Differentiation of insulin-producing cells from human umbilical cord mesenchymal stem cells infected by MAFA-PDX1 overexpressed lentivirus / 中国组织工程研究

BACKGROUND:Transplantation of stem cell-derived islet β cells has been considered effective for the treatment of type 1 diabetes.Human umbilical cord mesenchymal stem cell is an ideal cellular source,but with a low differentiation efficiency to islet β cells. OBJECTIVE:To explore the possibility of human umbilical cord mesenchymal stem cells modified by MAFA and PDX1 to differentiate into insulin-producing cells. METHODS:MAFA-PDX1 lentivirus expression vectors were constructed.The efficiency and potentiality of human umbilical cord mesenchymal stem cells differentiated into insulin-producing cells with three methods were compared by cell morphology,RT-qPCR,and dithizone staining[protocol A:Simple lentivirus group;protocol B:Drug(nicotinamide β-mercaptoethanol)induction followed by lentivirus group;protocol C:lentivirus and drug induction group]. RESULTS AND CONCLUSION:(1)Morphological change of cells:Cell morphology was all altered after the induction of three protocols.At day 11,human umbilical cord mesenchymal stem cells induced by protocol B showed the most cell clusters among the three protocols,appearing aggregated islet-like cell clusters.(2)Islet-related gene expression detected by RT-qPCR:Horizontal comparison of the three protocols at the same induction time point showed that the expression levels of MAFA and PDX1 genes were the highest in protocol C on day 5 of induction,and those in protocol B were the highest on day 11 of induction.Human umbilical cord mesenchymal stem cells induced by protocol B had the greatest expression of GCG gene at day 5,INS and GLUT2 genes at day 11.(3)Dithizone staining to identify zinc ions:parts of the post-induced cells were stained brownish red by dithizone on day 11.The partial small island cells were stained brownish red with a darker color(positive expression)in protocol B.(4)It is concluded that the overexpression of MAFA and PDX1 can promote the differentiation of human umbilical cord mesenchymal stem cells into insulin-producing cells.The combination of MAFA-PDX1 gene modification and drug induction is superior to the single gene modification.

Effects of perinatal exposure to benzo[a]pyrene on the expressionof PDX-1 and TFAM in pancreas and mitochondrial DNA copynumber in offspring rats / 预防医学

Objective@#To observe the effects of perinatal exposure to benzo[a]pyrene (B[a]P) on the expression of pancreatic duodenal homeobox-1 (PDX-1) and mitochondrial transcription factor A (TFAM) and mitochondrial DNA copy number in offspring mice, and to explore the role of maternal exposure to B[a]P in the pancreatic function damage of offspring mice.@*Methods@#Forty pregnant rats were randomly divided into the control group, the lowest dose group (2 μg/kg), the low dose group (200 μg/kg), medium dose group (800 μg/kg) and high dose group (1 600 μg/kg), with 8 rats in each group. From day 1 of pregnancy, each exposed group was given 0.2 mL/100 g body weight of B[a]P and corn oil mixture by gavage once a day until 3 weeks after delivery, while the control group was given the same dose of corn oil. The pancreatic tissue of three-week-old mice were collected after abdominal anesthesia for insulin immunohistochemical detection. The protein and mRNA expression levels of PDX-1 and TFAM, as well as mitochondrial DNA copy number were detected. Spearman rank correlation analysis was used to analyze the correlation between B[a]P exposure dose and the above indicators.@*Results@#The insulin-positive area ratio and average optical density of insulin in the medium and the high dose groups were significantly lower than those in the control group (all P<0.05). The insulin-positive area ratio and average optical density of insulin were negatively correlated with the B[a]P dose (rs=-0.862 and -0.858, both P<0.05). The protein expression levels of PDX-1 and TFAM in the high dose group were significantly lower than those in the control group (both P<0.05). The protein expression levels of PDX-1 and TFAM were negatively correlated with the B[a]P dose (rs=-0.756 and -0.799, both P<0.05). The mRNA expression levels of PDX-1 and mitochondrial DNA copy number in the medium and high dose groups were significantly lower than those in the control group, and the mRNA expression level of TFAM in the high dose group was significantly lower than that in the control group (all P<0.05). The mRNA expression levels of PDX-1, TFAM, and mitochondrial DNA copy number were negatively correlated with the B[a]P dose (rs=-0.722, -0.550 and -0.840, all P<0.05).@*Conclusion@#Perinatal exposure to B[a]P can induce the damage of islet β cells in offspring rats, which may be related to the decreased expression of PDX-1 and TFAM and the copy number of mitochondrial DNA.

Research status and prospect of basic application of islet organoid / 器官移植

Organoids are tissue structures, generated from pluripotent stem cells and cultured in vitro, which form self-organize and recapitulate tissues with similar structure and function to the original organs. Organoids have similar appearance and function to the original tissues, and have been widely applied in basic research and clinical trial. At present, the organoids of liver, kidney, islet, brain, intestine and other organs have been successfully cultivated. The use of islet organoid is a hotspot in the field of organoid research. However, islet organoid is currently applied in basic research because rejection after organ transplantation and other issues remain unresolved. In this article, the origin, development and basic application of islet organoid were reviewed, aiming to provide reference for the transformation from basic research of islet organoid into clinical application as well as the treatment of diabetes mellitus.

Effect of Zuoguiwan on Serum Related Indexes and Pancreatic PDX-1 Expression in GDM RatsWANG Chao-qun1，2, WANG Yong-hui2, LIU Tao2, XU Kai-xia2, ZHAO Le2, ZHOU Ran2*（1.HuBei University of Traditional Chinese Medicine（TCM），Wuhan 430065，China；2.Shanxi University of TCM，Taiyuan 030024，China） / 中国实验方剂学杂志

Objective:To investigate the effect of maternal intervention of Zuoguiwan on the improvement of serum and pancreatic index in rats of gestational diabetes mellitus(GDM). Method:The GDM pregnant rats were replicated by monogamous cage and introneal injection of streptozotocin (STZ). The pregnant rats were randomly divided into normal group, model group, insulin group (20 U·kg-1), and low, medium and high-dose Zuoguiwan groups (2.5，5.0，10.0 g·kg-1). The insulin group was given hypodermic injection of portal winter insulin, all drug groups were respectively administrated with corrsponding drugs for 2 weeks, once a day. Meanwhile, control group and model group were given physiological saline by gavage. After that, the levels of serum insulin, fasting plasma glucose（FBG）, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA), protein and gene expression levels of Pancreas-duodenal homologous box factor-1 (PDX-1) were respectively detected by Western blot and real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. Result:Compared with normal group, FBG, TNF-α and IL-6 levels of pregnant rats in model group increased significantly (P<0.01), the level of IL-8 increased uniformly (P<0.05), and serum insulin level, protein and gene expression levels of PDX-1 decreased significantly (P<0.01). Compared with model group, FBG and serum IL-8 levels of pregnant rats in insulin group were significantly lower (P<0.01), while serum insulin level, protein and gene expression levels of PDX-1 increased significantly(P<0.01). FBG, IL-8, IL-6 and TNF-α levels in serum of pregnant rats in medium-dose Zuoguiwan group decreased obviously, meanwhile serum insulin level, protein and gene expression levels of PDX-1 increased significantly (P<0.01). Conclusion:Zuoguiwan may promote the secretion of insulin by reducing the serum inflammatory factors in GDM rats, and at the same time up-regulate the expression of PDX-1 protein and gene, so as to restrain insulin resistance, reduce the damage of pancreatic cells and improve the blood glucose of rats.

Mechanism of lncRNA-PLUTO on promoting insulin biosynthesis and secretion / 中国药科大学学报

@#The aim of this study is to investigate the effect of long non-coding RNA PLUTO on β cell function and its underlying mechanism. Islet cells of db/db, ob/ob and HFD mice were isolated and extracted by enzymatic method to detect the expression of PLUTO in islet cells of different obesity model mice. Glucolipid toxicity was used to stimulate islet primary cells and Min6 cells, and the expression of PLUTO was detected to elucidate the reasons of PLUTO down-regulation induced by obesity factors. RT-qPCR and ELISA were performed to analyze the function of PLUTO on β cells; Western blot and rescue experiments were carried out to elucidate the regulatory mechanism of β cells by PLUTO. The results showed that the expression levels of PLUTO were significantly decreased in obesity model mice compared with control mice; over-expression PLUTO in primary islets and Min6 cells could improve insulin biosynthesis and secretion; Western blot analysis showed that PLUTO promoted insulin secretion and synthesis by up-regulating the transcription and translation of Pdx1, which is the adjacent gene of PLUTO.

Lupin gamma conglutin protein: effect on Slc2a2, Gck and Pdx-1 gene expression and GLUT2 levels in diabetic rats

ABSTRACT Recently, lupin seed (Lupinus albus L., Fabaceae) products have emerged as a functional food due to their nutritional and health benefits. Numerous reports have demonstrated the hypoglycemic effects of lupin's gamma conglutin protein; nonetheless, its mechanism of action remains elusive. To understand the role of this protein on glucose metabolism, we evaluated the effect of administering L. albus' gamma conglutin on Slc2a2, Gck, and Pdx-1 gene expression as well as GLUT2 protein tissue levels in streptozotocin-induced diabetic rats. While consuming their regular diet, animals received a daily gamma conglutin dose (120 mg/kg per body weight) for seven consecutive days. Serum glucose levels were measured at the beginning and at the end of the experimental period. At the end of the trial, we quantified gene expression in pancreatic and hepatic tissues as well as GLUT2 immunopositivity in Langerhans islets. Gamma conglutin administration lowered serum glucose concentration by 17.7%, slightly increased Slc2a2 and Pdx-1 mRNA levels in pancreas, up-regulated Slc2a2 expression in the liver, but it had no effect on hepatic Gck expression. After gamma conglutin administration, GLUT2 immunopositivity in Langerhans islets of diabetic animals resembled that of healthy rats. In conclusion, our results indicate that gamma conglutin up-regulates Slc2a2 gene expression in liver and normalizes GLUT2 protein content in pancreas of streptozotocin-induced rats.

Induction of Nestin Early Expression as a Hallmark for Mesenchymal Stem Cells Expression of PDX-1 as a Pre-disposing Factor for Their Conversion into Insulin Producing Cells

Diabetes constitutes a worldwide epidemic that affects all ethnic groups. Cell therapy is one of the best alternatives of treatment, by providing an effective way to regenerate insulin-producing cells lost during the course of the disease, but many issues remain to be solved. Several groups have been working in the development of a protocol capable of differentiating Mesenchymal Stem Cells (MSCs) into physiologically sound Insulin Producing Cells (IPCs). In order to obtain a simple, fast and direct method, we propose in this manuscript the induction of MSCs to express NESTIN in a short time period (2 h), proceeded by incubation in a low glucose induced medium (24 h) and lastly by incubation in a high glucose medium. Samples from cell cultures incubated in high glucose medium from 12 to 168 h were obtained to detect the expression of INSULIN-1, INSULIN -2, PDX-1 and GLUT-2 genes. Induced cells were exposed to a glucose challenge, in order to assess the production of insulin. This method allowed us to obtain cells expressing PDX-1, which resembles a progenitor insulin-producing cell.

Protective effect of salvianolic acid B on pancreatic islet cells in diabetic rats with fluctuating blood glucose / 中草药

Objective: To observe the protective effect of salvianolic acid B (Sal B) on pancreatic islet cells in diabetic rats with fluctuating blood glucose and the possible mechanisms implicated. Methods: Diabetes model in rats was established by feeding with high-sugar and high-fat diets combined with ip injection of streptozotocin (STZ). Then the rats were subjected to ip injection of insulin and/or ig administration of glucose at indicated time for 6 weeks to induce blood glucose fluctuation, with those in Sal B groups ig supplemented with Sal B 160 or 80 mg/kg. The contents of fasting blood glucose (FBG), fasting serum insulin (FINS), and glycosylated hemoglobin (GHb) and the levels of total anti-oxidant capacity (TAC), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) in both serum and pancreatic tissues were determined with commercially available kits. Pathological changes and cell apoptosis in pancreatic islets were evaluated by HE staining and TUNEL staining, respectively. Protein levels of PDX-1 in pancreatic tissues were examined by Western blotting analysis. Results: Compared with the control group, the contents of FBG, GHb, and MDA in diabetic rats were increased significantly, while the levels of FINS, TAC, and SOD activity were decreased markedly (P < 0.01). Pancreatic islets in diabetic rats became decreased in size and number, while cell apoptosis in islets increased notably (P < 0.01). Protein level of PDX-1 was significantly decreased in pancreas of diabetic rats (P < 0.01). Supplementation with Sal B resulted in a significant decrease in FBG, GHb, and MDA contents and increase in FINS, TAC, and SOD activity in diabetic rats (P < 0.05, 0.01). Sal B significantly attenuated pathological changes and reduced cell apoptosis in pancreatic islets of diabetic rats, with the expression of PDX-1 protein up-regulated evidently (P < 0.05 or 0.01). Conclusion: Sal B can significantly ameliorate pancreatic pathological changes and improve pancreatic islet function in diabetic rats with fluctuating blood glucose, which might be attributed to attenuation of oxidative stress, up-regulation of PDX-1 expression, and suppression of islet cell apoptosis.

Expression of pancreatic and duodenal homeobox1 (PDX1) protein in the interior and exterior regions of the intestine, revealed by development and analysis of Pdx1 knockout mice / 한국실험동물학회지

We developed pancreatic and duodenal homeobox1 (Pdx1) knockout mice to improve a compensatory hyperinsulinemia, which was induced by hyperplasia in the beta cells or Langerhans' islands, as the diabetic model mice. For targeting of Pdx1 gene by homologous recombination, ES cells derived from a 129(+Ter)/SvJclxC57BL/6JJcl hybrid mouse were electroporated and subjected to positive-negative selection with hygromycin B and ganciclovir. As these results, one of the three chimeric mice succeeded to produce the next or F1 generation. Then, the mouse fetuses were extracted from the mother's uterus and analyzed immunohistologically for the existence of a pancreas. The fetuses were analyzed at embryonic day 14.5 (E14.5) because Pdx1 knockout could not alive after birth in this study. Immunohistochemical staining revealed that 10 fetuses out of 26 did not have any PDX1 positive primordium of the pancreas and that the PDX1 expresses in both the interior and exterior regions of intestine. In particular, one the exterior of the intestine PDX1 was expressed in glands that would be expected to form the pancreas. The result of PCR genotyping with extracted DNA from the paraffin sections showed existence of 10 Pdx1-knockout mice and corresponded to results of immunostaining. Thus, we succeeded to establish a Pdx1-knockout (Pdx1-/-) mice.

Adenoviruses Expressing PDX-1, BETA2/NeuroD and MafA Induces the Transdifferentiation of Porcine Neonatal Pancreas Cell Clusters and Adult Pig Pancreatic Cells into Beta-Cells

BACKGROUND: A limitation in the number of insulin-producing pancreatic beta-cells is a special feature of diabetes. The identification of alternative sources for the induction of insulin-producing surrogate beta-cells is a matter of profound importance. PDX-1/VP16, BETA2/NeuroD, and MafA overexpression have been shown to influence the differentiation and proliferation of pancreatic stem cells. However, few studies have been conducted using adult animal pancreatic stem cells. METHODS: Adult pig pancreatic cells were prepared from the non-endocrine fraction of adult pig pancreata. Porcine neonatal pancreas cell clusters (NPCCs) were prepared from neonatal pigs aged 1-2 days. The dispersed pancreatic cells were infected with PDX-1/VP16, BETA2/NeuroD, and MafA adenoviruses. After infection, these cells were transplanted under the kidney capsules of normoglycemic nude mice. RESULTS: The adenovirus-mediated overexpression of PDX-1, BETA2/NeuroD and MafA induced insulin gene expression in NPCCs, but not in adult pig pancreatic cells. Immunocytochemistry revealed that the number of insulin-positive cells in NPCCs and adult pig pancreatic cells was approximately 2.6- and 1.1-fold greater than those in the green fluorescent protein control group, respectively. At four weeks after transplantation, the relative volume of insulin-positive cells in the grafts increased in the NPCCs, but not in the adult porcine pancreatic cells. CONCLUSION: These data indicate that PDX-1, BETA2/NeuroD, and MafA facilitate the beta-cell differentiation of NPCCs, but not adult pig pancreatic cells. Therefore PDX-1, BETA2/NeuroD, and MafA-induced NPCCs can be considered good sources for the induction of pancreatic beta-cells, and may also have some utility in the treatment of diabetes.

Decreased Expression and Induced Nucleocytoplasmic Translocation of Pancreatic and Duodenal Homeobox 1 in INS-1 Cells Exposed to High Glucose and Palmitate

BACKGROUND: Type 2 diabetes mellitus (T2DM) is often accompanied by increased levels of circulating fatty acid. Elevations in fatty acids and glucose for prolonged periods of time have been suggested to cause progressive dysfunction or apoptosis of pancreatic beta cells in T2DM. However, the precise mechanism of this adverse effect is not well understood. METHODS: INS-1 rat-derived insulin-secreting cells were exposed to 30 mM glucose and 0.25 mM palmitate for 48 hours. RESULTS: The production of reactive oxygen species increased significantly. Pancreatic and duodenal homeobox 1 (Pdx1) expression was down-regulated, as assessed by reverse transcription-polymerase chain reaction and Western blot analyses. The promoter activities of insulin and Pdx1 were also diminished. Of note, there was nucleocytoplasmic translocation of Pdx1, which was partially prevented by treatment with an antioxidant, N-acetyl-L-cysteine. CONCLUSION: Our data suggest that prolonged exposure of beta cells to elevated levels of glucose and palmitate negatively affects Pdx1 expression via oxidative stress.

Expression of PDX1 in human umblical cord mesenchymal stem cells mediated by adenovirus vector / 中华内分泌外科杂志

ObjectiveTo construct recombinant adenovirus vector containing human pancreatic and duodenal homeobox factor 1 (PDX1) and detect its expression in human umblical cord mesenchymal stem cells (HUCMSCs). MethodsPDX1 obtained by BgⅢ/XhoI enzyme digestion from pUC57-PDX1 was ligated into the recombinant shuttle vector pShuttle-GFP-CMV to obtain the recombinant shuttle plasmid pShuttle-GFP-CMVPDX1. pShuttle-GFP-CMV- PDX1 was shifted to pAdxsi vector to obtain pAdxsi-GFP-PDX1 virus plasmid. The recombinant plasmid was packaged and amplified in 293 cells. The expression of PDX1 gene and protein in HUCMSCs was detected by fluorescence microscopy, RT-PCB, immunofluorescence, immunohistochemistry, and Western Blot. ResultsPDX1 gene was inserted correctly into shuttle plasmid and the recombinant adenovirus vector was successfully constructed according to the results of sequence and enzyme digestion identification. The adenovirus was effectively transfected into HUCMSCs. RT-PCR verified that PDX1 mRNA was positively expressed in HUCMSCs. Expression of PDX1 protein in the nuclear of HUCMSCs was found by immunofluorescence assay, immunohistochemistry and Western Blot. ConclusionThe adenovirus vector containing PDX1 gene is successfully constructed and effectively expressed in HUCMSCs.

Effect of epigenetic modification on Pdx-1 gene transcription in different mouse cells / 中国病理生理杂志

AIM: To investigate the role of epigenetic modification in Pdx-1 gene transcription and expression, and to compare the differences between epigenetic modifications of Pdx-1 gene promoter in various cell types of mice. METHODS: The promoter DNA methylation and histone modification status of Pdx-1 and MLH1 genes in NIT-1 cells, NIH3T3 cells and mouse embryonic stem cells were measured by chromatin immunoprecipitation-real time PCR method. The expression levels of these genes in the three cell lines were measured by real time RT-PCR. The relation between epigenetic modifications and gene expression was analyzed. RESULTS: (1) Compared to mES cells, there was lower DNA methylation and higher H3K4m3 modification levels in the promoter of Pdx-1 gene in NIT-1 cells (P<0.05). DNA methylation, H3 acetylation, H3K4m3 and H3K9m3 modification levels in the promoter of Pdx-1 gene in NIH3T3 cells were distinctly increased (P<0.05). (2) Pdx-1 gene transcription expressed only in NIT-1 cells. The Spearman's rho between Pdx-1 gene expression and DNA methylation (r=-0.802,P<0.01) was observed. The Pearson correlation between Pdx-1 gene expression and H3K4m3 modification (r=0.997,P<0.01) was also found. The Spearman's rho between Pdx-1 gene expression and H3K9m3 modification (r=-0.879,P<0.01) was observed. (3) No correlation between housekeeper MLH1 gene expression and epigenetic modification was found. CONCLUSION: DNA methylation, H3K4m3 and H3K9m3 modification coordinated participate to regulate and control the expression of Pdx-1 gene. It is of great significance to the differentiation of β cells from ES cells.

siRNA Specific to Pdx-1 Disturbed the Formation of the Islet in Early Zebrafish Embryos / 华中科技大学学报（医学）（英德文版）

Pdx-1, an important transcription factor highlighting in the early pancreatic development,islet functions and pancreatic disorders, needs to be more investigated in zebrafish, and siRNA is still seldom applied in zebrafish embryo-related research.Our aim was to explore the role of pdx-1 in pan-creatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse tran-scriptase-PCR (RT-PCR), in situ hybridization and immunofluorescent staining were used in this re-search, and the morphology of the islet in normal zebrafish embryos, and in those treated with the siRNA specific to pdx-1 (siPDX-1) or siGFP was observed and compared. The expression of pdx-1 was detected in the stages of 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, 16-hour by RT-PCT. The in situ hy-bridization and immunofluorescent staining results showed that siPDX-I disturbed the formation of the islet in zebrafish embryos. Pdx-1 played multiple roles in maintaining the phenotype of the islet during embryogenesis in zebrafish.

Effect of Pdx1 Expression after Sox9 Depletion in the Early Pancreatic Development in African Clawed Frog (Xenopus laevis) / 대한해부학회지

The pancreas is a mixed exocrine and endocrine gland involved in the control of many homeostatic functions.During embryogenesis,the pancreas arises from dorsal and ventral evaginations of the foregut,which subse- quently fuse into a single organ.The characterization of early genes expressed in the developing pancreas is critical to understand its specification and differentiation.Pdx1 is one of the earliest markers of pancreatic development and a key molecule in its development.Sox proteins form a large class of transcriptional regulators implicated in the control of a variety of developmental processes.One member of this family,Sox9,is expressed in the developing pancreas, but little is known about the function of Sox9 in the developing pancreas.We further investigated Sox9 function during pancreatic development in Xenopus .Using a hormone-inducible inhibitory mutant of Sox9 ,we found that Pdx1 expres- sion was reduced in the ventral pancreatic buds in Sox9-depleted embryos.We suggest that Sox9 gene expression may be involved in pancreatic development in Xenopus.

Effects of hyperglycemia on the Pdx-1 gene in fetal rat islets in vitro / 重庆医科大学学报

Objective:To investigate the effects of hyperglycemia on the pancreas and duodenal homeobox gene-1 (Pdx-1) and insulin gene in fetal rat islets in vitro.Methods:Fetal rat islets were digested by collagenase and underwent culture at 5.5 mmol/L, 11.1 mmol/L and 33.3 mmol/L glucose concentration respectively.The insulin content of fetal rat islets and insulin releasing test were measured by radioimmunoassay.The expression of Pdx-1 protein and mRNA were evaluated by indirect immunofluorescence cytochemistry staining and reverse transcriptase-polymerase chain reaction (RT-PCR).Results:The results showed that the insulin content and stimulated index of hyperglycemia group 1 ( 11.1 mmol/L) were significantly higher than that of controls ( 5.5 mmol/L) and hyperglycemia group 2 ( 33.3 mmol/L).The majority of Pdx-1 positive cells in control group were confined to the nuclear periphery, but the majority of Pdx-1 positive cells in hyperglycemia groups were translocated to the nucleoplasm, and the expression of Pdx-1 mRNA was also elevated in hyperglycemia groups.However,the expression of insulin mRNA decresed in hyperglycemia group 2.Conclusion:Hyperglycemia may regulate Pdx-1 in fetal rat islets by increasing its protein level and translocation to the nucleus.

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells / 中国病理生理杂志

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5? was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5? was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.

The expression of stem cell pdx-1 in different purity pancreatic islets before and after transdifferentiated culture / 重庆医科大学学报

Objective:To obtain the stem cells in different pancreatic islets purity utilizing free-serum culture medium and investigate the PDX-1 mRNA expression distinction before and after transdifferentiation culture.Methods:Pancreatic islets of male SD rats(islet donors)were isolated by injection of collagenase into pancreatic ducts,and maintained in serum Ham's F-10 for 36h.Then the cells were kept in free-serum Ham's F-10 for 14 days.The last stage was to shift culture media from free-serum Ham's F-10 to free-serum DMEM/F12 that contained KGF,BSA,glucose and ITS.On the 28d,Dithizon was added into the culture flask for observation of 3D cell.The PDX-1 positive cells was investigated by immunocytochemistry.Results:After transdifferentiation culture,some 3D cells were red.The pdx-1 was remarkable in cell nucleus.Conclusion:After transdifferentiation culture,some pancreatic stem cell display the character of islet cell.The daughter cells of pancreatic stem cells have character of stem cells after transdifferentiation.KGF may have effect on promoting the expression of PDX-1.