RESUMO
BACKGROUND: Nocardiosis is a rare and life-threatening opportunistic infection in immunocompromised patients. Myasthenia gravis (MG) patients are potentially at risk of nocardia infection because of the use of immunosuppressive agents. To date, only 7 patients with MG have been reported to have nocardiosis. Disseminated nocardiosis with ocular involvement has not been reported in MG patients. CASE PRESENTATION: A 66-year-old man with MG who was receiving treatment with methylprednisolone and azathioprine was found to have a respiratory infection. He also had heterogeneous symptoms with skin, brain and ocular manifestations. Nocardia bacteria verified by the culture of puncture fluid, and a diagnosis of disseminated nocardiosis was made. Except for left eye blindness, the patient completely recovered from the disease with combination antibiotic therapy. To further understand nocardiosis in patients with MG, we reviewed the previous relevant literature. According to the literature, this is the first report of disseminated nocardiosis with ocular involvement in an MG patient. CONCLUSIONS: MG patients with immunosuppressant treatments are potentially at risk of a rare nocardia infection, and a favourable prognosis can be achieved through early diagnosis and appropriate antibiotic therapy.
Assuntos
Hospedeiro Imunocomprometido , Miastenia Gravis/imunologia , Nocardiose/imunologia , Idoso , Antibacterianos/uso terapêutico , Oftalmopatias/microbiologia , Humanos , Imunossupressores/administração & dosagem , Masculino , Miastenia Gravis/complicações , Miastenia Gravis/tratamento farmacológico , Nocardia , Nocardiose/tratamento farmacológico , Nocardiose/patologiaRESUMO
Traumatic brain injury (TBI) is one of the major causes of death and disability worldwide. Novel and effective therapy is needed to prevent the secondary spread of damage beyond the initial injury. The aim of this study was to investigate whether berberine has a neuroprotective effect on secondary injury post-TBI, and to explore its potential mechanism in this protection. The mice were randomly divided into Sham-saline, TBI-saline and TBI-Berberine (50 mg/kg). TBI was induced by Feeney's weight-drop technique. Saline or berberine was administered via oral gavage starting 1 h post-TBI and continuously for 21 days. Motor coordination, spatial learning and memory were assessed using beam-walking test and Morris water maze test, respectively. Brain sections were processed for lesion volume assessment, and expression of neuronal nuclei (NeuN), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), 8-hydroxy-2-deoxyguanosine (8-OHdG), ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) were detected via immunohistochemistry and immunofluorescence. There were statistically significant improvement in motor coordination, spatial learning and memory in the TBI-Berberine group, compared to the TBI-saline group. Treatment with berberine significantly reduced cortical lesion volume, neuronal loss, COX-2, iNOS and 8-OHdG expression in both the cortical lesion border zone (LBZ) and ipsilateral hippocampal CA1 region (CA1), compared to TBI-saline. Berberine treatment also significantly decreased Iba1- and GFAP-positive cell number in both the cortical LBZ and ipsilateral CA1, relative to saline controls. These results indicated that berberine exerted neuroprotective effects on secondary injury in mice with TBI probably through anti-oxidative and anti-inflammatory properties.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Berberina/farmacologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Exposure to a novel environment enhances the extinction of contextual fear through the "tagging-and-capture" process. However, the underlying molecular mechanisms of novelty-induced enhancement of fear extinction are still unclear. NMDA receptor activity was recently revealed to be required for the enhancement of fear extinction caused by exposure to novelty. Src family kinases (SFKs) act as a molecular hub for regulation of NMDA receptors. We hypothesized that SFKs might be involved in novelty-induced enhancement of fear extinction. We found that the enhancement of fear extinction induced by novelty exposure is accompanied by Src kinase phosphorylation and activation in a restricted time window. Furthermore, intrahippocampal infusion of SFKs inhibitor PP2 inhibits Src kinase phosphorylation and activation, attenuates the activation of NR2B-containing NMDA receptors, and thereby reverses the enhancement of fear extinction induced by novelty exposure. These results suggested that Src kinase may serve as a behavioral tag in the procedural enhancement of fear extinction by novelty exposure.
Assuntos
Ativação Enzimática , Comportamento Exploratório , Medo/fisiologia , Hipocampo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Quinases da Família src/metabolismo , Animais , Masculino , Fosforilação , Ratos Sprague-DawleyRESUMO
BACKGROUND AND HYPOTHESIS: Our previous studies have found that functional changes in the hippocampal circuit from dentate gyrus (DG) to cornu ammonis 3 and 1 (CA3, CA1) are highly associated with schizophrenia (SZ). However, no studies have explored the genetic expression across the three and two human hippocampal subfields (DG-CA3-CA1 and CA3-CA1) between subjects with SZ and healthy controls (CT). STUDY DESIGN: We matched cohorts between CT (n = 13) and SZ (n = 13). Among SZ, 6 subjects were on antipsychotics (AP) while 7 were off AP. We combined RNA-seq data from all three and two hippocampal subfields and performed differentially expressed gene analyses across DG-CA3-CA1 and CA3-CA1 affected by either SZ or AP. STUDY RESULTS: We found that differentially expressed genes (DEGs) from effects of SZ and AP across DG-CA3-CA1 and CA3-CA1 were highly associated with gene ontology terms related to hormonal and immune signaling, cellular mitosis and apoptosis, ion and amino acid transports, and protein modification and degradation. Additionally, we found that multiple genes related to solute-carrier family and immune signaling were significantly upregulated across DG-CA3-CA1 and CA3-CA1 in patients with SZ relative to CT, and AP consistently and robustly repressed the expression of these upregulated genes in the DG-CA3-CA1 and CA3-CA1 from subjects with SZ. CONCLUSIONS: Together, these data suggest that the upregulated solute-carrier family genes in the hippocampus might have important roles in the pathophysiology of SZ, and that AP may reduce the symptoms of psychosis in SZ via rescuing the solute-carrier gene expression.
RESUMO
As global aging becomes more prominent, neurocognitive disorders (NCD) incidence has increased. Patients with NCD usually have an impairment in one or more cognitive domains, such as attention, planning, inhibition, learning, memory, language, visual perception, and spatial or social skills. Studies indicate that 50-80% of these adults will develop neuropsychiatric symptoms (NPS), such as apathy, depression, anxiety, disinhibition, delusions, hallucinations, and aberrant motor behavior. The progression of NCD and subsequent NPS requires tremendous care from trained medical professionals and family members. The behavioral symptoms are often more distressing than cognitive changes, causing caregiver distress/depression, more emergency room visits and hospitalizations, and even earlier institutionalization. This signifies the need for early identification of individuals at higher risk of NPS, understanding the trajectory of their NCD, and exploring treatment modalities. In this case report and review, we present an 82-year-old male admitted to our facility for new-onset symptoms of depression, anxiety, and persecutory delusions. He has no significant past psychiatric history, and his medical history is significant for extensive ischemic vascular disease requiring multiple surgeries and two episodes of cerebrovascular accident (CVA). On further evaluation, the patient was diagnosed with major NCD, vascular subtype. We discuss differential diagnoses and development of NPS from NCD in order to explain the significance of more thorough evaluation by clinicians for early detection and understanding of NCD prognosis.
Assuntos
Delusões , Doenças Vasculares , Idoso de 80 Anos ou mais , Humanos , Masculino , Delusões/etiologia , Depressão/etiologia , Alucinações , Transtornos Neurocognitivos , Doenças Vasculares/complicaçõesRESUMO
Charcot-Marie-Tooth (CMT) disease, also known as hereditary motor sensory neuropathy, is a group of rare genetically heterogenous diseases characterized by progressive muscle weakness and atrophy, along with sensory deficits. Despite extensive pre-clinical and clinical research, no FDA-approved therapy is available for any CMT type. We previously identified C1ORF194, a novel causative gene for CMT, and found that both C1orf194 knock-in (I121N) and knockout mice developed clinical phenotypes similar to those in patients with CMT. Encouraging results of adeno-associated virus (AAV)-mediated gene therapy for spinal muscular atrophy have stimulated the use of AAVs as vehicles for CMT gene therapy. Here, we present a gene therapy approach to restore C1orf194 expression in a knockout background. We used C1orf194-/- mice treated with AAV serotype 9 (AAV9) vector carrying a codon-optimized WT human C1ORF194 cDNA whose expression was driven by a ubiquitously expressed chicken ß-actin promoter with a CMV enhancer. Our preclinical evaluation demonstrated the efficacy of AAV-mediated gene therapy in improving sensory and motor abilities, thus achieving largely normal gross motor performance and minimal signs of neuropathy, on the basis of neurophysiological and histopathological evaluation in C1orf194-/- mice administered AAV gene therapy. Our findings advance the techniques for delivering therapeutic interventions to individuals with CMT.
Assuntos
Doença de Charcot-Marie-Tooth , Humanos , Camundongos , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Fenótipo , Administração Intravenosa , MutaçãoRESUMO
Although both the heat shock protein 70 (HSP70) and the activating transcription factor 5 (ATF5) have been shown to promote cell survival of transformed cells but not survival of non-transformed cells, the relationship of the two molecules is unknown. Here we show that HSP70 and ATF5 are concomitantly up-regulated upon transient but down-regulated over prolonged cellular stress and apoptotic stimulation in the rat C6 glioma and human U87 glioma cells. HSP70 interacts strongly with the N-terminal activation domain of ATF5, which is expected to be rigid and uniquely structured under physiological conditions because of extraordinary high concentration (over 25%) of proline residues. Binding of HSP70 to ATF5 is an ATP-driven process and requires functional ATPase on the nucleotide binding domain of the HSP70 molecule. Overexpression of HSP70 dramatically stabilizes the ATF5 protein, which is otherwise subject to rapid degradation, facilitated by both proteasome-dependent and caspase-dependent processes, whereas HSP70 depletion leads to acceleration of ATF5 degradation and transcription repression of Bcl-2 and Egr-1, which are downstream targets of ATF5 in C6 and U87 glioma cells. Our data reveal an essential role for HSP70 in maintaining high levels of ATF5 expression in glioma cells and support the conclusion that ATF5 is an important substrate protein of HSP70 that mediates HSP70-promoted cell survival in glioma cells.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Apoptose , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Fatores Ativadores da Transcrição/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Glioma/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Ligação Proteica/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RatosRESUMO
Amniotic fluid-derived stem cells have attracted considerable attention in the field of regenerative medicine. Approach of genetic modification probably enhances their regenerative potential. In this work, we wanted to determine whether baculovirus as a new gene vector could efficiently and safely transduce mouse amniotic fluid-derived stem cells (mAFSs). Cells were isolated from mouse amniotic fluid and cultured in vitro. These cells were analyzed by examining phenotypes and differentiation potential. They were further transduced with baculovirus. Baculovirus-transduced mAFSs were induced to differentiate into adipogenic, osteogenic, myogenic, and neurogenic lineages. Mouse amniotic fluid-derived stem cells were successfully isolated and cultured in vitro. They were positive for CD29 and Sca-1, but negative for CD34, CD45, or CD11b. Furthermore, they could differentiate into adipocytes, osteocytes, myocytes, and neurocytes in vitro. Baculovirus could efficiently transduced mAFSs. More importantly, baculovirus-transduced mAFSs retained differentiation potential. Thus, baculovirus vector effective and safe transduction is an attractive promise for genetic modification of mAFSs. Baculovirus genetically modified mAFSs will probably be more suitable as vehicles for regenerative medicine.
Assuntos
Líquido Amniótico/citologia , Baculoviridae/genética , Diferenciação Celular , Células-Tronco/citologia , Células-Tronco/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Separação Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Neurônios/citologia , Osteogênese , FenótipoRESUMO
Astrocytic differentiation is developmentally impaired in patients with childhood-onset schizophrenia (SCZ). To determine why, we used genetic gain- and loss-of-function studies to establish the contributions of differentially expressed transcriptional regulators to the defective differentiation of glial progenitor cells (GPCs) produced from SCZ patient-derived induced pluripotent cells (iPSCs). Negative regulators of the bone morphogenetic protein (BMP) pathway were upregulated in SCZ GPCs, including BAMBI, FST, and GREM1, whose overexpression retained SCZ GPCs at the progenitor stage. SMAD4 knockdown (KD) suppressed the production of these BMP inhibitors by SCZ GPCs and rescued normal astrocytic differentiation. In addition, the BMP-regulated transcriptional repressor REST was upregulated in SCZ GPCs, and its KD similarly restored normal glial differentiation. REST KD also rescued potassium-transport-associated gene expression and K+ uptake, which were otherwise deficient in SCZ glia. These data suggest that the glial differentiation defect in childhood-onset SCZ, and its attendant disruption in K+ homeostasis, may be rescued by targeting BMP/SMAD4- and REST-dependent transcription.
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Diferenciação Celular , Neuroglia/metabolismo , Proteínas Repressoras/metabolismo , Esquizofrenia/metabolismo , Transdução de Sinais , Proteína Smad4/metabolismo , Adolescente , Adulto , Linhagem Celular , Criança , Feminino , Humanos , Masculino , Neuroglia/patologia , Proteínas Repressoras/genética , Esquizofrenia/genética , Esquizofrenia/patologia , Proteína Smad4/genéticaRESUMO
BACKGROUND: Human mesenchymal stem cells (MSCs) have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. Since most human diseases models are murine, mouse MSCs should have been studied in detail. The mdx mouse - a Duchenne muscular dystrophy model - was produced by introducing a point mutation in the dystrophin gene. To understand the role of dystrophin in MSCs, we compared MSCs from mdx and C57BL/10 mice, focusing particularly on the aspects of light and electron microscopic morphology, immunophenotyping, and differentiation potential. RESULTS: Our study showed that at passage 10, mdx-MSCs exhibited increased heterochromatin, larger vacuoles, and more lysosomes under electron microscopy compared to C57BL/10-MSCs. C57BL/10-MSCs formed a few myotubes, while mdx-MSCs did not at the same passages. By passage 21, mdx-MSCs but not C57BL/10-MSCs had gradually lost their proliferative ability. In addition, a significant difference in the expression of CD34, not Sca-1 and CD11b, was observed between the MSCs from the 2 mice. CONCLUSION: Our current study reveals that the MSCs from the 2 mice, namely, C57BL/10 and mdx, exhibit differences in proliferative and myogenic abilities. The results suggest that the changes in mouse MSC behavior may be influenced by lack of dystrophin protein in mdx mouse.
Assuntos
Antígenos CD34/biossíntese , Distrofina/genética , Células-Tronco Mesenquimais/fisiologia , Animais , Antígenos CD34/metabolismo , Proliferação de Células , Separação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Distrofina/deficiência , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Desenvolvimento Muscular/genética , Distrofia Muscular de Duchenne/genética , Mutação/genética , Especificidade da EspécieRESUMO
BACKGROUND: Schwann cells (SC) which are myelin-forming cells in peripheral nervous system are very useful for the treatment of diseases of peripheral nervous system and central nervous system. However, it is difficult to obtain sufficient large number of SC for clinical use, so alternative cell systems are desired. RESULTS: Using a procedure similar to the one used for propagation of neural stem cells, we could induce rat adipose-derived stem cells (ADSC) into floating neurospheres. In addition to being able to differentiate into neuronal- and glial-like cells, neurospheres could be induced to differentiate into SC-like cells. SC-like cells were bi- or tri-polar in shape and immunopositive for nestin and SC markers p75, GFAP and S-100, identical to genuine SC. We also found that SC-like cells could induce the differentiation of SH-SY5Y neuroblastoma cells efficiently, perhaps through secretion of soluble substances. We showed further that SC-like cells could form myelin structures with PC12 cell neurites in vitro. CONCLUSION: These findings indicated that ADSC could differentiate into SC-like cells in terms of morphology, phenotype and functional capacities. SC-like cells induced from ADSC may be useful for the treatment of neurological diseases.
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Adipócitos/citologia , Células de Schwann/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Técnicas de Cocultura , Indução Embrionária , Citometria de Fluxo , Imuno-Histoquímica , Neuroblastoma/patologia , Células PC12 , Ratos , Ratos Sprague-Dawley , Esferoides CelularesRESUMO
The use of stem cells will lead to novel treatments for a wide range of diseases due to their properties of self-renewing, pluripotent, and undifferentiated state, and the stem cells are usually genetically modified for cell and gene therapy. If the baculovirus, as a new gene vector, can be effectively transduced into various mammalian bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, it will be a better gene vector to genetically modify the stem cells. The aim of the present study is to investigate the transduction efficiency of recombinant baculovirus (BacV-CMV-EGFP), which expressed a reporter gene encoding enhanced green fluorescent protein (EGFP) under a cytomegalovirus immediate early (CMV-IE) promoter, into various mammalian BMSCs. The BMSCs of mouse, rat, porcine, rhesus, and human were cultured primarily in vitro. After more than three passages, the mammalian BMSCs were seeded into dishes and cultured in a humidified incubator at 37 °C with 5% CO(2). When the cells reached about 80% confluence, the complete medium was removed by aspiration. The cells were transduced with recombinant baculovirus at a multiplicity of infection (MOI) of 200 vector genomes/cell with 500 µL PBS at 25 °C for 4 h. At the end of baculovirus transduction, cells were washed and incubated with 2 mL complete medium, and baculovirus-transduced mammalian BMSCs were cultured in a humidified incubator for 2 d. Then, the inverted fluorescent microscope was used to observe GFP expressions in different mammalian BMSCs, and flow cytometry was used to detect the transduction efficiency of baculovirus in various mammalian BMSCs. After more than three passages, the BMSCs of mouse, rat, porcine, rhesus, and human showed a homogeneous spindle-shaped morphology. Compared with the BMSCs of mouse, rat and porcine, the inverted fluorescent microscope observations showed that there were more BMSCs expressing GFP and greater mean fluorescence intensity in rhesus and human transduced with baculovirus. The baculovirus could efficiently transduce into the BMSCs of mouse, rat, porcine, rhesus and human, and the transduction efficiency was (20.21±3.02)%, (22.51±4.48)%, (39.13±5.79)%, (71.16±5.36)% and (70.67±3.74)%, respectively. In conclusion, baculovirus displays different transduction efficiency into various mammalian BMSCs. Due to the high transduction efficiency for primate and human BMSCs, baculovirus is possibly a more suitable gene vector to genetically modify BMSCs of human and primates.
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Baculoviridae , Vetores Genéticos , Células-Tronco Mesenquimais/citologia , Transdução Genética , Animais , Células da Medula Óssea/citologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Macaca mulatta , Camundongos , Regiões Promotoras Genéticas , Ratos , SuínosRESUMO
OBJECTIVE: To construct the recombinant plasmid containing human microdystrophin cDNA, and study the microdystrophin expression in vivo and in vitro. METHODS: Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I, the product was inserted into plasmid pVAX1, resulting in pAMICDYS. And then 3T3 cells were transfected with pAMICDYS. Forty-eight hours after transfection, the expression of the microdystrophin was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Finally, TA muscles of mdx mice were injected with the recombinant plasmid pAMICDYS through i.m. and the pathological change of TA was evaluated by histology, and the expression of microdystrophin in mdx TA was detected by immunohistochemical analysis. RESULTS: The recombinant plasmid containing human microdystrophin cDNA was constructed successfully. The recombinant plasmid was proved to be able to express microdystrophin protein both in vivo and in vitro. Moreover, treatment of the TA of mdx mice with the recombinant plasmid could decrease the number of centrally nucleated myofibers. CONCLUSION: Recombinant plasmid containing the microdystrophin gene was constructed successfully, and it could express microdystrophin protein both in vivo and in vitro. It provides basis for further study on microdystrophin as a target gene to treat Duchenne muscular dystrophy (DMD) by electrotransfer, i.v, arterial injection and combining with other exogenous gene to enhance microdystrophin expression.
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DNA Recombinante/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Plasmídeos/genética , Animais , Clonagem Molecular , Enzimas de Restrição do DNA/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , DNA Recombinante/metabolismo , Expressão Gênica , Engenharia Genética , Terapia Genética , Vetores Genéticos/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Distrofia Muscular de Duchenne/metabolismo , Células NIH 3T3 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
In this study, PCR-SSCP analysis was used to identify genetic variation in IGFBP-3 gene in Chinese Merino and Kazakh sheep. A PCR product of 178 bp corresponding to partial intron1 illustrated three unique binding patterns by SSCP analysis. Frequencies of the genotype AA, AB, BB and allele A, B in Chinese Merino sheep were 0.70, 0.24, 0.06, and 0.82, 0.18 respectively , and they were 0.87, 0.13, 0.00, and 0.93, 0.07 respectively in Kazaka sheep. Sequence analysis revealed a G/T transversion at position 122 of the fragment. This polymorphic locus of IGFBP-3 gene was at Hardy-Weinberg dis-equilibrium (P<0.01) in the two breeds. Different genotypes slightly affected several wool traits of Chinese Merino sheep. The individuals of genotype AA, AB, and BB had no significant difference in post-shearing weight and clean wool rate. Sta-ple length (SL) was decreased with the genotype of AA, AB, and BB, and the difference between AA and AB was significant (P<0.01). Greasy fleece weight (GFW) and follicle density in individuals of genotype AA was significantly lower than that in individuals of genotype AB (P<0.01) and BB (P<0.05); Average fiber diameter (AFD) in individuals of genotype AA was significantly higher than that in individuals of genotype AB (P<0.01) and BB (P<0.05).
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Genótipo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Polimorfismo Genético , Carneiro Doméstico/genética , Lã/economia , Alelos , Animais , DNA/análise , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismoRESUMO
OBJECTIVE: To construct the recombinant adenovirus containing herpes simplex virus-1 virion protein (VP) 22 and human microdystrophin gene, then the adenovirus was transfected into C2C12 myoblast and studied on the property of protein transduction with VP22-mediated microdystrophin in C2C12 myoblast. METHODS: The full-length VP22 cDNA was obtained from recombinant plasmid pSINrep5-VP22 with PCR, and the product was directionally inserted into pShuttle-CMV to acquire the plasmid pCMV-VP22. Microdystrophin cDNA was obtained from recombinant plasmid pBSK-micro digested with restrictive endonuclease NotI, and the product was directionally inserted into pCMV-VP22 to acquire the plasmid pCMV-VP22-MICDYS. The plasmid of pCMV-VP22-MICDYS was lined with Pme I, and the fragment containing VP22-microdystrophin was reclaimed and transfected into E1 coli BJ5183 with plasmid pAdeasy-1. After having been screened by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the cytopathic effect of cells and by PCR method to acquire the recombinant adenovirus Ad-VP22-MICDYS. Finally, the C2C12 myoblast were transfected with the recombinant adenovirus Ad-VP22-MICDYS and Ad-MICDYS, and the expression of microdystrophin was detected by RT-PCR, Western blot and immunocytochemistry. RESULTS: The recombinant adenovirus including VP22 and microdystrophin gene was successfully constructed. VP22 transferred VP22-microdystrophin fused protein from infected C2C12 myoblast into uninfected cells and enhance the expression of microdystrophin in myoblast. CONCLUSIONS: Recombinant adenovirus containing VP22 and microdystrophin gene was constructed successfully. VP22 can enhance the expression with microdystrophin in myoblast. It lays the foundation for further studying on VP22-mediated recombinant including microdystrophin gene to cure Duchenne muscular dystrophy.
Assuntos
Adenoviridae/genética , Distrofina/genética , Vetores Genéticos/genética , Transdução Genética , Proteínas Estruturais Virais/genética , Adenoviridae/fisiologia , Animais , Linhagem Celular , Distrofina/metabolismo , Vetores Genéticos/metabolismo , Humanos , Camundongos , Mioblastos/metabolismo , Mioblastos/virologia , Simplexvirus/genética , Simplexvirus/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/genética , Vírion/metabolismoRESUMO
OBJECTIVE: To compare the transduction efficiencies of adenoviral vector, adeno-associated viral vector, baculoviral vector, and plasmid vector in human bone-marrow-derived mesenchymal stem cells (hBMSCs). METHODS: The hBMSCs were cultured in vitro and transducted with the adenoviral vector, adeno-associated viral vector, baculoviral vector, and plasmid vector. The expression of target protein was observed by inverted fluorescent microscopy and flow cytometry. RESULTS: Inverted fluorescent microscopy showed that some of the hBMSCs after transduction expressed the green fluorescent protein (GFP) and the hBMSCs transducted with baculoviral vector expressed more GFP than those of other three vectors. Flow cytometry showed that the transduction efficiencies and mean fluorescence intensities of the adenoviral vector, adeno-associated viral vector, and plasmid vector were 42%, 37%, and 22% and 158, 115, and 77, respectively, which were significantly lower than those of baculoviral vector (70%, P < 0.01; 212, P < 0.05; respectively). CONCLUSION: Compared with the adenoviral vector, adeno-associated viral vector, and plasmid vector, the baculoviral vector has higher transduction efficiency in hBMSCs and therefore may be a more suitable gene vector for research in human gene therapy.
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Células da Medula Óssea/virologia , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/virologia , Transdução Genética/métodos , Adenoviridae/genética , Adenoviridae/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Dependovirus/genética , Dependovirus/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Plasmídeos/genética , Plasmídeos/metabolismoRESUMO
OBJECTIVE: To investigate whether the recombinant baculovirus (Bac-CMV-EGFP) can effectively transduce into rhesus Bone-marrow derived Mesenchymal Stem Cells (rBMSCs) in vitro, and whether there are some efficiency to the rBMSCs of viability, proliferational and differentiational capacity after recombinant baculovirus transducing. METHODS: The rBMSCs were cultured in vitro. After passaged more than three times, the rBMSCs were transduced with various dose of baculovirus (Multiplicity Of Infection, MOI, the MOI is 50, 100, 200, 300, and 500 vector genome (vg)/cell, respectively). We used flow cytometry to detect different transductive efficiency of various dose of baculovirus to rBMSCs. Under a suitable dose of baculovirus (300 v.g/cell), we studied cell viability, proliferation and differentiation capacity, and compared results with the control. RESULTS: Baculovirus could be transduced into rBMSCs in vitro. The transductive efficiency reached about 80% when the MOI was 300 v.g/cell, at 25 degrees C, and incubated for 4 h. Furthermore, under a higher transductive efficiency of baculovirus, there were no obvious influence to the rBMSCs of viability, proliferation and differentiation capacity compared with that of the control. CONCLUSION: The baculovirus can be safely and effectively transduced into rBMSCs in vitro, without any negative efficiency to cell viability, proliferation and differentiation capacity.
Assuntos
Baculoviridae/genética , Células da Medula Óssea/citologia , DNA Recombinante/genética , Macaca mulatta , Células-Tronco Mesenquimais/metabolismo , Transdução Genética/métodos , Animais , Baculoviridae/fisiologia , Proliferação de Células , Sobrevivência Celular , Células-Tronco Mesenquimais/citologiaRESUMO
In this study, we investigated whether intrinsic glial dysfunction contributes to the pathogenesis of schizophrenia (SCZ). Our approach was to establish humanized glial chimeric mice using glial progenitor cells (GPCs) produced from induced pluripotent stem cells derived from patients with childhood-onset SCZ. After neonatal implantation into myelin-deficient shiverer mice, SCZ GPCs showed premature migration into the cortex, leading to reduced white matter expansion and hypomyelination relative to controls. The SCZ glial chimeras also showed delayed astrocytic differentiation and abnormal astrocytic morphologies. When established in myelin wild-type hosts, SCZ glial mice showed reduced prepulse inhibition and abnormal behavior, including excessive anxiety, antisocial traits, and disturbed sleep. RNA-seq of cultured SCZ human glial progenitor cells (hGPCs) revealed disrupted glial differentiation-associated and synaptic gene expression, indicating that glial pathology was cell autonomous. Our data therefore suggest a causal role for impaired glial maturation in the development of schizophrenia and provide a humanized model for its in vivo assessment.
Assuntos
Quimera/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Neuroglia/patologia , Esquizofrenia/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Comportamento , Diferenciação Celular/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Bainha de Mielina/metabolismo , Neuroglia/metabolismo , Fenótipo , Esquizofrenia/genéticaRESUMO
The insulin/insulin-like growth factor signaling (IIS) pathway is a major conserved regulator of aging. Nematode, fruit fly and mouse mutants with reduced IIS signaling exhibit extended lifespan. These mutants are often dwarfs leading to the idea that small body mass correlates with longevity within species. However, when different species are compared, larger animals are typically longer-lived. Hence, the role of IIS in the evolution of life history traits remains unresolved. Here we used comparative approach to test whether IGF1R signaling changes in response to selection on lifespan or body mass and whether specific tissues are involved. The IGF1R levels in the heart, lungs, kidneys, and brains of sixteen rodent species with highly diverse lifespans and body masses were measured via immunoblot after epitope conservation analysis. We report that IGF1R levels display strong negative correlation with maximum lifespan only in brain tissue and no significant correlations with body mass for any organ. The brain-IGF1R and lifespan correlation holds when phylogenetic non-independence of data-points is taken into account. These results suggest that modulation of IGF1R signaling in nervous tissue, but not in the peripheral tissues, is an important factor in the evolution of longevity in mammals.
Assuntos
Encéfalo/metabolismo , Receptor IGF Tipo 1/metabolismo , Roedores/classificação , Roedores/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Epitopos , Dados de Sequência Molecular , Receptor IGF Tipo 1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Roedores/fisiologia , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Although Schwann cell (SC) transplantation can enhance peripheral and central nerve repair experimentally, it is difficult to generate sufficient SC quickly for clinical application. So alternative cell systems for SC are desired. SC-like cells induced from adipose-derived stem cells (ADSC) may be one of the ideal alternative cell systems for SC. However, myelin-forming ability, which is the most important characteristics and function of SC, has not been investigated in SC-like cells from ADSC up to now. In this experiment, ADSC were harvested from rat inguinal fat pad. Rat ADSC were fibroblast-like in shape, almost all the cells expressed mesodermal marker fibronectin, and only few cells expressed neural stem cell marker nestin. A mixture of glial growth factors (Heregulin, bFGF, PDGF and forskolin) could induce rat ADSC into SC-like cells. SC-like cells were spindle-like in shape and expressed glial markers GFAP and S100, similar to genuine SC. When intracellular cAMP was increased, SC-like cells could express myelin protein p0. More importantly, when co-cultured with rat pheochromocytoma cell line (PC12 cells), SC-like cells could induce the differentiation of PC12 cells rapidly and form myelin structures with PC12 cells in vitro. Our data further demonstrated that SC-like cells from ADSC were able to form myelins and these cells may benefit the treatment of peripheral and central nerve injuries.