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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 267-274, 2020 Feb.
Artículo en Chino | MEDLINE | ID: mdl-32027288

RESUMEN

OBJECTIVE: To investigate the effects of human amniotic mesenchymal stem cell(AMSC) on acute graft-versus-host disease (aGVHD) in xenotransplatation. METHODS: NPG mice were injected with human PBMNC via tail vein to establish a xenografted aGVHD model. The mice in the experimental group were divided into PBMNC infusion group and PBMNC+AMSC co-infusion group, the general condition, survival time and manifestations of aGVHD were observed, the body weight and blood routine indicators were detected, the pathological changes of aGVHD target organs (lung, liver, spleen, small intestine) were observed by HE staining, and the levels of human T cells in peripheral blood, tissues and organs of mice was detected by flow cytometry. RESULTS: The manifestations of aGVHD (lassitude hunchback, shrub, weight reduction, etc.) and the pathological damage of the target organs (lung, liver, spleen, intestine) in PBMNC+AMSC co-infusion group were lighter than those in PBMNC infusion group. Moreover, the PBMNC and AMSC co-infusion significantly reduced the implantion proportion of human T lymphocytes (CD3+, CD45+) in mice and increased the ratio of CD4+/CD8+. CONCLUSION: Infusion of human-derived AMSC can attenuate the manifestations of aGVHD in mouse xenografts to a certain level, and improve the pathological damage of receptor target organs.


Asunto(s)
Enfermedad Injerto contra Huésped , Células Madre Mesenquimatosas , Enfermedad Aguda , Animales , Xenoinjertos , Humanos , Ratones , Linfocitos T , Trasplante Heterólogo
2.
Plast Reconstr Surg ; 145(2): 409-418, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31985633

RESUMEN

BACKGROUND: Irradiated allogeneic costal cartilage is an alternative option of cartilage graft in patients with insufficient autologous cartilage. However, complications can occur during long-term follow-up. This study investigated whether Tutoplast-processed cartilage, one of the irradiated allogeneic costal cartilages, acts as a scaffold for adipose-derived stem cells and chondrogenesis. METHODS: In vitro setting, human adipose-derived stem cells seeded onto Tutoplast-processed cartilage were cultured in chondrogenic medium and observed using a scanning electron microscope. Next, 3 types of irradiated cartilage-including Tutoplast-processed cartilage, undifferentiated stem cells on Tutoplast-processed cartilage (undifferentiated group), and chondrogenic differentiated stem cells on Tutoplast-processed cartilage (chondrogenic group)-were implanted subcutaneously into nude mice. Gross, histologic, and gene expression analyses of Tutoplast-processed cartilages were performed at postoperative weeks 2 and 4. RESULTS: Human adipose-derived stem cells subjected to in vitro three-dimensional culture differentiated into chondrocytes and expressed cartilage-specificgenes. Adipose-derived stem cells seeded onto Tutoplast-processed cartilage were differentiated into chondrocytes in chondrogenic medium. In the chondrogenic group, the chondrogenic-differentiated cells attached to the surface of the Tutoplast-processed cartilage were maintained during the follow-up and were distinct from the existing Tutoplast-processed cartilage. Moreover, the chondrogenic group had higher expression of cartilage-specific genes compared with the undifferentiated group. CONCLUSIONS: Adipose-derived stem cells seeded onto Tutoplast-processed cartilage underwent chondrogenic differentiation, generating new cartilage, which was maintained after implantation without critical complications. The findings are clinically valuable in terms of overcoming the limitations of irradiated allogeneic costal cartilage, and broaden the surgical options for treatments requiring cartilage.


Asunto(s)
Cartílago/fisiología , Condrogénesis/fisiología , Células Madre Mesenquimatosas/fisiología , Agrecanos/metabolismo , Animales , Biomarcadores/metabolismo , Cartílago/efectos de la radiación , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno Tipo X/metabolismo , Femenino , Humanos , Técnicas In Vitro , Inyecciones Subcutáneas , Músculos Intercostales , Trasplante de Células Madre Mesenquimatosas/métodos , Ratones Desnudos , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Modelos Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Trasplante Heterólogo , Trasplante Homólogo
3.
Chem Commun (Camb) ; 56(13): 1956-1959, 2020 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-31956868

RESUMEN

Herein, we report a pH stimulus-disaggregated BODIPY sensitizer (PTS) with low background-toxicity for achieving activated photodynamic/photothermal tumor therapy. Both the photodynamic and photothermal properties of PTS can be activated under acidic conditions, and PTS exhibits excellent antitumor properties, which is revealed by both in vitro and in vivo tests.


Asunto(s)
Compuestos de Boro/química , Fármacos Fotosensibilizantes/química , Animales , Compuestos de Boro/farmacología , Compuestos de Boro/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Luz , Ratones , Ratones Endogámicos BALB C , Neoplasias/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Fototerapia , Trasplante Heterólogo
4.
Nat Commun ; 11(1): 550, 2020 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-31992716

RESUMEN

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.


Asunto(s)
Ingeniería Genética , Glioblastoma/genética , Glioblastoma/patología , Células Madre Pluripotentes/patología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Diferenciación Celular , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Genoma , Glioblastoma/metabolismo , Glioma/genética , Glioma/patología , Humanos , Ratones , Ratones SCID , Mutación , Trasplante de Neoplasias , Células Madre Neoplásicas/patología , Neurofibromina 1/genética , Fosfohidrolasa PTEN/genética , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/genética
5.
Chemistry ; 26(1): 33-48, 2020 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-31599057

RESUMEN

The ability to modify biologically active molecules such as antibodies with drug molecules, fluorophores or radionuclides is crucial in drug discovery and target identification. Classic chemistry used for protein functionalisation relies almost exclusively on thermochemically mediated reactions. Our recent experiments have begun to explore the use of photochemistry to effect rapid and efficient protein functionalisation. This article introduces some of the principles and objectives of using photochemically activated reagents for protein ligation. The concept of simultaneous photoradiosynthesis of radiolabelled antibodies for use in molecular imaging is introduced as a working example. Notably, the goal of producing functionalised proteins in the absence of pre-association (non-covalent ligand-protein binding) introduces requirements that are distinct from the more regular use of photoactive groups in photoaffinity labelling. With this in mind, the chemistry of thirteen different classes of photoactivatable reagents that react through the formation of intermediate carbenes, electrophiles, dienes, or radicals, is assessed.


Asunto(s)
Preparaciones Farmacéuticas/química , Proteínas/química , Animales , Anticuerpos/química , Línea Celular Tumoral , Radioisótopos de Cobre/química , Reacción de Cicloadición , Humanos , Marcaje Isotópico , Ligandos , Metano/análogos & derivados , Metano/química , Ratones , Ratones Desnudos , Neoplasias/diagnóstico por imagen , Tomografía Computarizada de Emisión de Fotón Único , Trasplante Heterólogo , Rayos Ultravioleta
7.
Plant Mol Biol ; 102(1-2): 213-223, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31845303

RESUMEN

KEY MESSAGE: Transcriptome landscape reveals the molecular mechanisms involved in the improvement of fruit traits by the grafting of watermelon and bottle gourd. Grafting has been used as a sustainable alternative for watermelon breeding to control soil-borne pathogens and to increase tolerance to various abiotic stresses. However, some reports have shown that grafting can negatively affect the quality of fruits. Despite several field studies on the effects of grafting on fruit quality, the regulation of this process at the molecular level has not been revealed. The aim of this study was to elucidate various molecular mechanisms involved in different tissues of heterografted watermelon and bottle gourd plants. Grafting with bottle gourd rootstock increased the size and rind thickness of watermelon fruits, whereas that with watermelon rootstock produced bottle gourd fruits with higher total soluble solid content and thinner rinds. Correspondingly, genes related to ripening, softening, cell wall strengthening, stress response and disease resistance were differentially expressed in watermelon fruits. Moreover, genes associated mainly with sugar metabolism were differentially expressed in bottle gourd fruits. RNA-seq revealed more than 400 mobile transcripts across the heterografted sets. More than half of these were validated from PlaMoM, a database for plant mobile macromolecules. In addition, some of these mobile transcripts contained a transfer RNA-like structure. Other RNA motifs were also enriched in these transcripts, most with a biological role based on GO analysis. This transcriptome study provided a comprehensive understanding of various molecular mechanisms underlying grafted tissues in watermelon.


Asunto(s)
Citrullus/metabolismo , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Transcriptoma , Trasplante Heterólogo , Metabolismo de los Hidratos de Carbono , Citrullus/genética , Resistencia a la Enfermedad/genética , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Fitomejoramiento , Raíces de Plantas/metabolismo , ARN Mensajero/metabolismo , ARN de Planta , Análisis de Secuencia , Estrés Fisiológico
8.
Chem Commun (Camb) ; 55(100): 15101-15104, 2019 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-31782436

RESUMEN

Herein, we have designed bifunctional composite nanospheres for carcinoembryonic antigen (CEA) sensing and targeted drug delivery, based on carbon dot loaded silica nanoparticles coated with DNA-cross-linked hydrogels. As a result, highly sensitive and selective CEA detection was achieved in vitro, and an effective cytotoxic effect was realized in vivo after loading doxorubicin.


Asunto(s)
Antígeno Carcinoembrionario/análisis , ADN/química , Hidrogeles/química , Nanopartículas/química , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/uso terapéutico , Carbono/química , Doxorrubicina/química , Doxorrubicina/uso terapéutico , Portadores de Fármacos/química , Células Hep G2 , Humanos , Ratones , Ratones Desnudos , Microscopía Confocal , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Puntos Cuánticos/química , Dióxido de Silicio/química , Trasplante Heterólogo
9.
Chem Commun (Camb) ; 55(98): 14844-14847, 2019 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-31768507

RESUMEN

Ultrasmall sub-10 nm nanoparticles of Prussian blue analogues incorporating GdIII ions at their periphery revealed longitudinal relaxivities above 40 mM-1 s-1 per GdIII regardless of the nature of the core and the polymer coating. Large T1-weighted contrast enhancements were achieved in addition to a highly efficient photothermal effect and in vivo photoacoustic imaging in tumors.


Asunto(s)
Ferrocianuros/química , Imagen por Resonancia Magnética/métodos , Nanopartículas/química , Nanomedicina Teranóstica , Animales , Línea Celular Tumoral , Medios de Contraste/química , Gadolinio/química , Humanos , Ratones , Neoplasias/diagnóstico por imagen , Trasplante Heterólogo
10.
Chem Commun (Camb) ; 55(98): 14852-14855, 2019 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-31769450

RESUMEN

A self-delivery system consisting of lonidamine and a self-assembling peptide was designed for the selective killing of phosphatase-overexpressing cancer cells, which was mediated by both enhanced cellular uptake of LND-peptide and enzyme-triggered intracellular fiber formation, thereby providing a generalized strategy to develop cancer-targeting systems of drug conjugates.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Antineoplásicos/química , Indazoles/química , Péptidos/química , Fosfatasa Alcalina/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Hidrogeles/química , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Trasplante Heterólogo
11.
Chem Commun (Camb) ; 55(98): 14789-14792, 2019 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-31761922

RESUMEN

We have reported rational design of a polymeric NO delivery micelle as a cytosol-selective NO bomb. Protected NO-donors are released from the micelle under endolysosomal conditions, and then deprotected by cytosolic glutathione. Cytosol-selective NO delivery facilitates significant tumor regression without the aid of other therapeutic modalities even in intravenous administrations.


Asunto(s)
Antineoplásicos/química , Citosol/metabolismo , Micelas , Óxido Nítrico/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Glutatión/metabolismo , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Óxido Nítrico/uso terapéutico , Polímeros/química , Trasplante Heterólogo
12.
Enzymes ; 46: 11-22, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31727272

RESUMEN

In vivo experimental models are still essential for advancing our understanding of cancer and developing novel therapeutic strategies, despite rapid and remarkable developments in cellular and molecular technologies. Multiple patient-derived tumor xenograft (PDX) models, in which primary cancer tissues or cells are transplanted into immunodeficient mice, have been developed. PDX models are widely used in the field of precision cancer medicine. The purpose of this chapter is to introduce the chick embryo xenograft model, which has a longer history than the athymic nude mouse model.


Asunto(s)
Trasplante de Neoplasias , Neoplasias/patología , Animales , Embrión de Pollo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Desnudos , Trasplante Heterólogo
13.
Nat Genet ; 51(12): 1691-1701, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31740836

RESUMEN

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation.


Asunto(s)
Cadherinas/genética , Cromatina/genética , Síndrome de Down/patología , Células Madre Pluripotentes Inducidas/citología , Neuronas/fisiología , Adulto , Animales , Astrocitos/citología , Astrocitos/fisiología , Encéfalo/citología , Encéfalo/embriología , Diferenciación Celular , Línea Celular , Síndrome de Down/genética , Regulación de la Expresión Génica , Histonas/genética , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Ratones , Persona de Mediana Edad , Neuronas/citología , Regiones Promotoras Genéticas , Ratas , Análisis de la Célula Individual , Médula Espinal/citología , Médula Espinal/trasplante , Trasplante Heterólogo
14.
Invest Ophthalmol Vis Sci ; 60(14): 4904-4914, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31770435

RESUMEN

Purpose: Uveal melanoma is a common primary intraocular malignancy accompanied by high mortality. Previous evidence has highlighted the implication of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in uveal melanoma. Accordingly, we further uncovered the possible role of lncRNA plasmacytoma variant translocation 1 gene (PVT1) and microRNA-17-3p (miR-17-3p) in uveal melanoma. Methods: A series of experiments were performed to examine the relationship among lncRNA PVT1, miR-17-3p, and murine double minute clone 2 oncoprotein (MDM2). Afterward, gain- and loss-of-function approaches were used with uveal melanoma cells to verify the role of lncRNA PVT1, miR-17-3p, and MDM2 in the tumorigenesis and development of uveal melanoma. Results: Highly expressed lncRNA PVT1 and MDM2, yet lowly expressed miR-17-3p, were identified in ocular uveal melanoma tissues versus normal adjacent tissues. Then, dual luciferase reporter gene assay, RNA binding protein immunoprecipitation, and RNA pull-down assays showed that lncRNA PVT1 specifically bound to miR-17-3p, and that MDM2 was a target gene of miR-17-3p. Gain- and loss-of-function studies elucidated that silencing of lncRNA PVT1 or overexpression of miR-17-3p resulted in decreased MDM2 expression and increased transcriptional activity of p53, in addition to inhibiting uveal melanoma cell proliferation, migration, and invasion, yet promoted cell apoptosis in vitro. In addition, lncRNA PVT1 silencing or miR-17-3p overexpression was noted to inhibit tumor growth in vivo. Conclusions: Downregulation of lncRNA PVT1 could potentially promote miR-17-3p expression to suppress tumorigenesis and development of uveal melanoma by activating the p53 signaling pathway through binding to MDM2.


Asunto(s)
Silenciador del Gen/fisiología , Melanoma/prevención & control , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Largo no Codificante/genética , Neoplasias de la Úvea/prevención & control , Animales , Western Blotting , Movimiento Celular/fisiología , Supervivencia Celular , Cartilla de ADN/química , Técnica del Anticuerpo Fluorescente Indirecta , Genes Reporteros , Hibridación Fluorescente in Situ , Masculino , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Activación Transcripcional , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Neoplasias de la Úvea/metabolismo
15.
Cell Biochem Funct ; 37(8): 618-624, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31710117

RESUMEN

The aim of this study was to investigate the effect of vaccinia virus expressing IL-37 (VV-IL-37) on cell proliferation, migration and invasion of hepatocellular carcinoma (HCC) and its possible underlying molecular mechanisms. In this study, we constructed a cancer-targeted vaccinia virus carrying the IL-37 gene knocked in the region of the viral thymidine kinase (TK) gene. Human HCC cell lines were assayed in vitro for cell proliferation, migration and invasion. Serum level, relative mRNA level and protein level of IL-37 in HCC cell lines SMMC7721 and Bel7402 were tested by ELISA assay, qRT-PCR and western blot, respectively. The levels of IL-2, IFN-γ and TNF-α in HCC tumor tissues were also analyzed by ELISA. STAT3 and p-STAT3 expression in tumor tissues were determined by western blot. Our results showed that VV-IL-37 efficiently infected and inhibited HCC cells proliferation, migration and invasion via decreasing STAT3 phosphorylation. In vivo, VV-IL-37 expressed IL-37 at a high level in the transplanted tumor, reduced STAT3 activity, and eventually inhibited tumor growth. In conclusion, we demonstrate that VV-IL-37 promotes antitumor immune responses in HCC.


Asunto(s)
Carcinoma Hepatocelular/patología , Interleucina-1/metabolismo , Neoplasias Hepáticas/patología , Virus Vaccinia/fisiología , Animales , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-1/genética , Interleucina-2/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Factor de Transcripción STAT3/metabolismo , Trasplante Heterólogo , Virus Vaccinia/genética
16.
Chemotherapy ; 64(3): 119-128, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31661694

RESUMEN

OBJECTIVE: To investigate whether TRIAP1inhibition affects the ovarian cancer cell resistance to cisplatin (DDP) via the Cyt c/Apaf-1/caspase-9 pathway by in vitro and in vivo experiments. METHODS: CCK8 assay was performed to find out how treatment with both TRIAP1 siRNA and DDP affects the cell viability of SKOV3 cells and DDP-resistant human ovarian carcinoma cell line SKOV3/DDP. SKOV3/DDP cells were transfected with control siRNA or TRIAP1 siRNA before 24 h of treatment with DDP (5 µg/mL). Flow cytometry was employed to detect cell apoptosis and Western blot to examine the expressions of Cyt c/Apaf-1/caspase-9 pathway-related proteins. SKOV3/DDP cells transfected with control siRNA or TRIAP1 siRNA were subcutaneously injected into BALB/c-nu/nu nude mice followed by the intraperitoneal injection of DDP (4 mg/kg). Cyt c/Apaf-1/caspase-9 pathway in transplanted tumors was detected by immunohistochemistry. RESULTS: TRIAP1 expression declined in SKOV3 cells when compared with SKOV3/DDP cells. The proliferation rate was lower in SKOV3/DDP cells transfected with TRIAP1 siRNA combined with treatment of DDP (1, 2, 4, 6, 8, 16, 32 µg/mL) than in those transfected with control siRNA. Moreover, the TRIAP1 siRNA group had an increased SKOV3/DDP cell apoptosis rate with the activation of the Cyt c/Apaf-1/caspase-9 pathway. During DDP treatment, nude mice in TRIAP1 siRNA group had slower growth and smaller size of transplanted tumor than those in control siRNA group, with increased expression of Cyt c, Apaf-1, and caspase-9. CONCLUSION: TRIAP1 inhibition may enhance the sensitivity of SKOV3/DDP cells to cisplatin via activation of the Cyt c/Apaf-1/caspase-9 pathway.


Asunto(s)
Cisplatino/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular Tumoral , Cisplatino/uso terapéutico , Citocromos c/genética , Citocromos c/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Trasplante Heterólogo
17.
Nat Commun ; 10(1): 4730, 2019 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-31628330

RESUMEN

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.


Asunto(s)
Sistemas CRISPR-Cas , Diferenciación Celular/genética , Proliferación Celular/genética , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Animales , Electroporación/métodos , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante Heterólogo
19.
Nat Commun ; 10(1): 4479, 2019 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-31578323

RESUMEN

Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level ß-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented ß-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and ß-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.


Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Globinas beta/genética , Anemia de Células Falciformes/genética , Animales , Antígenos CD34/metabolismo , Vectores Genéticos/genética , Humanos , Macaca mulatta , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Reproducibilidad de los Resultados , Trasplante Heterólogo , Globinas beta/metabolismo
20.
Chemistry ; 25(61): 13994-14002, 2019 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-31506999

RESUMEN

Quinone methide (QM) as a latent trapping unit has been widely explored in activity-based self-immobilizing reagents. However, further application of this strategy has been largely hampered by the limited labeling efficiency to proteins. In this study, a thorough investigation on the labeling efficiency and the structure of QM-based trapping unit is presented, from which a QM with multiple leaving groups was identified as an optimal trapping unit. An alkaline phosphatase (ALP) immobilizing reagent featured with this multiple-labeling trapping unit exhibited lower nonspecific binding and, remarkably, a significantly higher labeling efficiency over other immobilizing reagents upon enzymatic activation. The utility of this imaging reagent was further demonstrated with the in vitro and in vivo visualization of the ALP activities. Furthermore, the multiple functional trapping unit may find greater value in the other activity-based immobilizing probes.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Indolquinonas/química , Fosfatasa Alcalina/química , Animales , Colorantes Fluorescentes/química , Células HEK293 , Células HeLa , Humanos , Indolquinonas/metabolismo , Ratones , Ratones Desnudos , Microscopía Fluorescente , Imagen Óptica , Coloración y Etiquetado , Trasplante Heterólogo
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