Isolation of genes involved in pancreas regeneration by subtractive hybridization

2010 ◽  
Vol 391 (9) ◽  
Author(s):  
Jong-Ho Choi ◽  
Min-Young Lee ◽  
Yoolee Kim ◽  
Jeong-Yun Shim ◽  
Sang-Moon Han ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Embryonic Stem ◽  
Subtractive Hybridization ◽  
Marker Genes ◽  
Mrna Levels ◽  
Genetic Therapy ◽  
Rt Pcr ◽  
Β Cells ◽  
Paracrine Factors ◽  
Pancreas Regeneration

AbstractThe deterioration of β cells in the pancreas is a crucial factor in the progression of diabetes mellitus; therefore, the recovery of β cells is of vital importance for effective diabetic therapeutic strategies. Partially pancreatectomized rats have been used for the investigation of pancreatic regeneration. Because it was determined that tissue extract from the partially-dissected pancreas induces pancreatic differentiation in embryonic stem cells, paracrine factors were thought to be involved in the regeneration. In this study, we screened for genes that had higher mRNA levels 2 days after 60%-pancreatectomy. The genes were isolated using subtractive hybridization and DNA sequencing. Twelve genes (adipsin,Aplp2,Clu,Col1a2,Glul,Krt8,Lgmn, LOC299907, LOC502894,Pla2g1b,Reg3αandXbp1) were identified, and RT-PCR and real-time PCR analyses were performed to validate their expression levels. Among the genes identified, three genes (Glul,LgmnandReg3a) were selected for further analyses. Assays revealed thatGlulandReg3αenhance cell growth.Glul,LgmnandReg3αchange the expression level of islet marker genes, whereNEUROD,NKX2.2,PAX4andPAX6are up-regulated andsomatostatinis down-regulated. Thus, we believe thatGlul,LgmnandReg3acan serve as novel targets in diabetes mellitus genetic therapy.

scholarly journals Analysis of mRNA expression of CNN3, DCN, FBN2, POSTN, SPARC and YWHAQ genes in porcine foetal and adult skeletal muscles

2008 ◽  
Vol 53 (No. 5) ◽  
pp. 181-186 ◽  
Author(s):  
K. Bílek ◽  
A. Knoll ◽  
A. Stratil ◽  
K. Svobodová ◽  
P. Horák ◽  
...  
Keyword(s):  
Skeletal Muscles ◽  
Muscle Growth ◽  
Biceps Femoris ◽  
Subtractive Hybridization ◽  
Postnatal Growth ◽  
Prenatal Development ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Muscle Biology ◽  
Fetal Muscle

Skeletal muscle growth is determined by the number of prenatally formed fibres and by the degree of their postnatal hypertrophy; i.e. prenatal development may influence the postnatal growth. Suppression subtractive hybridization (SSH) was used to identify genes more expressed in fetal hind limb muscles of Piétrain pigs (44 days of gestation) compared to the adult biceps femoris. Six potential functional candidate genes (<i>CNN3</i>, <i>DCN</i>, <i>FBN2</i>, <i>POSTN</i>, <i>SPARC</i> and <i>YWHAQ</i>) were selected to verify the SSH results using real-time RT-PCR. Expression levels of the studied genes were significantly higher (<i>P</i>< 0.05) in the fetal muscle compared to the adult muscle. <i>FBN</i>2 and <i>POSTN</i> exhibited the highest mRNA levels (mean relative ratios were 182.7 and 121.6, respectively). The studied genes may play an important role in muscle biology and may be candidates for muscling traits.

The WNT receptor FZD7 contributes to self-renewal signaling of human embryonic stem cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Kai Melchior ◽  
Jonathan Weiß ◽  
Holm Zaehres ◽  
Yong-mi Kim ◽  
Carolyn Lutzko ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Marker Genes ◽  
Specific Marker ◽  
Mrna Levels ◽  
Es Cell ◽  
Specific Expression ◽  
Self Renewal ◽  
Human Es Cells

Abstract A number of recent studies identified nuclear factors that together have the unique ability to induce pluripotency in differentiated cell types. However, little is known about the factors that are needed to maintain human embryonic stem (ES) cells in an undifferentiated state. In a search for such requirements, we performed a comprehensive meta-analysis of publicly available SAGE and microarray data. The rationale for this analysis was to identify genes that are exclusively expressed in human ES cell lines compared to 30 differentiated tissue types. The WNT receptor FZD7 was found among the genes with an ES cell-specific expression profile in both SAGE and microarray analyses. Subsequent validation by quantitative RT-PCR and flow cytometry confirmed that FZD7 mRNA levels in human ES cells are up to 200-fold higher compared to differentiated cell types. ShRNA-mediated knockdown of FZD7 in human ES cells induced dramatic changes in the morphology of ES cell colonies, perturbation of expression levels of germ layer-specific marker genes, and a rapid loss of expression of the ES cell-specific transcription factor OCT4. These findings identify the WNT receptor FZD7 as a novel ES cell-specific surface antigen with a likely important role in the maintenance of ES cell self-renewal capacity.

scholarly journals miR-410 enhanced hESC-derived pancreatic endoderm transplant to alleviate gestational diabetes mellitus

2015 ◽  
Vol 55 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Yang Mi ◽  
Na Guo ◽  
Tongqiang He ◽  
Jing Ji ◽  
Zhibin Li ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Gestational Diabetes ◽  
Gestational Diabetes Mellitus ◽  
Embryonic Stem ◽  
Late Pregnancy ◽  
Β Cells ◽  
Pregnant Females ◽  
Pancreatic Progenitors ◽  
First Time

Gestational diabetes mellitus (GDM) is a condition commonly encountered during mid to late pregnancy with pathologic manifestations including hyperglycemia, hyperinsulinemia, insulin resistance, and fetal mal-development. The deficit and dysfunction of insulin secreting β-cells are signature symptoms for GDM. Pancreatic progenitors derived from human embryonic stem cells (hESCs) were shown to be able to effectively treat diabetes in mice. In this study, we first identified that microRNA-410 (miR-410) directly targets lactate dehydrogenase A (LDHA), a gene selectively repressed in normal insulin secreting β-cells. hESCs that can be induced to express miR-410 hence keeping LDHA levels in check were then differentiatedin vitrointo pancreatic endoderm, followed by transplantation intodb/+mouse model of GDM. The transplant greatly improved glucose metabolism and reproductive outcome of the pregnant females suffering from GDM. Our findings describe for the first time the method of combining miRNA with hESCs, providing proof of concept by employing genetically modified stem cell therapy for treating GDM.

scholarly journals Localization and regulation of pancreatic selenoprotein P

2012 ◽  
Vol 50 (1) ◽  
pp. 31-42 ◽  
Author(s):  
Holger Steinbrenner ◽  
Anna-Lena Hotze ◽  
Bodo Speckmann ◽  
Antonio Pinto ◽  
Helmut Sies ◽  
...  
Keyword(s):  
Real Time ◽  
High Glucose ◽  
Sodium Selenate ◽  
Selenoprotein P ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Β Cells ◽  
Β Cell ◽  
Gene Reporter Assay ◽  
Insulinoma Cells

Progressive loss of pancreatic β-cell mass is a crucial feature of type 2 diabetes mellitus. As β-cells express very low amounts of the antioxidant enzymes catalase and glutathione peroxidase (GPx), they appear to be particularly vulnerable to oxidative damage in the pathogenesis of diabetes. Here, we investigated the pancreatic expression pattern and regulation of selenoprotein P (Sepp1), which may serve as an additional antioxidant enzyme inside and outside of cells. Sepp1 was detected in rodent pancreas by immunofluorescence and real-time RT-PCR. Regulation of Sepp1 biosynthesis in INS-1 rat insulinoma cells was investigated by real-time RT-PCR, luciferase gene reporter assay, and immunoblotting. Sepp1 and Gpx1 gene expressions in rat pancreas were 58 and 22% respectively of the liver values. Pancreatic Sepp1 expression was restricted to the endocrine tissue, with Sepp1 being present in the α- and β-cells of mouse islets. In INS-1 insulinoma cells, Sepp1 expression was stimulated by the selenium compound sodium selenate and diminished in the presence of high glucose (16.7 vs 5 mM) concentrations. Sepp1 mRNA stability was also lowered at 16.7 mM glucose. Moreover, Sepp1 mRNA levels were decreased in isolated murine islets cultured in high-glucose (22 mM) medium compared with normal glucose (5.5 mM) medium. Pancreatic Sepp1 expression was elevated upon treatment of mice with the β-cell toxin streptozotocin. This study shows that pancreatic islets express relatively high levels of Sepp1 that may fulfill a function in antioxidant protection of β-cells. Downregulation of Sepp1 expression by high glucose might thus contribute to glucotoxicity in β-cells.

scholarly journals A Insight into (β) Cell Development to Rebuild Bonafide β-Cells: Reprogramming Verses Differentiation of Stem Cells

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Misbah Sultana ◽  
Saima Jadoon ◽  
Arif Malik
Keyword(s):  
Diabetes Mellitus ◽  
Stem Cells ◽  
Pathological Condition ◽  
Methodological Approach ◽  
Embryonic Stem ◽  
Β Cells ◽  
Β Cell ◽  
Control Diabetes

Background: Pancreatic β-cells for insulin secretion are the main regulators of mammalian metabolic process. Diabetes and hyperglycemia make the patient dependable on exogenous insulin, which is due to the lack of functional β-cells. Oxidative stress also play an important role in the aggravation of this pathological condition. Recent insights into the development of β-cells along with the pluripotent stem cells discovery have opened new ways to generate β-cells that can help in the screening of various drugs and also in the transplantation therapy since, these pluripotent Embryonic Stem Cells (ESCs) can develop in any type of cell, due to which any defective tissue can be substituted. Objectives: The objective of this study was to evaluate current strategies which can help to control diabetes mellitus. Methodology: Methodological approach of this review was based on the comparison of theoretical studies and researches related to diabetes mellitus. Results: In vitro or in vivo replication might be repeated based on proteins or through small molecules. Efforts have been made through which differentiated cells can be converted into β-cells by transcriptional regulators that are the significant players for the development, as well as responsible to identify conditions that cause replication of β-cell both in vitro and in vivo. The recent strategies can be applied for new β-cells generation and also highlights the future aspects regarding the mechanisms that govern later differentiation stages. Conclusion: This review provides an update on generating β-cells from different strategies and also brief about the development and function of β-cells that how they could help to control diabetes.

Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

2019 ◽  
Vol 19 (2) ◽  
pp. 120-126
Author(s):  
J. Wei ◽  
Y. Yu ◽  
Y. Feng ◽  
J. Zhang ◽  
Q. Jiang ◽  
...  
Keyword(s):  
Negative Correlation ◽  
Hepg2 Cells ◽  
Serum Levels ◽  
Significant Negative Correlation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Apolipoprotein M ◽  
Protein Mass ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

scholarly journals Protective Effect of Psoralea corylifolia L. Seed Extract against Palmitate-Induced Neuronal Apoptosis in PC12 Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Yunkyoung Lee ◽  
Hee-Sook Jun ◽  
Yoon Sin Oh
Keyword(s):  
Pc12 Cells ◽  
Molecular Mechanisms ◽  
Neuronal Cell ◽  
Marker Genes ◽  
Heme Oxygenase 1 ◽  
Mrna Levels ◽  
Protective Effects ◽  
Psoralea Corylifolia ◽  
Antioxidant Genes ◽  
Bax Protein

The extract of Psoralea corylifolia seeds (PCE) has been widely used as a herbal medicine because of its beneficial effect on human health. In this study, we investigated the protective effects and molecular mechanisms of PCE on palmitate- (PA-) induced toxicity in PC12 cells, a neuron-like cell line. PCE significantly increased cell viability in PA-treated PC12 cells and showed antiapoptotic effects, as evidenced by decreased expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, and bax protein as well as increased expression of bcl-2 protein. In addition, PCE treatment reduced PA-induced reactive oxygen species production and upregulated mRNA levels of antioxidant genes such as nuclear factor (erythroid-derived 2)-like 2 and heme oxygenase 1. Moreover, PCE treatment recovered the expression of autophagy marker genes such as beclin-1 and p62, which was decreased by PA treatment. Treatment with isopsoralen, one of the major components of PCE extract, also recovered the expression of autophagy marker genes and reduced PA-induced apoptosis. In conclusion, PCE exerts protective effects against lipotoxicity via its antioxidant function, and this effect is mediated by activation of autophagy. PCE might be a potential pharmacological agent to protect against neuronal cell injury caused by oxidative stress or lipotoxicity.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals Synergistic Effects of Milk-Derived Exosomes and Galactose on α-Synuclein Pathology in Parkinson’s Disease and Type 2 Diabetes Mellitus

2021 ◽  
Vol 22 (3) ◽  
pp. 1059
Author(s):  
Bodo C. Melnik
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Dopaminergic Neurons ◽  
Vagal Nerve ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
The Brain

Epidemiological studies associate milk consumption with an increased risk of Parkinson’s disease (PD) and type 2 diabetes mellitus (T2D). PD is an α-synucleinopathy associated with mitochondrial dysfunction, oxidative stress, deficient lysosomal clearance of α-synuclein (α-syn) and aggregation of misfolded α-syn. In T2D, α-syn promotes co-aggregation with islet amyloid polypeptide in pancreatic β-cells. Prion-like vagal nerve-mediated propagation of exosomal α-syn from the gut to the brain and pancreatic islets apparently link both pathologies. Exosomes are critical transmitters of α-syn from cell to cell especially under conditions of compromised autophagy. This review provides translational evidence that milk exosomes (MEX) disturb α-syn homeostasis. MEX are taken up by intestinal epithelial cells and accumulate in the brain after oral administration to mice. The potential uptake of MEX miRNA-148a and miRNA-21 by enteroendocrine cells in the gut, dopaminergic neurons in substantia nigra and pancreatic β-cells may enhance miRNA-148a/DNMT1-dependent overexpression of α-syn and impair miRNA-148a/PPARGC1A- and miRNA-21/LAMP2A-dependent autophagy driving both diseases. MiRNA-148a- and galactose-induced mitochondrial oxidative stress activate c-Abl-mediated aggregation of α-syn which is exported by exosome release. Via the vagal nerve and/or systemic exosomes, toxic α-syn may spread to dopaminergic neurons and pancreatic β-cells linking the pathogenesis of PD and T2D.

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