胰腺-十二指肠同源盒1基因(pancreatic and duodenal homeobox 1,PDX1)是决定胰腺分化和发育,调控胰岛素的关键基因。PDX1的缺失及下调可诱发糖尿病的发生和发展,而对其上调则能够调节胰腺发育、促β细胞分化和胰岛素分泌,发挥治疗糖尿...胰腺-十二指肠同源盒1基因(pancreatic and duodenal homeobox 1,PDX1)是决定胰腺分化和发育,调控胰岛素的关键基因。PDX1的缺失及下调可诱发糖尿病的发生和发展,而对其上调则能够调节胰腺发育、促β细胞分化和胰岛素分泌,发挥治疗糖尿病作用。PDX1已然成为糖尿病治疗药物开发的新靶点。越来越多的天然产物被发现具有上调PDX1的作用且表现出抗糖尿病活性。然而,目前尚未有相关文献进行总结报道。本文通过检索CNKI、Pubmed等数据库,对调控PDX1的天然产物抗糖尿病作用的相关研究进行归纳和总结,以期为靶向调控PDX1的新型抗糖尿病药物开发提供思路和参考。展开更多
AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem c...AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.展开更多
AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,prog...AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,progenitor cells within the pancreatic epithelium cannot be characterized in vitro,thoughβ-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition(EMT)may generateβ-cell progenitors.Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth(dexamethasone,epidermal growth factor,3,5,3’-triiodo-l-thyronine,bovine brain extract).Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation.Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter.In combination with lentiviral tracing,cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry(BD fluorescence activated cell sorting),immunostaining and real-time polymerase chain reaction(PCR)(7900HT Fast Realtime PCR System).RESULTS:In the presence of 10%serum MSCs rapidly expand in vitro while the epithelial cell population declines.The percentage of vimentin+cells increased from 22%±5.83%to 80.43%±3.24%(14 d)and99.00%±0.0%(21 d),and the percentage of epithelial cells decreased from 74.71%±8.34%to 26.57%±9.75%(14 d)and 4.00%±1.53%(21 d),P<0.01 for all time points.Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT.Cells expanded in our epithelial medium contained significantly less mesenchymal cells(vimentin+)compared to controls(44.87%±4.93%vs 95.67%±1.36%;P<0.01).During cell differentiation lentiviral reporting demonstrated that,PDX1+and insulin+cells were localized within adherent epithelial cell aggregates compared to controls.Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1,insulin,PAX4 and RFX(P<0.05).CONCLUSION:PDX1+cells were confined to adherent epithelial cell aggregates and not vimentin+cells(mesenchymal),suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.展开更多
The hepatopancreatic duct (HPD) system links the liver and pancreas to the intestinal tube and is composed of the extrahepatic biliary duct, gallbladder, and pancreatic duct. Haematopoietically expressed-homeobox (Hhe...The hepatopancreatic duct (HPD) system links the liver and pancreas to the intestinal tube and is composed of the extrahepatic biliary duct, gallbladder, and pancreatic duct. Haematopoietically expressed-homeobox (Hhex) protein plays an essential role in the establishment of HPD;however, the molecular mechanism remains elusive. Here, we show that zebrafish hhex-null mutants fail to develop the HPD system characterized by lacking the biliary marker Annexin A4 and the HPD marker sox9b. The hepatobiliary duct part of the mutant HPD system is replaced by an intrahepatic intestinal tube characterized by expressing the intestinal marker fatty acid-binding protein 2a (fabp2a). Cell lineage analysis showed that this intrahepatic intestinal tube is not originated from hepatocytes or cholangiocytes. Further analysis revealed that cdx1b and pdx1 are expressed ectopically in the intrahepatic intestinal tube and knockdown of cdx1b and pdx1 could restore the expression of sox9b in the mutant. Chromatin-immunoprecipitation analysis showed that Hhex binds to the promoters of pdx1 and cdx1b genes to repress their expression. We therefore propose that Hhex, Cdx1b, Pdx1, and Sox9b form a genetic network governing the patterning and morphogenesis of the HPD and digestive tract systems in zebrafish.展开更多
文摘胰腺-十二指肠同源盒1基因(pancreatic and duodenal homeobox 1,PDX1)是决定胰腺分化和发育,调控胰岛素的关键基因。PDX1的缺失及下调可诱发糖尿病的发生和发展,而对其上调则能够调节胰腺发育、促β细胞分化和胰岛素分泌,发挥治疗糖尿病作用。PDX1已然成为糖尿病治疗药物开发的新靶点。越来越多的天然产物被发现具有上调PDX1的作用且表现出抗糖尿病活性。然而,目前尚未有相关文献进行总结报道。本文通过检索CNKI、Pubmed等数据库,对调控PDX1的天然产物抗糖尿病作用的相关研究进行归纳和总结,以期为靶向调控PDX1的新型抗糖尿病药物开发提供思路和参考。
基金Supported by National Institute of Genetic Engineering and Biotechnology,Ministry of Science Research and Technology,Tehran,Iran
文摘AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.
基金Supported by Canadian Institutes of Health Research,No.MOP8030the Alberta Diabetes Institute
文摘AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,progenitor cells within the pancreatic epithelium cannot be characterized in vitro,thoughβ-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition(EMT)may generateβ-cell progenitors.Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth(dexamethasone,epidermal growth factor,3,5,3’-triiodo-l-thyronine,bovine brain extract).Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation.Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter.In combination with lentiviral tracing,cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry(BD fluorescence activated cell sorting),immunostaining and real-time polymerase chain reaction(PCR)(7900HT Fast Realtime PCR System).RESULTS:In the presence of 10%serum MSCs rapidly expand in vitro while the epithelial cell population declines.The percentage of vimentin+cells increased from 22%±5.83%to 80.43%±3.24%(14 d)and99.00%±0.0%(21 d),and the percentage of epithelial cells decreased from 74.71%±8.34%to 26.57%±9.75%(14 d)and 4.00%±1.53%(21 d),P<0.01 for all time points.Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT.Cells expanded in our epithelial medium contained significantly less mesenchymal cells(vimentin+)compared to controls(44.87%±4.93%vs 95.67%±1.36%;P<0.01).During cell differentiation lentiviral reporting demonstrated that,PDX1+and insulin+cells were localized within adherent epithelial cell aggregates compared to controls.Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1,insulin,PAX4 and RFX(P<0.05).CONCLUSION:PDX1+cells were confined to adherent epithelial cell aggregates and not vimentin+cells(mesenchymal),suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.
基金the Ministry of Science and Technology of the People's Republic of China (2015CB942802 and 2017YFA0504501)the National Natural Science Foundation of China (http://www.nsfc.gov.cn/)(31330050 and 31571495).
文摘The hepatopancreatic duct (HPD) system links the liver and pancreas to the intestinal tube and is composed of the extrahepatic biliary duct, gallbladder, and pancreatic duct. Haematopoietically expressed-homeobox (Hhex) protein plays an essential role in the establishment of HPD;however, the molecular mechanism remains elusive. Here, we show that zebrafish hhex-null mutants fail to develop the HPD system characterized by lacking the biliary marker Annexin A4 and the HPD marker sox9b. The hepatobiliary duct part of the mutant HPD system is replaced by an intrahepatic intestinal tube characterized by expressing the intestinal marker fatty acid-binding protein 2a (fabp2a). Cell lineage analysis showed that this intrahepatic intestinal tube is not originated from hepatocytes or cholangiocytes. Further analysis revealed that cdx1b and pdx1 are expressed ectopically in the intrahepatic intestinal tube and knockdown of cdx1b and pdx1 could restore the expression of sox9b in the mutant. Chromatin-immunoprecipitation analysis showed that Hhex binds to the promoters of pdx1 and cdx1b genes to repress their expression. We therefore propose that Hhex, Cdx1b, Pdx1, and Sox9b form a genetic network governing the patterning and morphogenesis of the HPD and digestive tract systems in zebrafish.
