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 pancreatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse transcriptase-PCR (RT-PCR), in situ hybridization and immunofluorescent staining were used in this research, 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 hybridization and immunofluorescent staining results showed that siPDX-1 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.
Embryo, Nonmammalian ; Homeodomain Proteins/genetics ; Homeodomain Proteins/*metabolism ; Islets of Langerhans/cytology ; Islets of Langerhans/*embryology ; Islets of Langerhans/metabolism ; RNA Interference ; RNA, Small Interfering/*genetics ; Trans-Activators/genetics ; Trans-Activators/*metabolism ; Zebrafish

The regional distribution and relative frequency of insulin-, glucagon-, somatostatin- and pancreatic polypeptide (PP)-producing endocrine cells in the pancreas of BALB/c mouse were investigated by immunohistochemical method. The pancreas of mice was divided into two portions; pancreatic islets and exocrine portions, and pancreatic islets were further subdivided into two regions (central and peripheral regions) and the relative frequency and regional distribution of immunoreactive cells against insulin, glucagon, somatostatin and PP antisera were monitored. In the pancreatic islet portions, insulin-immunoreactive cells were located in the central regions and they were randomly dispersed in the whole pancreatic islets in some case of the small islets. Quite different from those of other mammals, glucagon-immunoreactive cells were dispersed throughout central to peripheral regions in case of large islets and in the smaller ones, most of these cells were situated in the peripheral regions. Somatostatin-immunoreactive cells were detected in the peripheral regions with various frequencies. Although some cells were demonstrated in the central regions of pancreatic islets, most of PP-immunoreactive cells were located in the peripheral regions. In the exocrine portions, all four types of immunoreactive cells were demonstrated in the BALB/c mouse. Some peculiar distributional patterns of pancreatic endocrine cells were found in BALB/c mouse, especially in case of glucagon-immunoreactive cells.
Animals ; Female ; Glucagon/*metabolism ; Immunohistochemistry/veterinary ; Insulin/metabolism ; Islets of Langerhans/cytology/*metabolism ; Male ; Mice ; Mice, Inbred BALB C/*metabolism ; Pancreatic Polypeptide/metabolism ; Somatostatin/metabolism

The regional distribution and relative frequency of the pancreatic endocrine cells in the C57BL/6 mouse were studied by immunohistochemical method using four types of specific mammalian antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (PP). The pancreas of mouse could be divided into three portions; pancreatic islets, pancreatic duct and exocrine portions, and pancreatic islets were further subdivided into three regions (central, mantle and peripheral regions) according to their located types of immunoreactive cells and pancreatic duct portions were also subdivided into two regions (epithelial and connective tissue regions). In the pancreatic islet portions, although some cells were also demonstrated in the mantle regions, most of insulin-immunoreactive cells were located in the central regions and they were randomly dispersed in the whole pancreatic islets. Glucagon-immunoreactive cells were detected in the mantle and peripheral regions. Their relative frequencies in the peripheral regions were somewhat numerous than those of the mantle regions. Somatostatin-immunoreactive cells were detected in the mantle and peripheral regions. However, no PP-immunoreactive cells were demonstrated in the pancreatic islets of C57BL/6 mouse. In the pancreatic duct portions, rare glucagon-immunoreactive cells were situated in the epithelial regions. Cell clusters that consisted of glucagon- or somatostatin- immunoreactive cells were found in some case of connective tissue regions of pancreatic ducts. However, insulin- and PP-immunoreactive cells were not detected in the epithelial nor connective tissue regions. In the exocrine portions, all four types of immunoreactive cells except for PP cells were demonstrated in the C57BL/6 mouse. However, no PP-immunoreactive cells were demonstrated. In conclusion, regional distribution of endocrine cells in the pancreas of C57BL/6 mouse was similar to that of mammals, especially other rodents except for topographically different distribution of endocrine cells compared to that of other rodents.
Animals ; Female ; Glucagon/metabolism ; Immunohistochemistry ; Insulin/metabolism ; Islets of Langerhans/cytology/*metabolism ; Male ; Mice ; Mice, Inbred C57BL/*metabolism ; Pancreatic Polypeptide/metabolism ; Somatostatin/metabolism

OBJECTIVE: To explore the effect of high glucose and lipid intervention on islet cell apoptosis through the inhibition of prostate apoptosis response factor-4 (Par-4) expression and the underlying mechanisms. METHODS: The mice islet β cells (NIT-1 cells) were randomly divided into a control group, a Par-4 inhibited group, a glucose and fatty acid intervented group and a glucose and fatty acid intervented+Par-4 inhibited group. Cell apoptosis was detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), and the protein expression levels of Par-4 and glucose regulated protein 78 (GRP78) were detected by Western blot. RESULTS: Compared with the control group [(3.14 ± 1.08)%], the apoptosis rate of islet beta cell [(33.82 ± 3.15)%] in the glucose and fatty acid intervented group was significantly increased accompanied by the dramatically elevated Par-4 and GRP78 expression (both P<0.05). Compared with the glucose and fatty acid intervented group, the apoptosis rate in glucose and fatty acid intervented+Par-4 inhibit group [(18.3 4 ± 2.11)%] was significantly decreased concomitant with the significantly decreased Par-4 and GRP78 expression (both P<0.05). CONCLUSION The glucose and fatty acid-induced apoptosis of mice islet β cells could be improved through the inhibition of Par-4 expression, which might be related to reduction of endoplasmic reticulum stress.
Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Cell Line ; Fatty Acids ; Gene Expression Regulation ; Glucose ; Heat-Shock Proteins ; metabolism ; In Situ Nick-End Labeling ; Islets of Langerhans ; cytology ; Mice

OBJECTIVETo establish an semi-automated effective method for large-scale purification of islet cells from human pancreas.

METHODSHuman pancreas tissue was digested with collagenase P using a semi-automated pancreas-digestion system followed by purification in a HCA-Ficoll continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity was evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release assay in vitro.

RESULTSThe number of the islets collected from each pancreas averaged 38 6201-/+78 219 islet equivalents (IEQ) before purification, and 231 420-/+28 054 IEQ after the purification with discontinuous gradient centrifugation. From each gram of the pancreatic tissue, 3148-/+317 IEQ were obtained with an average purity of (62.81-/+2.68) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 2.53-fold increase of insulin secretion over the basal level (3.3 mmol/l, P<0.001).

CONCLUSIONThe established semi-automated method can be applicable for large-scale purification of fully functional islet cells from human pancreas.

Cell Count ; Cell Separation ; instrumentation ; methods ; Cell Survival ; Glucose ; pharmacology ; Humans ; Insulin ; secretion ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Reproducibility of Results

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Diabetes Mellitus ; therapy ; Immunohistochemistry ; Islets of Langerhans ; cytology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Polymerase Chain Reaction ; Radioimmunoassay ; Rats ; Rats, Sprague-Dawley

DNase I footprinting assay using liver nuclear extracts revealed six protected regions between nucleotide -600 and +110 and hence named Box I-VI. Upstream promoter element (UPE), a DNA element playing crucial role in transcriptional control of the tissue specific expression of pancreatic beta-cell, has been detected within the proximal region of rat GLUT2 promoter. This region is included in Box VI. The protein-DNA interaction in this region (Box VI) was confirmed by mobility shift assay using liver nuclear extracts. Deletion of the region between -585 bp and -146 bp resulted in dramatic changes in promoter activity when they were expressed in liver and beta-cell derived cell line. When -585/-146 construct was expressed in liver, the activity was decreased to 46%, whereas the activity in beta-cell line, HIT-T15 cell, was increased by 84% when compared to -146/+190 construct. These opposing phenomena can be explained by the fact that beta-cell specifically expresses the UPE binding protein. Assuming that there may be Box VI-binding protein playing negative roles both in hepatocyte and beta-cell, and that the protein acts as a negative regulator of GLUT2 gene, the UPE binding protein in the beta-cell may overcome the inhibition by binding to the protein.
Animal ; Binding Sites ; Cell Line ; Comparative Study ; DNA Footprinting ; Deoxyribonuclease I ; Gene Expression Regulation ; Islets of Langerhans/metabolism* ; Islets of Langerhans/cytology ; Liver/metabolism* ; Liver/cytology ; Monosaccharide Transport Proteins/genetics ; Monosaccharide Transport Proteins/biosynthesis* ; Promoter Regions (Genetics)* ; Protein Binding ; Rats ; Transcription Factor AP-1

OBJECTIVETo investigate the role of T cell and its subsets in the induction of insulitis and type 1 diabetes mellitus (T1DM) in BALB/c mice.

METHODSAutoimmune diabetes mellitus was developed by intraperitoneal injection of 40 mg/kg streptozotocin (STZ) daily for 5 consecutive days in BALB/c mice as sources of donor cells. Spleen cells from diabetic mice were then cultured for 7 days in the stimulation of interleukin-2 (IL-2) to harvest diabetogenic T cells, which were subsequently transferred into normal BALB/c mice recipients. MTT, ELISA, and HE staining were used to analyze the lymphocyte proliferation, cytokine (IL-2, interferon-gamma, IL-4, and IL-10) levels, and pathological changes in pancreatic islets.

RESULTSAs few as 3 x 10(6) diabetogenic T cells successfully induced diabetes mellitus in recipients pretreated with STZ twice, whereas transfer of equal amount of normal splenocytes, T cell-depleted diabetogenic splenocytes, or diabetogenic CD4+ T cells alone in recipients receiving STZ twice pretreatment was proved not to induce diabetes mellitus either. A markedly increased lymphocyte proliferation, high levels of interferon-gamma and IL-2 in the supernatants of diabetogenic T cells were observed. In addition, a markedly enhanced lymphocyte proliferation, a high level of interferon-gamma secretion in serum, and numerous lymphocytes infiltration in pancreatic islets were detected in the diabetic mice induced by diabetogenic T cells transfer.

CONCLUSIONSA novel T1DM murine model is established in STZ-pretreated BALB/c mice by adoptive transfer of diabetogenic T cells. CD4+ T cells with interferon-gamma may promote the onset of diabetes mellitus.

Animals ; Blood Glucose ; metabolism ; Cytokines ; immunology ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Diabetes Mellitus, Type 1 ; immunology ; pathology ; Disease Models, Animal ; Islets of Langerhans ; cytology ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; cytology ; immunology

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microgram/microliter cDNA and 25 microliter effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.
Animals ; Body Weight ; Cell Survival ; Diabetes Mellitus, Experimental/*complications/metabolism ; Disease Models, Animal ; Glucose/pharmacology ; Glucose Tolerance Test ; Hyperglycemia/complications/*metabolism/*therapy ; Insulin/secretion ; Islets of Langerhans/blood supply/cytology/secretion ; *Islets of Langerhans Transplantation ; Liposomes/*administration & dosage/chemistry ; Male ; Mice ; Mice, Inbred BALB C ; Neovascularization, Physiologic ; RNA, Messenger/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Streptozocin ; Transfection ; Vascular Endothelial Growth Factors/biosynthesis/*genetics/*metabolism/secretion

This study was to explore the induced differentiation of human mesenchymal stem cells (MSCs) modified by pancreatic and duodenal homeobox factor 1 (Pdx1) gene into insulin-producing cells in vitro. After recombined adenovirus vector with Pdx1 gene infected MSCs for 7 d, cells were induced by induction factors. The genes' expressions related to islet beta cells such as Pdx1, insulin, glucose transporter-2 (Glut2), were detected with RT-PCR, immunocytochemistry and Western blot. The levels of insulin and C peptide secretion were examined with chemiluminescence immunoassay. Insulin(+) cell rate was detected by flow cytometry. After infected by recombined adenovirus with Pdx1 and combined with induction factors, MSCs were aggregated and islet-like cell clusters formed. Dithizone staining of these cells was positive. The genes' expression related to islet beta cells, such as Pdx1, insulin, Glut2, could be detected. After induction, the islet-like cell clusters secreted insulin and C peptide. The levels of insulin and C peptide secretion increased with glucose stimulation. Insulin(+) cell rate was (11.61 +/- 4.83)%. It could be concluded that Pdx1 gene modified MSCs from human umbilical cord could be induced to differentiate into islet beta-like cells.
Adenoviridae ; genetics ; metabolism ; Cell Differentiation ; genetics ; Cells, Cultured ; Genetic Vectors ; genetics ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Islets of Langerhans ; cytology ; Mesenchymal Stromal Cells ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Trans-Activators ; biosynthesis ; genetics ; Umbilical Cord ; cytology