β-cell differentiation in human embryonic stem cells. Biochemical and Biophysical Research Communications, 2008, 366:129-134.
[1].Tyden G,Bolinder J,Solders G,et al.Improved survival in patients with insulin-dependedt diabetes mellitus and end-stage diabetic nephropathy 10years after combined pancreas and kidney transplantation.Transplantation,1999,67:645-648.
[2].Shapiro AM,Lakey JR,Ryan EA,et al.Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.N Engl J Med,2000,343:230-238.
[3].Ryan EA,Lakey JRT,Rajotte RV,et al.Clinical outcomes and insulin secretion after islet transplantation with the Endmonton protocol.Diabetes,2001,50:710-719.
[4].Ryan EA,Lakey J R,Paty BW,et al.Successful islet transplantation:continued insulin reserve provides long-term glycemic control.Diabetes,2002,51(7):2148-2157.
[5].Ryan EA,Paty BW,Senior PA,et al.Five-year follow-up after clinical islet transplantation.Diabetes,2005,54(7):2060-2069.
[6].Matsumoto S and Tanaka K.Pancreatic islet cell transplantation using non-heart-beating donors(NHBDs).J Hepa Pancreat Surg,2005,12:227-230.
[7].Voros P,Farkas G,Lengyel Z,et al.Albuminuria after fetal pancreatic islet transplantation:a 10-year follow-up.Nephrol Dial Transplant,1998,13(11):2899-2904.
[8].王海慧,陈兵,张平,等.人胎胰岛细胞的分离培养及其意义.重庆医学, 2006, 35(9): 783-784.
[9]. Yoon KH, Quikel RR, Tatarkieuicz K, et al. Differentiation and expansion of beta cell mass in porcine neonatal pancreatic cell clusters transplanted into nude mice. Cell Transplant, 1999, 8:673-677.
[10].Van Der Laan LJ, Lockey C, Griffeth BC, et al. Infection by procine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature, 2000,407:29-31.
[11]. Ramiya VK, Maraist M, Arfors K E, et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med, 2000, 6(3):278-282.
[12]. Bonner-Weir S, Taneja M, Weir G C, et al. In vitro cultivation of human islets from expanded ductal tissue. PNAS, 2000, 97 (14): 7999-8004.
[13]. Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-postive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, excrine, and hepaticphenotypes. Diabetes, 2001, 50:521-533.
[14]. Kim SY, Lee SH, Kim BM, et al. Activation of nestin-positive duct stem (NPDS) cells in pancreas upon neogenic motivation and possible cytodifferentiation into insulin-secreting cells from NPDS cells. Dev Dyn, 2004, 230(1): 1-11.
[15]. Huang H, Tang X. Phenotypic determination and characterizationof nestin-positive precursors derived from human fetal pancreas. Lab Invest, 2003, 83 (4):539-547.
[16]. Yang L J, Li S W, Hatch H, et al. In vitro transdifferentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. PNAS, 2002, 99(12):8078-8083.
[17]. Zalzman M, Gupta S, Giri R K, et al. Reversal of hyperglycemia in mice by using human expandable insulin-producing cell sdifferentiated from fetal liver progenitor cells. PNAS, 2003, 100 (12):7253-7258.
[18]. Tamar S, Keren S, Irit M L, et al. Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells. PNAS, 2005, 102(22):7964-7969.
[19]. Imai J, Katagiri H, Yamada T, Ishigaki Y, Ogihara T, Uno K, et al. Constitutively active PDX1 induced efficient insulin production in adult murine liver. Biochem Biophys Res Commun, 2005, 326:402-409.
[20]. Oh SH, Muzzonigro TM, Bae S H, et al. Adult bone marrow-derived cells trans- differentiating into insulin-producing cells for the treatment of type 1 diabetes. Lab Invest, 2004, 84: 607-617.
[21]. Tang DQ, Cao LZ, Burkhardt BR, et al. In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes, 2004, 53(7): 1721-1732.
[22]. Jahr H, Bretzel RG. Insulin-positive cells in vitro generated from rat bone marrow stromal cells. Transplant Proc, 2003, 35: 2140-2141.
[23].贾延劼,钟乐,宋建辉,等。体外诱导大鼠骨髓间充质干细胞分化为胰岛素分泌细胞.中国当代儿科杂志,2003,5(5):393-397。
[24].李艳华,白慈贤,谢超,等。成人骨髓间充质干细胞体外定向诱导分化为胰岛样细胞团的研究.自然科学进展,2003,13(6):593-597.
[25].陆琰,张洹,迟作华.体外诱导人骨髓间充质干细胞向胰岛β样细胞分化的研究.暨南大学学报(医学版),2005,26(6):729-733.
[26].Burns CJ,Minger SL,Hall S,et al.The in vitro differentiation of rat neural stem cells into an insulin-expressing phenotype.Biochem Biophys Res Commun,2005,326(3):570-577.
[27].Hori Y,Gu X,Xie X,et al.Differentiation of insulin-producing cells from human neural progenitor cells.PLOS Medicine,2005,2(4):347-356.
[28].Kodama S,Kuhtreiher W,Fujimura S,et al.Islet regeneration during the reversal of autoimmune diabetes in NOD mice.Science,2003,302:1223-1226.
[29].Kojima H,Nakamura T,Fujita Y,et al.Combined expression of pancreatic duodenal homeobox-1 and islet factor 1 induces immature entereeytes to produce insulin.Diabetes,2002,51(5):1398-1408.
[30].Ende N,Chen R F,Reddi A S.Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice.Biochem Biophys Res Commun,2004,325(3):665-669.
[31].Ende N,Chen R F and Reddi A S.Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice.Biochem Biophys Res Commun,2004,321(1):168-171.
[32].Soria B,Roche E,Berna G,et al.Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000,49:157-162.
[33].Suheir A,Gila M,Michal At,et al.Insulin Production by Human Embryonic Stem Cells.Diabetes,2001,50:1691-1697.
[34].Silva DG,Petrovsky N,Socha L,et al.Mechanisms of accelerated immune-mediated diabetes resulting from islet beta cell expression of a Fas ligand transgene.J Immunol,2003,170:4996-5002.
[35].Ichim TE,Zhong R,MinWP.Prevention of allograft rejection by in vitro generated tolerogenic dendritic cells.Transplant Immunology,2003,11:295-306.
[1].Shapiro AMJ,Lakey JRT,and Ryan EA.Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immuno-suppressive regimen.N Engl J Med,2000,343:230-238.
[2].Soria B,Skoudy A,and Martin F.From stem cells to beta cells:new strategies in cell therapy of diabetes mellitus.Diabetologia,2001,44:407-415.
[3].Soria B,Roche E,Berna G,et al.Insulin secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes,2000,49:157-162.
[4].Suheir A,Gila M,Michal A,et al.Insulin Production by Human Embryonic Stem Cells.Diabetes,2001,50:1691-1697.
[5].Blyszczuk P,Czyz J,Kania G,et al.Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulinproducing cells.PNAS,2003,100:998-1003.
[6].Satsuki M,Eiji Y,and Jun-ichi M Regulated Expression of pdx-1 Promotes In Vitro Differentiation of Insulin- producing Cells From Embryonic Stem Cells.Diabetes,2004,53:1030-1037.
[7].Akira S,Shigehiko U,Yukiteru O,et al.Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer.World J Gastroenterol,2005,11(27):4161-4166.
[8].Vaca P,Berna' G,Marti'n F,and Sofia B.Nicotinamide Induces Both Proliferation and Differentiation of Embryonic Stem Cells Into Insulin-Producing Cells.Transplantation Proceedings,2003,35:2021-2023.
[9].张妍,郭希民,王常勇,等.硒促进小鼠胚胎干细胞分化为胰岛细胞的实验研究.军事医学科学院院刊,2004,28(2):126-129.
[11].Brole'n KC,Nico H,Josefina E,et al.Signals From the Embryonic Mouse Pancreas Induce Differentiation of Human Embryonic Stem Cells Into Insulin-Producing β-Cell-Like Cells.Diabetes,2005,54:2867-2874.
[12].Vaca P,Martin F,Vegara-Meseguer JM,et al.Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors.Stem Cells,2006,24(2):258-265.
[13].Moritoh Y,Yamato E,Yasui Y,et al.Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells.Diabetes,2003,52:1163-1168.
[14].Leon-Quinto T,Jones J,Skoudy A,et al.In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells.Diabetologia,2004,47:1442-1451.
[15].Lumelsky N,Blondel O,Laeng P,et al.Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets.Science,2001:292:1389-1394.
[16].周光纪,屈纪富,徐海伟,等.无血清培养法诱导小鼠胚胎干细胞分化为胰岛素分泌细胞的实验条件优化选择.中国临床康复,2005,9(34):23-26.
[17].Linda B.L,Hung-Chih K,Laura A,et al.Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes.Reproductive Biology and Endocrinology,2004,2:42-46.
[18].Masahide Y,Hiroshi Y,Hiroshi F,et al.Identification of Insulin-Producing Cells Derived from Embryonic Stem Cells by Zinc-Chelating Dithizone.Stem Cells,2002,20:284-292.
[19].Rajagopal J,Anderson,WJ,Kume S,et al.Insulin staining of ES cell progeny from insulin uptake.Science,2003,299:363.
[20].Hansson M,Tonning,A,Frandsen,U.et al.Artifactual insulin release from differentiated embryonic stem cells.Diabetes,2004,53:2603-2609.
[21].Enrique R,Pilar S,Juan AR,et al.Ectodermal commitment of insulinproducing cells derived from mouse embryonic stem cells.FASEB,2005,19(10):1341-1343.
[22].Xu X,Kahan B,Forgianni A,et al.Endoderm and pancreatic islet lineage differentiation from human embryonic stem cells.Cloning Stem Cells,2006,8(2):96-107.