Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38
Filtrar
1.
Protein Expr Purif ; 217: 106445, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38342386

RESUMO

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Cricetinae , Animais , Edição de Genes/métodos , Cricetulus , Células CHO , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , DNA Ribossômico , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo
2.
Mol Biol Rep ; 50(7): 6019-6027, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37286776

RESUMO

BACKGROUND: Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms. METHODS AND RESULTS: In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively. CONCLUSIONS: This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.


Assuntos
Anticorpos Monoclonais , Sirtuínas , Cricetinae , Animais , Cricetulus , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/genética , Sobrevivência Celular/genética , Células CHO , Apoptose/genética , Imunoglobulina G , Sirtuínas/genética , Proteínas Recombinantes/genética
3.
Cell Biochem Funct ; 41(4): 434-449, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37017290

RESUMO

Despite significant advancements in tissue engineering and regenerative medicine during the last two decades, the fabrication of proper scaffolds with appropriate cells can still be considered a critical achievement in this field. Hypoxia is a major stumbling block to chronic wound healing, which restrains tissue engineering plans because a lack of oxygen may cause cell death. This study evaluated the cocultured human keratinocytes and human adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatin/PU. The scaffold was characterized using Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) methods. Flow cytometry confirmed mesenchymal stem cells, and then the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and DAPI staining were used to assess the in vitro biocompatibility of the scaffold. The experimental results showed that the multilayer electrospun scaffold containing 2.5% SPC could efficiently produce oxygen. Furthermore, according to cell viability results, this structure makes a suitable substrate for the coculture of keratinocytes and AMSCs. Gene expression analysis of various markers such as Involucrin, Cytokeratin 10, and Cytokeratin 14 after 14 days confirmed that keratinocytes and AMSCs coculture on PU/PCL.SPC-gelatin/PU electrospun scaffold promotes dermal differentiation and epithelial proliferation compared to keratinocytes single-cell culture. Therefore, our study supports using oxygen-releasing scaffolds as a potential strategy to hasten skin tissue regeneration. Based on the results, this structure is suggested as a promising candidate for cell-based skin tissue engineering. Given that the developed oxygen-generating polymeric electrospun scaffolds could be used as part of a future strategy for skin tissue engineering, the PU/PCL.SPC-gelatin/PU hybrid electrospun multilayer scaffold in combination with keratinocyte/AMSC coculture is proposed as an effective substrate for skin tissue engineering and regenerative medicine platforms.


Assuntos
Células-Tronco Mesenquimais , Alicerces Teciduais , Masculino , Humanos , Técnicas de Cocultura , Alicerces Teciduais/química , Gelatina/metabolismo , Prepúcio do Pênis , Oxigênio/farmacologia , Oxigênio/metabolismo , Queratinócitos/metabolismo , Células-Tronco Mesenquimais/metabolismo
4.
Prep Biochem Biotechnol ; 53(3): 239-246, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-35579623

RESUMO

Several monoclonal antibodies targeting the CD20 have been produced and Ofatumumab is a case in point. Although whole antibodies target cancer cells effectively, their applications are restricted in some ways. Single-chain fragment variable antibodies, rather than employing the entire structure of antibodies, have proven a practical approach for creating completely functional antigen-binding fragments. In current research, the DNA coding sequence of VL and VH of the wild and mutant forms of ofatumumab were joined with a flexible linker (GGGGS)3 separately. Using the E. coli BL21 (DE3) expression system, the VL-linker-VH genes were cloned into the pET-28 a (+), and the associated recombinant proteins were produced. Purified and refolded scFvs (scFv-C and scFv-V3) represented a concentration of around 0.7 mg/ml from 1 L of initial E. coli culture with a molecular weight of about 27 kDa. Affinity measurement disclosed anti-CD20 scFv-V3 possesses a higher affinity constant compared to anti-CD20 scFv-C. The recombinant scFvs exclusively attach to Raji cells but not to Jurkat cells, according to a cell-ELISA analysis. The MTT test signified anti-CD20 scFvs could affect cell viability in Raji cells but had no impact on Jurkat cells and also, Raji cells viability was affected more significantly by anti-CD20 scFv-V3.


Assuntos
Antígenos CD20 , Anticorpos de Cadeia Única , Humanos , Antígenos CD20/genética , Antígenos CD20/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos de Cadeia Única/genética
5.
Drug Metab Rev ; 54(4): 386-400, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36031813

RESUMO

Anti-angiogenic therapy is a practical approach to managing diseases with increased angiogenesis, such as cancer, maculopathies, and retinopathies. Considering the fundamental gaps in the knowledge of the vital pathways involved in angiogenesis and its inhibition and the insufficient efficiency of existing angiogenesis inhibitors, there is an increasing focus on the emergence of new therapeutic strategies aimed at inhibiting pathological angiogenesis. Angiogenesis is forming a new vascular network from existing vessels; endothelial cells (ECs), vascular lining cells, are the main actors of angiogenesis in physiological or pathological conditions. Switching from a quiescent state to a highly migratory and proliferative state during new vessel formation called "angiogenic switch" is driven by a "metabolic switch" in ECs, angiogenic growth factors, and other signals. As the characteristics of ECs change by altering the surrounding environment, they appear to have a different metabolism in a tumor microenvironment (TME). Therefore, pathological angiogenesis can be inhibited by targeting metabolic pathways. In the current review, we aim to discuss the EC metabolic pathways under normal and TME conditions to verify the suitability of targeting them with novel therapies.


Assuntos
Células Endoteliais , Neoplasias , Humanos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias/metabolismo , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Inibidores da Angiogênese/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Microambiente Tumoral
6.
Mol Biol Rep ; 49(5): 3461-3468, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35076847

RESUMO

BACKGROUND: The increasing need for therapeutic monoclonal antibodies (mAbs) entails the development of innovative and improved expression strategies. Chromatin insulators have been utilized for the enhancement of the heterologous proteins in mammalian cells. METHODS AND RESULTS: In the current study the Ccnb1ip1 gene insulator element was utilized to construct a novel vector system for the expression of an anti-CD52 mAb in Chinese hamster ovary (CHO) cells. The insulator containing (pIns-mAb) and control (pmAb) vectors were generated and stable cell pools were established using these constructs. The expression level in the cells created with pIns-mAb vector was calculated to be 233 ng/mL, and the expression rate in the control vector was 210 ng/mL, which indicated a 10.9% increase in mAb expression in pIns-mAb pool. In addition, analysis of mAb expression in clonal cells established from each pool showed a 10% increase in antibody productivity in the highest mAb producing clone derived from the pIns-mAb pool compared to the clone isolated from pmAb pool. CONCLUSIONS: More studies are needed to fully elucidate the effects of Ccnb1ip1 gene insulator on recombinant therapeutic protein expression in mammalian cells. The combination of this element with other chromatin-modifying elements might improve its augmentation effect which could pave the way for efficient and cost-effective production of therapeutic drugs.


Assuntos
Anticorpos Monoclonais , Cromatina , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Mol Biol Rep ; 48(5): 4405-4412, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34089466

RESUMO

Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors.


Assuntos
Anticorpos Monoclonais/imunologia , DNA Satélite/genética , Vetores Genéticos , Imunoglobulina G/imunologia , Pró-Proteína Convertase 9/imunologia , Animais , Células CHO , Cricetulus , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Plasmídeos , Transfecção
8.
Dermatol Ther ; 34(4): e15028, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34145697

RESUMO

Dermal fibroblasts are the most accessible cells in the skin that have gained significant attention in cell therapy. Applying dermal fibroblasts' regenerative capacity can introduce new patterns to develop cell-based therapies to treat skin disorders. Dermal fibroblasts originate from mesenchymal cells and are located within the dermis. These cells are mainly responsible for synthesizing glycosaminoglycans, collagens, and components of extracellular matrix supporting skin's structural integrity. Preclinical studies suggested that allogeneic and autologous dermal fibroblasts provide widespread and beneficial applications for wound healing, burn ulcers, and inherited skin disorders. In this regard, generating induced pluripotent stem cells (iPSCs) from fibroblasts and gene-edited fibroblasts are promising approaches for treating skin disorders. Here, we aimed to review literature about ongoing and completed clinical trials that applied fibroblasts and bioengineered fibroblasts as therapeutic agents for various skin disorders. This review explores cell therapy protocols from the earliest phase of allogeneic and autologous fibroblasts development in different benches to translating them into bedside-level treatment for skin disorders, particularly recessive dystrophic epidermolysis bullosa.


Assuntos
Epidermólise Bolhosa Distrófica , Dermatopatias , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Fibroblastos , Humanos , Pele , Cicatrização
9.
J Cell Biochem ; 120(1): 940-950, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30160776

RESUMO

CD20 molecule, a phosphoprotein with 297 amino acids and four transmembrane domains, is a member of MS4A protein family. Anti-CD20 antibodies such as ofatumumab, which have been developed for cancer treatment and has demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia, are among the most successful therapies to date. Rational engineering methods can be applied with reasonable success to improve functional characteristics of antibodies. Considering the importance of this issue, we have used in silico modeling approach for the improvement of ofatumumab monoclonal antibody. Four mutated variants of ofatumumab were developed and expressed in Chinese hamster ovary (CHO) cells along with the unmodified antibody. Analysis of affinity of the purified antibodies with CD20 showed significant improvement in antigen-binding characteristics of one of the variants compared with the control antibody. This study represents the first step toward development of the second generation ofatumumab antibody with improved affinity.


Assuntos
Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Afinidade de Anticorpos/imunologia , Antígenos CD20/imunologia , Desenho de Fármacos , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD20/química , Linfócitos B/imunologia , Células CHO , Cricetulus , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Cinética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Mutantes , Mutação , Plasmídeos/genética , Plasmídeos/imunologia , Ressonância de Plasmônio de Superfície , Transfecção
10.
Prep Biochem Biotechnol ; 49(8): 822-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156045

RESUMO

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.


Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Dosagem de Genes , Humanos , Proteínas Recombinantes/genética , Transdução Genética
11.
Curr Microbiol ; 75(1): 107-115, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29189942

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) is a microbial adaptive immune system. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which has revolutionized the genome editing approaches through multiple distinct features such as using T-rich protospacer-adjacent motif, applying a short guide RNA lacking trans-activating crRNA, introducing a staggered double-strand break, and possessing RNA processing activity in addition to DNA nuclease activity. In the present review, we attempt to highlight most recent advances in CRISPR-Cpf1 (CRISPR-Cas12a) system in particular, considering ground expeditions of the nature and the biology of this system, introducing novel Cpf1 variants that have broadened the versatility and feasibility of CRISPR-Cpf1 system, and lastly the great impact of the CRISPR-Cpf1 system on the manipulation of the genome of prokaryotic, mammalian, and plant models is summarized. With regard to recent developments in utilizing the CRISPR-Cpf1 system in genome editing of various organisms, it can be concluded with confidence that this system is a reliable molecular toolbox of genome editing approaches.


Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Edição de Genes , Bactérias/enzimologia , Bactérias/metabolismo , Proteínas de Bactérias/genética , Endonucleases/genética
12.
Prep Biochem Biotechnol ; 48(2): 160-164, 2018 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-29313429

RESUMO

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.


Assuntos
Anticorpos Monoclonais/genética , Vetores Genéticos/genética , Imunoglobulina G/genética , Animais , Células CHO , Clonagem Molecular/métodos , Cricetulus , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética
13.
Appl Microbiol Biotechnol ; 101(11): 4371-4385, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28497204

RESUMO

Photobacterium species are Gram-negative coccobacilli which are distributed in marine habitats worldwide. Some species are unique because of their capability to produce luminescence. Taxonomically, about 23 species and 2 subspecies are validated to date. Genomes from a few Photobacterium spp. have been sequenced and studied. They are considered a special group of bacteria because some species are capable of producing essential polyunsaturated fatty acids, antibacterial compounds, lipases, esterases and asparaginases. They are also used as biosensors in food and environmental monitoring and detectors of drown victim, as well as an important symbiont.


Assuntos
Proteínas de Bactérias/genética , Photobacterium/genética , Photobacterium/fisiologia , Técnicas Biossensoriais , DNA Bacteriano , Ecossistema , Genoma Bacteriano , Luminescência , Photobacterium/crescimento & desenvolvimento , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA , Simbiose
14.
World J Microbiol Biotechnol ; 33(1): 4, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27837408

RESUMO

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system.


Assuntos
Células Eucarióticas/metabolismo , Glicoproteínas/metabolismo , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Animais , Glicosilação , Humanos , Leishmania/metabolismo , Proteínas Recombinantes/metabolismo
15.
Malays J Med Sci ; 23(2): 6-13, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27547109

RESUMO

BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s). METHODS: CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells. RESULTS: Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level. CONCLUSION: The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.

16.
Curr Stem Cell Res Ther ; 19(3): 351-366, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37073662

RESUMO

Exosomes are extracellular vesicles (EVs) that originate from endocytic membranes. The transfer of biomolecules and biological compounds such as enzymes, proteins, RNA, lipids, and cellular waste disposal through exosomes plays an essential function in cell-cell communication and regulation of pathological and physiological processes in skin disease. The skin is one of the vital organs that makes up about 8% of the total body mass. This organ consists of three layers, epidermis, dermis, and hypodermis that cover the outer surface of the body. Heterogeneity and endogeneity of exosomes is an advantage that distinguishes them from nanoparticles and liposomes and leads to their widespread usage in the remedy of dermal diseases. The biocompatible nature of these extracellular vesicles has attracted the attention of many health researchers. In this review article, we will first discuss the biogenesis of exosomes, their contents, separation methods, and the advantages and disadvantages of exosomes. Then we will highlight recent developments related to the therapeutic applications of exosomes in the treatment of common skin disorders like atopic dermatitis, alopecia, epidermolysis bullosa, keloid, melanoma, psoriasis, and systemic sclerosis.


Assuntos
Exossomos , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Exossomos/metabolismo , Pele , Comunicação Celular , RNA
17.
Iran Biomed J ; 27(5): 269-79, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37873643

RESUMO

Background: CD20 is a differentiation-related antigen exclusively expressed on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders, making it an ideal target for immunotherapy of hematological malignancies and autoimmune diseases. MAb-based therapies targeting CD20 have a principal role in the treatment of several immune-related disordes and cancers, including CLL. Fc gamma receptors mediate CD20 internalization in hematopoietic cells; therefore, this study aimed to establish non-hematopoietic stable cell lines overexpressing full-length human CD20 antigen as an in vitro model for CD20-related studies. Methods: CD20 gene was cloned into the transfer vector. The lentivirus system was transfected to packaging HEK 293T cells, and the supernatants were harvested. CHO-K1 cells were transduced using recombinant viruses, and a stable cell pool was developed by the antibiotic selection. CD20 expression was confirmed at the mRNA and protein levels. Results: Simultaneous expression of GFP protein facilitated the detection of CD20-expressing cells. Immunophenotyping analysis of stable clones demonstrated expression of CD20 antigen. In addition, the mean fluorescence intensity was significantly higher in the CD20-CHO-K1 clones than the wild-type CHO-K1 cells. Conclusion: This study is the first report on using second-generation lentiviral vectors for the establishment of a non-hematopoietic cell-based system, which stably expresses full-length human CD20 antigen. Results of stable CHO cell lines with different levels of CD20 antigen are well suited to be used for CD20-based investigations, including binding and functional assays.


Assuntos
Antígenos CD20 , Vetores Genéticos , Cricetinae , Animais , Humanos , Antígenos CD20/genética , Células CHO , Cricetulus , Vetores Genéticos/genética
18.
Biomed Pharmacother ; 167: 115505, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37716113

RESUMO

Mesenchymal stem cells (MSCs), as self-renewing multipotent stromal cells, have been considered promising agents for cancer treatment. A large number of studies have demonstrated the valuable properties of MSC-based treatment, such as low immunogenicity and intrinsic tumor-trophic migratory properties. To enhance the potency of MSCs for therapeutic purposes, equipping MSCs with targeted delivery functions using genetic engineering is highly beneficial. Genetically engineered MSCs can express tumor suppressor agents such as pro-apoptotic, anti-proliferative, anti-angiogenic factors and act as ideal delivery vehicles. MSCs can also be loaded with nanoparticle drugs for increased efficacy and externally moderated targeting. Moreover, exosomes secreted by MSCs have important physiological properties, so they can contribute to intercellular communication and transfer cargo into targeted tumor cells. The precise role of genetically modified MSCs in tumor environments is still up for debate, but the beginning of clinical trials has been confirmed by promising results from preclinical investigations of MSC-based gene therapy for a wide range of malignancies. This review highlights the advanced techniques of engineering/nano-engineering and MSC-derived exosomes in tumor-targeted therapy.

19.
Monoclon Antib Immunodiagn Immunother ; 42(4): 140-144, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37624609

RESUMO

During the past decades, tremendous advances have occurred in manufacturing recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. Nevertheless, the production of stable high-producing cell lines has remained a major obstacle in the development process of the CHO cell line. It has been shown that genomic regulatory elements can promote cell line development efficiency by improving transgenes' productivity and stability. Such elements include insulators, ubiquitous chromatin opening elements, scaffold/matrix attachment regions, and antirepressors. In addition, tDNA elements are shown to act as insulators in mammalian cells. This study examines the effect of the tDNA insulator on stable expression of a vascular endothelial growth factor receptor-Fc fusion protein.


Assuntos
Elementos Isolantes , Fator A de Crescimento do Endotélio Vascular , Animais , Cricetinae , Células CHO , Cricetulus , Anticorpos Monoclonais , Receptores de Fatores de Crescimento do Endotélio Vascular
20.
Bioimpacts ; 12(3): 219-231, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35677672

RESUMO

Introduction: Migration of fibroblast cells in wound areas is a critical aspect of the wound healing process. Employment of enhanced green fluorescent protein (EGFP) labeled fibroblast cells facilitates real-time monitoring and functional evaluation of these cells in both in vitro and in vivo settings. Plasma rich in growth factor (PRGF) is a potent accelerator of wound healing; therefore, in this study, a novel method to fabricate an electrospun bioactive scaffold containing PRGF was employed to induce in vitro cell proliferation and migration. Methods: First, the EGFP reporter gene was integrated into the AAVS1 locus of fibroblast cells using CRISPR/Cas9 system. Then, PRGF was obtained from platelet-rich plasma, and a multi-layered scaffold was fabricated using polyurethane-cellulose acetate (PU-CA) fibers as the outer layers and PRGF-containing gelatin fibers were located in the internal layer like a central strip. Scanning electron microscopy (SEM), tensile, water contact angle, and FTIR tests were performed to assess the characteristics of the scaffolds. The EGFP targeted cells were cultured on scaffolds with or without PRGF to investigate their viability, toxicity, and migration pattern in response to the release profile. Results: Fluorescence images showed that the number of migrating cells on scaffold containing PRGF was more significant than PU-CA scaffold up to day 6. Increased expression of SGPL1, DDR2, and VEGF genes was also observed on the scaffold containing PRGF compared to PU-CA using real-time polymerase chain reaction (PCR) analysis with around 3-, 2-, and 2-fold enhancement, respectively. Conclusion: The current scaffold provides the appropriate template for cell attachment and migration. In addition, the present results highlight the potential of reporter gene targeting for the in vitro analysis of biological processes such as migration.

SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa