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1.
Braz. j. biol ; 84: e250151, 2024. tab, graf
Artigo em Inglês | MEDLINE, LILACS, VETINDEX | ID: biblio-1350306

RESUMO

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.


Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.


Assuntos
Humanos , Corpo Vítreo , Proliferação de Células , Desdiferenciação Celular , Tretinoína , Células Tumorais Cultivadas , Diferenciação Celular , Divisão Celular , Linhagem Celular
2.
In Vitro Cell Dev Biol Anim ; 58(1): 14-20, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35075604

RESUMO

Extensive usage of synthetic chemical pesticides has collateral effects in harming human and animal health and the environment and promoting the development of resistance in pests. The potential of plant compounds as bio insecticides has been described as a promising field of agricultural development. The present study involved the use of Artemisia annua essential oils to evaluate their cytotoxic activities against an established cell line of lesser mulberry pyralid. Five types of hemocytes were recognized (prohaemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes) in the primary cultures maintained in Ex-Cell media with 10% fetal bovine serum (FBS). Artemisia annua essential oils produced noticeable cytotoxicity against the insect cell lines. Applied at a concentration 500 ppm, oils extracted from the vegetative or flowering stages of A. annua produced 71% and 80% cell death, respectively. Nanoemulsions of EOs from the vegetative or flowering stages of A. annua killed 67 and 60% of the cells, respectively. This study has clearly shown significant bioactivities of A. annua secondary metabolites to insect cell lines.


Assuntos
Artemisia annua , Asteraceae , Mariposas , Óleos Voláteis , Animais , Artemisia annua/química , Linhagem Celular , Hemócitos , Óleos Voláteis/química , Óleos Voláteis/farmacologia
3.
PLoS Genet ; 18(1): e1009666, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35061661

RESUMO

Dynamic and temporally specific gene regulatory changes may underlie unexplained genetic associations with complex disease. During a dynamic process such as cellular differentiation, the overall cell type composition of a tissue (or an in vitro culture) and the gene regulatory profile of each cell can both experience significant changes over time. To identify these dynamic effects in high resolution, we collected single-cell RNA-sequencing data over a differentiation time course from induced pluripotent stem cells to cardiomyocytes, sampled at 7 unique time points in 19 human cell lines. We employed a flexible approach to map dynamic eQTLs whose effects vary significantly over the course of bifurcating differentiation trajectories, including many whose effects are specific to one of these two lineages. Our study design allowed us to distinguish true dynamic eQTLs affecting a specific cell lineage from expression changes driven by potentially non-genetic differences between cell lines such as cell composition. Additionally, we used the cell type profiles learned from single-cell data to deconvolve and re-analyze data from matched bulk RNA-seq samples. Using this approach, we were able to identify a large number of novel dynamic eQTLs in single cell data while also attributing dynamic effects in bulk to a particular lineage. Overall, we found that using single cell data to uncover dynamic eQTLs can provide new insight into the gene regulatory changes that occur among heterogeneous cell types during cardiomyocyte differentiation.


Assuntos
Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Análise de Célula Única/métodos , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , RNA-Seq
4.
Iran J Allergy Asthma Immunol ; 21(2): 178-188, 2022 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-35490271

RESUMO

Up-regulation of immune checkpoint ligands is considered as one of the most important immune escape mechanisms in acute myeloid leukemia (AML). Herein, we investigate a relationship between the inhibition of PI3K/Akt/mTOR signaling pathways and the regulation of immune checkpoint ligands in AML cells. The HL-60 cell line was treated with idelalisib as PI3K inhibitor, MK-2206 as Akt inhibitor, and everolimus as mTOR inhibitor either in a single or combined format. Cell viability and apoptosis were evaluated using MTT and flow cytometry assays, respectively. The relative expression of PD-L1, galectin-9, and CD155 was determined by real-time PCR. Our findings demonstrated decreased proliferation and increased apoptosis of HL-60 cells after treatment with idelalisib, MK-2206, and everolimus. As expected, the combined treatment showed a more inhibiting effect than the single treatment. Interestingly, our results elucidated that the expression of PD-L1 and Gal-9 but not MK-2206 decreased after treatment with idelalisib and everolimus. Regarding CD155, the expression of this molecule was downregulated after treatment with everolimus, but not idelalisib and MK-2206. However, combined treatment of HL-60 cells with two or three inhibitors decreased the expression levels of PD-L1, Gal-9, and CD155 checkpoint ligands. We showed that PI3K/Akt/mTOR pathway inhibitors not only serve as cytotoxic drugs but also regulate the expression of immune checkpoint ligands and interfere with the immune evasion mechanisms of AML leukemic cells. Combinational treatment approaches to block these pathways might be a promising and novel therapeutic strategy for AML patients via interfering in immune escape mechanisms.


Assuntos
Leucemia Mieloide Aguda , Fosfatidilinositol 3-Quinases , Inibidores da Angiogênese/farmacologia , Antígeno B7-H1/genética , Linhagem Celular , Everolimo/farmacologia , Everolimo/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ligantes , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologia , Serina-Treonina Quinases TOR/uso terapêutico
5.
Biol Pharm Bull ; 45(5): 664-667, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35491171

RESUMO

Hepatic stellate cells (HSCs) play a significant role in the development of chronic liver diseases. Hepatic damage activates HSCs and results in hepatic fibrosis. The functions of activated HSCs require an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt). However, the regulatory mechanisms underlying Ca2+ signaling in activated HSCs remain largely unknown. In the present study, functional analyses of Ca2+-sensing receptors (CaSRs) were performed using activated human HSCs, LX-2. Expression analyses revealed that CaSR proteins were expressed in α-smooth muscle actin-positive LX-2 cells. Extracellular Ca2+ restoration (from 0 to 2.2 mM) increased [Ca2+]cyt in these cells. The extracellular Ca2+-induced increase in [Ca2+]cyt was reduced by the CaSR antagonists, NPS2143 and Calhex 231. Furthermore, the growth of LX-2 cells was blocked by NPS2143 and Calhex 231 in concentration-dependent manners (IC50 = 6.0 and 9.5 µM, respectively). LX-2 cell proliferation was also attenuated by NPS2143 and Calhex 231. In conclusion, CaSRs are functionally expressed in activated HSCs and regulate Ca2+ signaling and cell proliferation. The present results provide insights into the molecular mechanisms underlying hepatic fibrosis and will contribute to the development of potential therapeutic targets.


Assuntos
Células Estreladas do Fígado , Transdução de Sinais , Linhagem Celular , Proliferação de Células , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática/patologia
6.
Dis Markers ; 2022: 7404813, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493304

RESUMO

Influenza pandemics are a global threat to human health, with existing vaccines and antiviral drugs providing limited protection. There is an urgent need for new prophylactic and treatment strategies. In this study, 12 short hairpin (sh)RNAs targeting conserved regions of influenza A virus (IAV) matrix protein (M)2, nucleocapsid protein (NP), nonstructural protein (NS), and polymerase acidic (PA) were synthesized, and their effects on IAV replication in cells were investigated using Madin-Darby canine kidney (MDCK) cells transfected with the shRNA plasmids. Additionally, mice were administered a polyethyleneimine PEI/pLKD-NP-391 complex in aerosol form and then infected with AIV, and viral particles in the mouse lung were detected. IAV production was markedly lower in MDCK cells transfected with pLKD-M-121, pLKD-M-935, pLKD-NP-391, pLKD-NP-1291, pLKD-PA-1365, and pLKD-PA-1645 plasmids than in control cells (p < 0.01). The viral load in MDCK cells was decreased by transfection of plasmids pLKD-M-121 (p < 0.05) and pLKD-M-935, pLKD-NP-391, pLKD-NP-1291, pLKD-PA-1365, and pLKD PA-1645 (p < 0.01) compared to an empty plasmid. The viral load was significantly lower in the lungs of mice transfected with pLKD-NP-391 than in the control plasmid and mock transfection groups (p < 0.01 and p < 0.05, respectively). Thus, IAV production was inhibited by shRNAs targeting matrix IAV components; moreover, inhalation of a PEI/pLKD-NP-391 complex in aerosol form suppressed IAV production in infected mice. Thus, these shRNAs can be effective for the prevention and treatment of influenza virus infection.


Assuntos
Vírus da Influenza A , Influenza Humana , Aerossóis/farmacologia , Animais , Linhagem Celular , Cães , Humanos , Vírus da Influenza A/genética , Pulmão , Camundongos , Plasmídeos , Polietilenoimina/farmacologia , RNA Interferente Pequeno/genética
7.
Front Cell Infect Microbiol ; 12: 855920, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493731

RESUMO

Genome rearrangement occurs to porcine circovirus type 2 (PCV2) during in vitro and in vivo infections, and a number of rearranged PCV2 genomes have been isolated and characterized. This study was conducted to investigate the role of the rearranged PCV2 (rPCV2) in PCV2 replication and the biological effect of rPCV2 in host cells. Two whole rPCV2 genome sequences (358 nt and 1125 nt in length) were synthesized and recombinant plasmids pBSK(+)-rPCV2 (pBSK(+)-1125 and pBSK(+)-358) were constructed. A novel virus-like agent (rPCV2-1125) was rescued by in vitro transfection of porcine kidney cell line (PK-15) and porcine alveolar macrophage 3D4/21 cells. The data indicate that rPCV2-1125 significantly enhanced PCV2 replication in vitro. Furthermore, rPCV2-1125 led to oxidative stress in host cells, as indicated by decreased intracellular glutathione (GSH) and total superoxide dismutase (SOD) activities, as well as increased malondialdehyde (MDA) levels. These results provide new insights into genome rearrangement of PCV2 and will contribute to future studies of PCV2 replication and associated mechanisms.


Assuntos
Circovirus , Animais , Linhagem Celular , Circovirus/genética , Malondialdeído , Oxirredução , Suínos , Replicação Viral
8.
STAR Protoc ; 3(2): 101288, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35496789

RESUMO

The FusX TALE Based Editor (FusXTBE) is a programmable base editing platform that can introduce specific TC-to-TT variations in the mitochondrial DNA (mtDNA). Here, we provide a protocol describing the synthesis and testing of the FusXTBE plasmids in cultured human cell lines. This tool is designed to be easily modified to work in diverse applications where editing of mitochondrial DNA is desired. For complete details on the use and execution of this protocol, please refer to Sabharwal et al. (2021) and Ma et al. (2016).


Assuntos
DNA Mitocondrial , Mitocôndrias , Linhagem Celular , DNA Mitocondrial/genética , Humanos , Mitocôndrias/genética
9.
Croat Med J ; 63(2): 187-196, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35505652

RESUMO

Vitamin D has been a focus of attention in liver cancer due to its direct and indirect antineoplastic effects. This review critically evaluates data from recently published basic and clinical studies investigating the role of vitamin D in liver cancer. Basic studies indicate that vitamin D plays an important role in liver cancer development by suppressing the activity of hepatic stellate cells and Kupffer cells. Furthermore, vitamin D has a direct anti-proliferative, anti-angiogenic, proapoptotic, and prodifferentiative effect on liver cancer cells. Recent investigation suggested several interesting mechanisms of these actions, such as inactivation of Notch signaling, p27 accumulation, and tyrosine-protein kinase Met/extracellular signal-regulated kinases inhibition. On the other hand, data from clinical observational studies, although promising, are still inconclusive. Unfortunately, studies on the effect of vitamin D supplementation were generally focused on short-term outcomes of chronic liver diseases (liver enzyme levels or elastographic finding); therefore, there are still no reliable data on the effect of vitamin D supplementation on liver cancer occurrence or survival.


Assuntos
Técnicas de Imagem por Elasticidade , Neoplasias Hepáticas , Linhagem Celular , Humanos , Transdução de Sinais , Vitamina D/farmacologia , Vitamina D/uso terapêutico
10.
Front Immunol ; 13: 849752, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493529

RESUMO

The innate immune system can limit the growth of invading pathogens by depleting micronutrients at a cellular and tissue level. However, it is not known whether nutrient depletion mechanisms discriminate between living pathogens (which require nutrients) and pathogen-associated molecular patterns (PAMPs) (which do not). We stimulated SHK-1 cells with different PAMPs (outer membrane vesicles of Piscirickettsia salmonis "OMVs", protein extract of P. salmonis "TP" and lipopolysaccharides of P. salmonis "LPS") isolated from P. salmonis and evaluated transcriptional changes in nutritional immunity associated genes. Our experimental treatments were: Control (SHK-1 stimulated with bacterial culture medium), OMVs (SHK-1 stimulated with 1µg of outer membrane vesicles), TP (SHK-1 stimulated with 1µg of total protein extract) and LPS (SHK-1 stimulated with 1µg of lipopolysaccharides). Cells were sampled at 15-, 30-, 60- and 120-minutes post-stimulation. We detected increased transcription of zip8, zip14, irp1, irp2 and tfr1 in all three experimental conditions and increased transcription of dmt1 in cells stimulated with OMVs and TP, but not LPS. Additionally, we observed generally increased transcription of ireg-1, il-6, hamp, irp1, ft-h and ft-m in all three experimental conditions, but we also detected decreased transcription of these markers in cells stimulated with TP and LPS at specific time points. Our results demonstrate that SHK-1 cells stimulated with P. salmonis PAMPs increase transcription of markers involved in the transport, uptake, storage and regulation of micronutrients such as iron, manganese and zinc.


Assuntos
Padrões Moleculares Associados a Patógenos , Salmão , Animais , Linhagem Celular , Lipopolissacarídeos/farmacologia , Macrófagos , Micronutrientes , Piscirickettsia
11.
J Vis Exp ; (182)2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35499335

RESUMO

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.


Assuntos
Aminoácidos , Desenvolvimento Ósseo , Linhagem Celular , Osteoblastos , Biossíntese de Proteínas
12.
Cancer Res ; 82(9): 1689-1691, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35502546

RESUMO

Among the relatively few established human prostate cancer cell lines, LNCaP cells are unique in their ability to model key stages of prostate cancer progression. Analyses of LNCaP cells and their derivatives have been invaluable for elucidating important translational aspects of prostate tumorigenesis, metastasis, and drug response, particularly in the context of androgen receptor signaling. Here, we present major highlights from a wealth of literature that has exploited LNCaP cells and their derivatives to inform on prostate cancer progression and androgen response for improving the treatment of patients with prostate cancer. See related article by Horoszewicz and colleagues, Cancer Res 1983;43:1809-18.


Assuntos
Próstata , Neoplasias da Próstata , Androgênios , Linhagem Celular , Transformação Celular Neoplásica , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/patologia
13.
PLoS One ; 17(5): e0267855, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35511922

RESUMO

Most of the AML patients in remission develop multidrug resistance after the first-line therapy and relapse. AML stem cells have gained attention for their chemoresistance potentials. Chemoresistance is a multifactorial process resulting from altered survival signaling pathways and apoptosis regulators such as MAPK, NF-κB activation and ROS production. We targeted the survival pathway p38 MAPK, NF-κB and ROS generation in human chemoresistant AML stem cell line KG1a, susceptible to enhance cell sensitivity to the chemotherapy drug 5-Fluorouridine, compared to the chemosensitive AML cell line HL60. After confirming the phenotypic characterization of KG1a and HL60 cells using flow cytometry and transcriptomic array analyses, cell treatment with the NF-κB inhibitor IKKVII resulted in a complete induction of apoptosis, and a few p38 MAPK inhibitor SB202190-treated cells underwent apoptosis. No change in the apoptosis status was observed in the ROS scavenger N-acetylcysteine-treated cells. The p38 MAPK pathway blockade enhanced the KG1a cell sensitivity to 5-Fluorouridine, which was associated with the upregulation of microribonucleic acid-(miR-)328-3p, as determined by the microarray-based miRNA transcriptomic analysis. The downregulation of the miR-210-5p in SB202190-treated KG1a cells exposed to FUrd was monitored using RT-qPCR. The miR-328-3p is known for the enhancement of cancer cell chemosensitivity and apoptosis induction, and the downregulation of miR-210-5p is found in AML patients in complete remission. In conclusion, we highlighted the key role of the p38 MAPK survival pathway in the chemoresistance capacity of the AML stem cells and potentially involved miRNAs, which may pave the way for the development of a new therapeutic strategy targeting survival signaling proteins and reduce the rate of AML relapse.


Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Apoptose , Linhagem Celular , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio , Recidiva , Células-Tronco/metabolismo , Uridina/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
J Nutr Sci Vitaminol (Tokyo) ; 68(2): 79-86, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35491208

RESUMO

Skeletal muscle plays crucial roles in locomotion, protein reservoir, and maintenance of metabolic homeostasis. Loss of muscle, known as muscle atrophy, causes the metabolic diseases such as type 2 diabetes mellitus, hypertension, and so on. Therefore, great efforts have been devoted to prevent the muscle atrophy. Policosanols are a mixture of long chain fatty alcohols extracted from various natural sources. They have long been used as functional foods to lower the level of serum lipids, including triacylglycerol and cholesterol, and to protect against inflammatory stress. In this study, we examine the protective effect and molecular mechanism of Cuban policosanol on skeletal muscle cell death and mitochondrial dysfunction using lipopolysaccharide-treated C2C12 cells. Our results demonstrated that policosanol significantly rescued cell survival (40% vs. 88%; LPS vs. LPS+policosanol) via activation of the Akt pathway, resulting in inhibition of apoptosis (p<0.05). Moreover, policosanol restored the LPS-induced repression of collagen by two fold (0.33±0.04 vs. 0.67±0.03 compared to that of control; LPS vs. LPS+policosanol) via activation of ERK-mTOR-p70S6K pathways. In addition, policosanol increased the mitochondrial fusion by regulating the activities of DRP1 and Mfn2, leading to ameliorate the mitochondrial dysfunction induced by LPS. Improved mitochondria function increased the oxygen consumption rate with glucose as fuel source, indicating that policosanol could shift the glucose metabolism from lactate fermentation, induced by lipopolysaccharide, to oxidative phosphorylation. Thus, policosanol is a promising agent for preventing the inflammation-induced muscle cell death and mitochondrial dysfunction.


Assuntos
Diabetes Mellitus Tipo 2 , Lipopolissacarídeos , Animais , Apoptose , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Álcoois Graxos/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Atrofia Muscular/metabolismo , Mioblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
15.
Mol Cells ; 45(5): 284-290, 2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35534190

RESUMO

Process of manufacturing therapeutics exosome development for commercialization. The development of exosome treatment starts at the bench, and in order to be commercialized, it goes through the manufacturing, characterization, and formulation stages, production under Good Manufacturing Practice (GMP) conditions for clinical use, and close consultation with regulatory authorities. Exosome, a type of nanoparticles also known as small extracellular vesicles are gaining attention as novel therapeutics for various diseases because of their ability to deliver genetic or bioactive molecules to recipient cells. Although many pharmaceutical companies are gradually developing exosome therapeutics, numerous hurdles remain regarding manufacture of clinical-grade exosomes for therapeutic use. In this mini-review, we will discuss the manufacturing challenges of therapeutic exosomes, including cell line development, upstream cell culture, and downstream purification process. In addition, developing proper formulations for exosome storage and, establishing good manufacturing practice facility for producing therapeutic exosomes remains as challenges for developing clinicalgrade exosomes. However, owing to the lack of consensus regarding the guidelines for manufacturing therapeutic exosomes, close communication between regulators and companies is required for the successful development of exosome therapeutics. This review shares the challenges and perspectives regarding the manufacture and quality control of clinical grade exosomes.


Assuntos
Exossomos , Vesículas Extracelulares , Técnicas de Cultura de Células , Linhagem Celular , Sistemas de Liberação de Medicamentos , Exossomos/metabolismo
16.
STAR Protoc ; 3(2): 101347, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35509972

RESUMO

Adjacent membrane receptors can show different cellular responses to ligand stimulation. Here, we describe a super-resolution microscopy imaging protocol for tracking the dynamics of two different membrane-bound receptors in single cells. We describe the transfection protocol by electroporation. We detail the imaging procedure for receptors in a single cell. We then outline the data analysis pipeline. We have applied this protocol to imaging of endocytosis of the LOX-1 and AT1 in CHO-K1 cells, but the protocol can be applied to a variety of membrane receptors in other cell lines. For complete details on the use and execution of this protocol, please refer to Takahashi et al. (2021).


Assuntos
Endocitose , Microscopia , Linhagem Celular , Microscopia/métodos
17.
Eur Rev Med Pharmacol Sci ; 26(8): 2712-2720, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35503616

RESUMO

OBJECTIVE: To investigate the protective effect and mechanism of ALDH2 on PC12 cells and brain nerve tissue injury under hypoxia. MATERIALS AND METHODS: The hypoxia model of PC12 cells with low ALDH2 expression was established and screened. The eukaryotic expression vector of wild type pEGFP-N1-ALDH2 and blank plasmid pEGFP-N1 were constructed and transfected into PC12 hypoxia cells respectively. After reoxygenation culture, the morphology, quantity, ALDH2 expression level and apoptosis rate of the two groups were observed, and the role of ALDH2 in cell hypoxia injury was analyzed. Eighty SD rats were randomly divided into model group (ischemia-reperfusion injury group), Alda-1 group (intraperitoneal injection of alda-1 12 hours before and after modeling), DMSO group (intraperitoneal injection of dimethyl sulfoxide) and sham operation group, with 20 rats in each group. The neurobehavioral score, apoptosis rate of nerve cells, the content and activity of ALDH2 in active cerebral cortex and hippocampal CA1 area were compared. RESULTS: The number of PC12 cells in hypoxia group was lower than that in control group. The expression level of ALDH2 protein in PC12 cells after 4 hours of hypoxia was lower than that in normal culture group. The number of PC12 cells transfected with wild-type recombinant plasmid was significantly more than that of blank plasmid group. Compared with the hypoxia group, the pre apoptotic and post apoptotic cells in wild type transfection group decreased after hypoxia treatment. Compared with sham operation group, nerve injury and apoptosis were increased in group M and DMSO, while ALDH2 activity and expression did not change significantly. Compared with M group and DMSO group, the nerve injury and apoptosis in Alda-1 group were improved, ALDH2 activity was increased, and ALDH2 expression was not significantly changed in Alda-1 group. CONCLUSIONS: Increasing the expression of ALDH2 or enhancing the activity of ALDH2 can improve the injury of neurons induced by hypoxia/reoxygenation.


Assuntos
Aldeído-Desidrogenase Mitocondrial , Encéfalo/metabolismo , Hipóxia/metabolismo , Neurônios/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Animais , Apoptose , Linhagem Celular , Dimetil Sulfóxido/farmacologia , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley
18.
Am J Vet Res ; 83(6)2022 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-35544415

RESUMO

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.


Assuntos
Carcinoma de Células Escamosas , Doenças do Gato , Neoplasias Bucais , Sarcoma , Animais , Sulfato de Cálcio , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/veterinária , Doenças do Gato/tratamento farmacológico , Gatos , Linhagem Celular , Hidrogéis , Neoplasias Bucais/veterinária , Sarcoma/tratamento farmacológico , Sarcoma/veterinária
19.
Am J Vet Res ; 83(6)2022 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-35544417

RESUMO

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.


Assuntos
Carcinoma de Células Escamosas , Doenças do Gato , Neoplasias Bucais , Sarcoma , Animais , Sulfato de Cálcio , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/veterinária , Doenças do Gato/tratamento farmacológico , Gatos , Linhagem Celular , Hidrogéis , Neoplasias Bucais/veterinária , Sarcoma/tratamento farmacológico , Sarcoma/veterinária
20.
STAR Protoc ; 3(1): 101165, 2022 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-35535161

RESUMO

DNA damage caused by genetic instability or extrinsic treatment can induce DNA leakage from the nucleus or mitochondria into the cytosol and activate innate and adaptive immunity. To enable characterization of these endogenous cytosolic DNAs and the mechanisms that produce them, we developed an approach for isolation of cytosolic DNA with no detectable mitochondrial contamination. Here we describe cytosolic compartment separation followed by DNA purification from colorectal cancer cells and illustrate how this may be expanded to other cell types.


Assuntos
DNA , Mitocôndrias , Linhagem Celular , Células Cultivadas , Citosol/metabolismo , DNA/genética , Mitocôndrias/genética
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