ABSTRACT
Limitation in the number of insulin-producing pancreatic b-cells is a typical feature of diabetes. It has been indicated thatactivating pancreatic transcription factors can promote the transformation of hepatocytes into insulin-secreting b-like cells,indicating that direct hepatocyte differentiation seems promising as a treatment for diabetes. Nevertheless, the reprogramming efficiency still remains low. Our previous study found that the expression of c-fos-induced growth factor (FIGF)was increased in the pancreatic tissues in partial pancreatectomy mice compared to that in normal mice. Here, we observedthat treatment with Ad-FIGF was found to enhance MafA and Ngn3-induced reprogramming of BNL CL.2 cells to b-likecells with the ability of secreting insulin. And FIGF overexpression increased the levels of histone H3/H4 acetylation atMafA and Ngn3 promoter regions in BNL CL.2 cells. Importantly, in vivo study further confirmed that forced expression ofFIGF facilitated the insulin expression and decreased the blood glucose levels in STZ mice. These results strengthen thepossibility of developing cell-based therapies for diabetes through utilizing b-like cells derived from non-insulin-secretingcells.
ABSTRACT
Objective To clarify the role of MafA gene in development of MODY (maturity onset diabetes of the young) by studying insulin production,gene expression,and serum glucose level in heterozygous MafA gene knockout mice.Methods C57BL/6J mice were used as control animals,MafA gene heterozygous mice were analyzed.The distribution curve of blood sugar levels over time and serum insulin of heterozygous mice were determined by using IPGTT.The sensitivity of the mice to insulin was examined by injecting insulin assay.The expression levels of genes of MafA,insulin,pdX1,Beta2,and other genes of heterozygous mice were analyzed by semi-quantitative RT-PCR.Morphological changes in pancreatic tissue and α-and β-cell counts were obtained by using immunofluorescence/histological examination.Results (1) Two weeks after birth,MafA gene heterozygous mice began to show that the blood glucose level was increased,weight was reduced,and the amount of insulin secretion was clearly decreased (P＜0.05 or P＜0.01) while the insulin sensitivity did not change significantly.(2)The islet volume in MafA gene heterozygous mice was increased significantly as compared with the control group.However there were no significant changes in the number of pancreatic cells,distribution patterns,and the ratio of α and β cell.(3) Semi-quantitative RT-PCR detection showed that,compared with the control group,MafA gene level,the amount of insulin and Beta2 gene in MafA gene heterozygous mice were significantly reduced(all P＜0.05),while no changes in the amount of glucagons and level of Pdx1 were found.Conclusions The blood glucose level of MafA gene heterozygous mice was raised early after birth.MafA gene is likely to be a new disease ralated gene of MODY.
ABSTRACT
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.
Subject(s)
Adult , Aged , Animals , Humans , Adenoviridae , Capsules , Gene Expression , Immunohistochemistry , Insulin , Kidney , Pancreas , Stem Cells , Swine , TransplantsABSTRACT
Objective To study the effect of the MafA therapy for blood slucose control in diabetic rats.Methods Rats were divided into 3 groups:treatment group,DM group and control group.In DM group,Wistar rats were rendered diabetic by intrvenous injection of streptozotocin(STZ).In treatment group,mixture of MafA and liposomes at the volume ratio of 1:1 was injected to the portal vein of the diabetic rats.Blood glucose change in the diabetic rats Was measured.Results ① For treatment group,blood slucose decreased from 20.6 mmol/L-22.8 mmol／L to 13.6 mmol／L-14.8 mmol／L and plasma insulin level was significantly elevated for a duration about 2 weeks.②After treatment,blood slucose level significantly decreased compared to DM group (P<0.05).③ Plasma insulin level Was significantly higher than that in DM group(P<0.05).④ Expression of MafA mRNA can be detected in the liver oftreatment group while it Was not found in DM group.Immunohistochemical analysis revealed that insulin expression Was found in the liver of treatment group while it Was not found in the liver of DM group.Conclusion Injection of MafA and liposomes at the volume ratio of 1:1 via the portal vein can effectively decrease blood glucose in diabetic rats.
ABSTRACT
Objective: To construct a recombinant retroviral vector harboring MafA and to establish a liver epithelial progenitor cell (LEPC) line for stable expression of MafA. Methods: MafA was amplified by PCR and suncloned into pBMN-Z-IRES-Neo vector to obtain pBMH-MafA-Neo vector. After introducing the pBMN-MafA-Neo into Phoenix package cells, the cell culture supernatant was used to infect LEPCs. LEPCs stably expressing MafA gene were screened out. RT-PCR was used to detect the influence of MafA on the phenotype of LEPCs. Results: We successfully constructed pBMH-MafA-Neo vector and obtained LEPCs which stably expressed MafA. Expression of GK and GLUT2 genes in LEPCs-MafA cells was higher than that in the LEPCs. Conclusion: We have successfully obtained LEPCs which can stably express MafA,laying a basis for studying the differentiation of LEPCs into pancreas cells.
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Objective:To construct eukaryotic expression vectors pEGFP-N2/MafA and pEGFP-C1/MafA,encoding the mouse musculus v-maf musculoaponeurotic fibrosarcoma oncogene family,protein A(MafA)gene,for the further research of its expression in HepG-2 cells.Methods:pRNA of the mouse MafA gene was distilled by RT-PCR from the mouse,and inserted into Hind Ⅲand SalⅠrestriction sites by PCR,then cloned into pEGFP-N2 and pEGFP-C1 vectors to obtain plasmids pEGFP-N2/MafA and pEGFP-C1/MafA.Human liver cancer cells(HepG-2) were transfected with formed plasmids by means of lipidosome.The MafA-EGFP fused protein was viewed directly with fluorensce microscope,and expression of MafA and insulin Ⅱ was detected by RT-PCR.Results:The mouse MafA gene was amplified through RT-PCR and successfully cloned into transfer vectors.The favorite gene sequence could be expressed and was completely consistent with that reported in genebank,but the insulin Ⅱ gene expression was not detected.Conclusion:The recombinant plasmids pEGFP-N2/MafA and pEGFP-C1/MafA were successfully constructed.But the insulin Ⅱgene was not expressed.
ABSTRACT
Objective:To construct a recombinant retroviral vector harboring MafA and to establish a liver epithelial progenitor cell(LEPC)line for stable expression of MafA.Methods:MafA was amplified by PCR and suncloned into pBMN-Z- IRES-Neo vector to obtain pBMH-MafA-Neo vector.After introducing the pBMN-MafA-Neo into Phoenix package cells,the cell culture supernatant was used to infect LEPCs.LEPCs stably expressing MafA gene were screened out.RT-PCR was used to detect the influence of MafA on the phenotype of LEPCs.Results:We successfully constructed pBMH-MafA-Neo vector and obtained LEPCs which stably expressed MafA.Expression of GK and GLUT2 genes in LEPCs-MafA cells was higher than that in the LEPCs.Conclusion:We have successfully obtained LEPCs which can stably express MafA,laying a basis for studying the differentiation of LEPCs into pancreas cells.