RESUMO
BACKGROUND: Neurogenin3 (Ngn3) and neurogenic differentiation 1 (NeuroD1), two crucial transcriptional factors involved in human diabetes (OMIM: 601724) and islet development, have been previously found to directly target to the E-boxes of the insulinoma-associated 2 (Insm2) gene promoter, thereby activating the expression of Insm2 in insulin-secretion cells. However, little is known about the function of Insm2 in pancreatic islets and glucose metabolisms. METHODS: Homozygous Insm2-/- mice were generated by using the CRISPR-Cas9 method. Glucose-stimulated insulin secretion and islet morphology were analyzed by ELISA and immunostainings. Expression levels of Insm2-associated molecules were measured using quantitative RT-PCR and Western blots. RESULTS: Fasting blood glucose levels of Insm2-/- mice were higher than wild-type counterparts. Insm2-/- mice also showed reduction in glucose tolerance and insulin/C-peptide levels when compared to the wild-type mice. RT-PCR and Western blot analysis revealed that expression of Insm1 was significantly increased in Insm2-/- mice, suggesting a compensatory response of the homolog gene Insm1. Similarly, transcriptional levels of Ngn3 and NeuroD1 were also increased in Insm2-/- mice. Moreover, Insm2-/- female mice showed a significantly decreased reproductive capacity. CONCLUSIONS: Our findings suggest that Insm2 is important in glucose-stimulated insulin secretion and is involved in the development pathway of neuroendocrine tissues which are regulated by the transcription factors Ngn3, NeuroD1 and Insm1.
Assuntos
Deleção de Genes , Intolerância à Glucose/genética , Secreção de Insulina , Fatores de Transcrição/genética , Animais , Sequência de Bases , Feminino , Genótipo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos Knockout , Modelos Biológicos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismoRESUMO
The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases. Graphical abstract Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 µm polycarbonate membrane filter to generate fusogenic plasma membrane vesicles (fPMVs), which contain bioactive molecules and organelles but not the nucleus. fPMVs can be endocytosed by target cells, while the cargo is released due to low-pH induced membrane fusion. These nucleus-free fPMVs are efficient at delivery of cytoplasmic proteins and mitochondria, leading to recovery of mitochondrial biogenesis and proliferative ability in mitochondria-deficient cells.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Núcleo Celular , DNA Mitocondrial/genética , Genômica , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , MicroRNAs/genética , Cimento de Policarboxilato/química , RNA Mensageiro/genética , Análise de Sequência de DNA , Vírus da Estomatite Vesicular IndianaRESUMO
OBJECTIVES: We previously found niacin receptor GPR109A was expressed in murine islet beta-cells, and signaling through GPR109A inhibited glucose stimulated insulin secretion (GSIS). However, the expression of GPR109A in human islets and its functional relevance is still not known. METHODS: The expression of GPR109A was examined by antibody staining and in situ hybridization on pancreatic paraffin sections. GPR109A was cloned and expressed in INS-1 islet beta-cells. Intracellular cAMP and GSIS were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of GPR109A was confirmed in murine islet beta-cells and further detected in human counterparts by using commercially available polyclonal antibodies. In situ hybridization study detected the transcripts of GPR109A, but not that of closely related GPR109B. Furthermore, GPR109A was significantly reduced in islets from diabetic individuals and animal model of db/db mice as compared to their respective controls. Further, GPR109A levels in insulinoma were also reduced dramatically as compared to islets found in corresponding non-tumor normal tissues. Quantitative RT-PCR analysis demonstrated that GPR109A transcripts were severely down-regulated in rodent insulinoma cell lines as compared to that of freshly isolated islets from mice. Finally, human and murine GPR109A expression cassettes were transfected into INS-1 cells, which resulted in reduced accumulation of cAMP and insulin secretion after incubation with niacin. The effect could be completely abrogated by pretreatment with pertussis toxin. CONCLUSIONS: These results demonstrate that GPR109A is functionally expressed in both human and murine islet beta-cells. However, the role of GPR109A in the prevention of diabetes or insulinoma needs further study.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Idoso , Animais , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Imunofluorescência , Glucose/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Our previous study showed an efficient targeting of islets of Langerhans by adenoviral injection via the celiac trunk. Unexpectedly, none of the endothelial cells was infected given the direct contact between adenoviruses and the capillary wall. The present study intended to provide an efficient approach for adenoviral targeting of the microcapillary endothelial cells in the pancreas. METHODS: We prepared microspheres of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with a size comparable to the diameter of capillary (5-10 µm). Scanning electron microscopy was applied to verify that adenoviruses carrying a green fluorescence protein gene were complexed with PHBHHx-microspheres after 30 min of co-incubation. The complexes were then injected into the pancreas of mice via the celiac trunk. RESULTS: Approximately 40% of endothelial cells in the pancreas were labeled 5 days after surgery. Islet cells were labeled occasionally, whereas labeling of the acinar and ductal tissues was barely detectable. Endothelium targeting was inefficient in other internal organs. Consistent with the reported superior tissue compatibility of PHBHHx, no discernable microspheres were found in all of the organs examined. Furthermore, splenocyte activation was dampened when adenoviruses were complexed with the microspheres. CONCLUSIONS: The present study has established an approach for efficient pancreatic capillary targeting by using microsphere-adenoviral complexes. This procedure could be invaluable for the treatment of capillary-related diseases.
Assuntos
Adenoviridae/genética , Embolização Terapêutica , Microesferas , Microvasos/patologia , Pâncreas/irrigação sanguínea , Poli-Hidroxialcanoatos/química , Transdução Genética , Adenoviridae/química , Adenoviridae/ultraestrutura , Animais , Doenças Cardiovasculares/terapia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/virologia , Fígado/metabolismo , Fígado/virologia , Camundongos , Camundongos Endogâmicos BALB C , Microvasos/metabolismo , Microvasos/virologia , Pâncreas/metabolismo , Pâncreas/patologia , Pâncreas/virologia , Tamanho da Partícula , Poli-Hidroxialcanoatos/síntese química , Rodaminas/química , Rodaminas/metabolismo , Baço/metabolismo , Baço/virologiaRESUMO
Gene therapy provides a promising approach to curing diabetes. However, an effective route for islet-specific targeting has yet to be established. Toward this end, the pancreatic blood circulation system in Balb/c mice was determined by the injection of rhodamine-containing beads. The efficiency of islet targeting was then measured by the injection of adenoviral vectors carrying a green fluorescence gene via the celiac trunk (C.T.). The results showed that >95% of islets and about 60% of ß cells within the pancreatic body and tail could be labeled 3 days after surgery. α-Cell labeling was not as efficient, whereas labeling of nonendocrine tissues was barely detectable. For proof of principle, adenoviral vectors carrying a Sirtuin transgene were injected similarly to test the islet protection effect in the streptozotocin (STZ)-induced type 1 diabetic model. The results demonstrated that overexpression of Sirtuin in STZ-treated mice reduced the level of ß-cell death and extent of glucose intolerance. This study reports on efficient islet-specific targeting by using adenoviral injection. This procedure could be invaluable to the treatment of diabetes and the study of islet biology.
Assuntos
Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Terapia Genética/métodos , Células Secretoras de Insulina/efeitos dos fármacos , Sirtuína 1/biossíntese , Estreptozocina/farmacologia , Adenoviridae/genética , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Intolerância à Glucose/terapia , Proteínas de Fluorescência Verde/genética , Injeções Intra-Arteriais , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular/métodos , Pâncreas/irrigação sanguínea , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Rodaminas , Sirtuína 1/genética , TransgenesRESUMO
Genetic lineage tracing is indispensable to unraveling the origin, fate, and plasticity of cells. However, the intrinsic leakiness in the CreER-loxP system raises concerns on data interpretation. Here, we reported the generation of a novel dual inducible CreER-loxP system with superior labeling characteristics. This two-component system consists of membrane localized CreER (mCreER: CD8α-FRB-CS-CreER) and TEV protease (mTEVp: CD8α-FKBP-TEVp), which are fusion proteins incorporated with the chemically induced dimerization machinery. Rapamycin and tamoxifen induce sequential dimerization of FKBP and FRB, cleavage of CreER from the membrane, and translocation into the nucleus. The labeling leakiness in Ad293 cells reduced dramatically from more than 70% to less than 5%. This tight labeling feature depends largely on the association of mCreER with HSP90, which conceals the TEV protease cutting site between FRB and CreER and thus preventing uninduced cleavage of the membrane-tethering CreER. Membrane-bound CreER also diminished significantly cytotoxicity. Our studies showed mCreER under the control of the rat insulin promoter increased labeling specificity in MIN6 islet beta-cells. Viability and insulin secretion of MIN6 cells remained intact. Our results demonstrate that this novel system can provide more stringent temporal and spatial control of gene expression and will be useful in cell fate probing.
RESUMO
BACKGROUND: Acetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury. METHODS: PMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated. RESULTS: The hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that the redox state and activities of aminotransferases were restored in APAP-treated HepG2 cells. CONCLUSIONS: The results suggest that PMVs from hUCMSCs could be used as a novel stem cell therapy for the treatment of APAP-induced liver injury.
Assuntos
Acetaminofen , Células-Tronco Mesenquimais , Acetaminofen/toxicidade , Diferenciação Celular , Membrana Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Hep G2 , Humanos , Cordão UmbilicalRESUMO
Enucleated mammalian cells (cytoplasts) have been widely used for studying differential roles of the cytoplasm and nucleus in various cellular processes. Here, we reported an improved enucleation protocol, in which cells were seeded in extracellular matrix (ECM)-coated 24-wells and spun at 4600â¯g and 35⯰C for 60â¯min in the presence of cytochalasin B and colchicine. When glass-bottom wells were used, cellular structures and organelles in cytoplasts could be examined directly by confocal microscopy. Nuclear envelope rupture did not occur probably due to mild centrifugation conditions used in this study. Addition of paclitaxel or doxorubicin completely blocked proliferation of residual nucleated cells; however, to our surprise, paclitaxel dramatically prolonged the survival of cytoplasts. Results from Annexin V and Propidium Iodide staining showed that cytoplasts died predominantly by apoptosis, which was partially inhibited by ECM and further by paclitaxel. Mitochondria were mostly rod-shaped and formed a connected network in paclitaxel-treated cytoplasts, indicating lack of fusion and fission dynamics. Moreover, paclitaxel increased mitochondrial membrane potential, suggesting that perturbation of mitochondria might be critical to the survival of cytoplasts. In conclusion, we had established an efficient and fast procedure for enucleation of adherent animal cells, which could facilitate the investigation of nucleocytoplasmic interaction.
Assuntos
Núcleo Celular/metabolismo , Colchicina/metabolismo , Citoplasma/metabolismo , Matriz Extracelular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/química , Colchicina/química , Citoplasma/química , Matriz Extracelular/química , Humanos , Imagem ÓpticaRESUMO
We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer's disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 µm pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.
Assuntos
Micropartículas Derivadas de Células/transplante , Citoplasma/transplante , Células-Tronco Mesenquimais/citologia , Animais , Fusão Celular , Membrana Celular/ultraestrutura , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos BALB C , Cimento de PolicarboxilatoRESUMO
The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.
Assuntos
DNA/genética , Membrana Eritrocítica/genética , Melhoramento Genético/métodos , Glicoproteínas/genética , Lentivirus/genética , Transfecção/métodos , Células 3T3 , Animais , DNA/administração & dosagem , Células HeLa , Humanos , CamundongosRESUMO
The aim of this study was to determine whether low dose doxycycline as an anti-inflammatory agent could improve glucose metabolism in diabetic animals. Therefore, doxycycline was supplemented in drinking water to 6-week-old male db/db mice for 10 weeks. Doxycycline reduced perirenal/epididymal fat, Lee's index, and liver cholesterol. Blood HDL-cholesterol increased, but total cholesterol and aspartate transaminase decreased. Glucose and insulin tolerances were improved, accompanying with reduced fasting blood glucose, insulin, HOMA-IR and advanced glycation end products. Islet number, ß-cell percentage and mass increased, while islet size decreased. Consistently, less apoptosis but more ß-cell proliferation were found in islets of treated mice. Freshly isolated islets from treated mice showed higher insulin content and enhanced glucose stimulated insulin secretion (GSIS). In addition, purified islets of Balb/c mice showed increased GSIS after cultivation in vitro with doxycycline, but not with chloramphenicol and levofloxacin. Inflammation markers, including lipopolysaccharides (LPS) and C-reactive protein (CRP) in serum as well as CD68-positive cells in treated islets, decreased significantly. Finally, LPS stimulated the production of inflammatory factors but inhibited GSIS of MIN6 cells; however, the effects were completely reversed by doxycycline. The results support further study of possible long-term usage of sub-antimicrobial doxycycline in diabetic patients.
Assuntos
Antineoplásicos/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Doxiciclina/uso terapêutico , Glucose/metabolismo , Inflamação/tratamento farmacológico , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Fígado/fisiologia , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos MutantesRESUMO
Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Muts) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.
Assuntos
DNA , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Pichia/genética , Transfecção , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Animais , Contagem de Células , Expressão Gênica , Vetores Genéticos , Células HeLa , Humanos , Pichia/química , Pichia/crescimento & desenvolvimento , Protoplastos , Proteínas Recombinantes/biossíntese , Transfecção/métodosRESUMO
OBJECTIVE: To investigate whether selected cytokines are detectable in the embryo culture medium (EM) of human preimplantation embryos (HPE) and what the relationship is of the cytokines with clinical outcomes. DESIGN: Cross-sectional study. SETTING: University-affiliated tertiary teaching hospital. PATIENT(S): Three-hundred and thirty infertile women who underwent fresh cycle in vitro fertilization (IVF) between January and December 2014. INTERVENTION(S): Collection on the day of transfer of the EM of each embryo that was transferred in all patients for measurement of cytokine levels. MAIN OUTCOME MEASURE(S): Measurement of 13 selected cytokines in the EM of day-3 HPE to analyze the relationship of the cytokine with embryo quality and clinical outcome. RESULT(S): Of the cytokines measured, only interleukin-8 (IL-8) was statistically significantly associated with clinical outcome. The rate of detectable IL-8 in the EM was 32.42%, and the pregnancy rate, implantation rate, and number of live births per in vitro fertilization (IVF) or intracytoplasmic sperm injection patient (N LBPP) were higher, and 0 IR was lower in patients for whom the medium from transferred embryos was positive for IL-8 (IL-8 positive group) compared with the patients for whom the medium tested negative for IL-8 (IL-8 negative group). Compared with the IL-8 negative group, a higher pregnancy rate was observed in the IL-8 positive group when the patients received equal good-ordinary quality embryos. CONCLUSION(S): In the EM from HPE, IL-8 is associated with higher pregnancy rates, higher IRs, and higher N LBPP, so IL-8 may be an independent predictor for pretransfer assessment of the embryo development potential in IVF patients.
Assuntos
Blastocisto/metabolismo , Meios de Cultivo Condicionados/metabolismo , Técnicas de Cultura Embrionária , Fertilização in vitro , Infertilidade Feminina/terapia , Interleucina-8/metabolismo , Adulto , Biomarcadores/metabolismo , Implantação do Embrião , Transferência Embrionária , Feminino , Fertilidade , Fertilização in vitro/efeitos adversos , Humanos , Infertilidade Feminina/diagnóstico , Infertilidade Feminina/fisiopatologia , Nascido Vivo , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Gravidez , Taxa de Gravidez , Resultado do Tratamento , Adulto JovemRESUMO
Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.
Assuntos
Células Secretoras de Insulina/citologia , Insulina/genética , Integrases/genética , Animais , Linhagem Celular , Imunofluorescência , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Células Secretoras de Insulina/metabolismo , Integrases/metabolismo , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Mutação Puntual , Regiões Promotoras Genéticas , Ratos , Coloração e Rotulagem , TransfecçãoRESUMO
OBJECTIVES: Chronic administration of nicotinic acid (NA), a potent antilipidemic compound, aggravates glycemic control in diabetic patients. It is not known if NA has direct effects on islet ß cells. METHODS: Real-time reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunofluorescence techniques were used to examine the expression of NA receptor PUMA-G, a member of the G protein-coupled receptor (G-PCR) family, in murine islet ß cells. Calcium transient was measured using confocal microscopy, whereas the intracellular cyclic adenosine monophosphate and glucose-stimulated insulin secretion (GSIS) from isolated islets were determined by the enzyme-linked immunosorbent assay. RESULTS: High levels of PUMA-G transcripts and protein were detected in all ß cells, and about 40% of α cells. PUMA-G transcripts increased more than 3-fold in islets incubated with interferon γ. Cyclic adenosine monophosphate accumulation, induced by IBMX/forskolin, was inhibited by NA; however, the inhibition was completely abolished by pretreatment of the culture with pertussis toxin. No calcium transient was detected in islet cells in the presence of NA. Static incubation of islets with NA led to an approximately 30% reduction of GSIS. CONCLUSIONS: The results indicated that PUMA-G stimulation by NA in islet ß cells inhibited GSIS likely via activation of the Gi signaling pathway.
Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Niacina/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Imunofluorescência , Hibridização In Situ , Técnicas In Vitro , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Interferon gama/farmacologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VasodilatadoresRESUMO
Islet transplantation represents an important alternative for the treatment of diabetes. However, the selection of suitable materials is critical for the success of such an implantation application. In this study, cellular migration, aggregation, and insulin production of a murine islet beta-cell line, NIT-1 cells on microbially produced polyesters poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) or polylactic acid (PLA) films were investigated. Spherical islet-like structures were only detected on PHBHHx films after 48 h cultivation. To understand the mechanism underlying the formation of cell aggregates, NIT-1-GFP, a stable transfectant of the green fluorescent protein was used in a time-lapse imaging study. Cell aggregation began on PHBHHx at 2 h, and became obvious at 4 h. Furthermore, cells on PHBHHx displayed higher metabolic activities measured by MTT assay than that on tissue culture plate. More importantly, insulin gene expression as well as extracellular secretion was upregulated after growth on PHBHHx for 72 h. Thus, PHBHHx can be a strong candidate for islet transplantation.
Assuntos
Ácido 3-Hidroxibutírico , Materiais Biocompatíveis , Caproatos , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Eletroporação , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Insulina/genética , Transplante das Ilhotas Pancreáticas , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Biopolyesters of polyhydroxyalkanoates (PHAs), including poly-3-hydroxybutyrate (PHB), co-polyester of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), and co-polyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) have been well investigated for their biocompatibility. For in vivo application, it is very important that the degradation products of PHAs, especially the oligomers, are not harmful to the cells and surrounding tissues. In this study, in vitro effects of oligo(3-hydroxybutyrate) (OHB), oligo(3-hydroxybutyrate-co-4-hydroxybutyrate) (O3HB4HB) and oligo(3-hydroxybutyrate-co-3-hydroxyhexanoate) (OHBHHx) on growth and differentiation of the murine beta cell line NIT-1 were investigated. Among the three oligo-hydroxyalkanoates (Oligo-HAs), cells treated with OHBHHx displayed higher viability, as measured by CCK-8 assay. Flow cytometric analysis of NIT-1 cells indicated that Oligo-HAs had an inhibitory effect on cell apoptosis. The cytosolic Ca(2+) transient of NIT-1 cells increased when fed with 0.04 g/l Oligo-HAs. For gap junction intercellular communication of cells, the effect of OHBHHx was the best among all materials tested. More importantly, extracellular insulin secretion was up-regulated after growing in OHBHHx for 48 h. The results demonstrated that the degradation products of PHAs, especially OHBHHx from PHBHHx, were not harmful to the beta cells. Therefore, PHBHHx warrant further study for application as a pancreatic tissue engineering material.
Assuntos
Butiratos/química , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Polímeros/química , Polímeros/farmacologia , Alicerces Teciduais/química , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Teste de Materiais , Camundongos , Engenharia TecidualRESUMO
Understanding the mechanisms of beta-cell dynamics in postnatal animals is central to cure diabetes. A major obstacle in evaluating the status of pancreatic cells is the lack of surface markers. Here we performed quantitative measurements of two internal markers to follow the developmental history of islets. One marker, cell-cycle activity, was established by measuring expression of Ki67 and the incorporation of 5-bromodeoxyuridine. The other marker, the aging process, was delineated by the determination of telomere length. Moreover, islet neogenesis, possibly from ductal precursors, was monitored by pancreatic duct labeling with an enhanced green fluorescence protein (EGFP) transgene. We found that islets from younger animals, on average, expressed higher Ki67 transcripts, displayed elevated 5-bromodeoxyuridine incorporation, and had longer telomeres. However, significant heterogeneity of these parameters was observed among islets from the same mouse. In contrast, the levels of proinsulin-1 transcripts in islets of different ages did not change significantly. Moreover, mitotic activities correlated significantly with telomere lengths of individual islets. Lastly, after 5.5 d pancreatic duct labeling, a few EGFP-positive islets could be identified in neonatal but not from adult pancreases. Compared with unlabeled control islets, EGFP-positive islets had higher mitotic activities and longer telomeres. The results suggest that islets are born at different time points during the embryonic and neonatal stages and imply that young islets might play an important role in the maintenance of islet mass in the adult pancreas.
Assuntos
Biomarcadores/análise , Ilhotas Pancreáticas/citologia , Pâncreas/metabolismo , Telômero/metabolismo , Envelhecimento , Animais , Antígeno Ki-67/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitose , Pâncreas/citologiaRESUMO
Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CD5. Here, we describe our studies with a representative CD5+ B-cell-derived natural autoantibody, the VH11Vkappa9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5-10% of the CD5+ B-cell subset, with a large portion accounted for by two novel BCRs, VH11Vkappa9 and VH12Vkappa4. We have found that the development of B-lineage cells with a VH11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i). one of the most common VH11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii). cells containing VH11- micro chains lacking N-addition are favored for progression to the B-cell stage, eliminating most bone marrow VH11 rearrangements; and (iii). only a subset of Vkappa-light chains combine with VH11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain VH11 generation to fetal development and may favor production of B cells with the prototype VH11Vkappa9 BCR.