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The aim of the present study was to establish a simple and efficient method for isolation and culture of primary rat glomerular microvascular endothelial cells in vitro. The bilateral kidneys were taken from 7-10-day old Sprague-Dawley rats, and the renal cortex was separated. Glomeruli were obtained by cutting and continuously passing 200-mesh and 300-mesh sieves. After type IV collagenase digestion for 15-20 min, renal microvascular globules were collected for inoculation and culture. The cultured cells were identified by cell morphology observation and immunocytochemical staining with factor VIII related antigen. The results showed that the renal microvascular globules were irregularly spherical, without cysts, and the capillary loop structure was clear; after 3 days of primary culture, short spindle-shaped cells crawled out around the renal microvascular globules and gradually formed cell colonies, showing an "island-like shape" distribution; 4-5 days later, the cell colonies fused with each other; 6 days later, the cells covered the bottom of the dish, showing a typical monolayer, paving stone-like, mosaic arrangement. The immunocytochemical staining of factor VIII related antigen showed that the cytoplasm was lightly stained brownish red, and factor VIII related antigen-positive rate of cells was nearly 100%. The above results suggested that this study successfully established a method combining continuous screening and collagenase digestion for culture of primary rat glomerular microvascular endothelial cells in vitro. It provides an important tool cell for studying the mechanism of the occurrence and development of diabetic nephropathy.
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Células Endoteliales , Animales , Células Cultivadas , Ratas , Ratas Sprague-DawleyRESUMEN
Hepatocellular carcinoma (HCC) is one of the most lethal and highly malignant tumors. Sonodynamic therapy (SDT) is a new cancer treatment method. One of its unique advantages lies in the treatment of deep tumors due to its excellent tissue penetration ability caused by ultrasound (US). However, most sonosensitizers suffer from weak sonodynamic activity and poor tumor-targeting ability. In addition, small interfering RNA (siRNA) is a promising anticancer drug, and the efficacy of siRNA-based gene therapy largely depends on the cell impermeability of the gene carrier. Here, we designed and synthesized a cationic polythiophene derivative (PT2) that can be used as a siRNA carrier for gene therapy. Moreover, PT2 could generate singlet oxygen (1O2) and hydroxyl radicals (O2â¢-) under US irradiation, which suggests that PT2 could be used for SDT. Our study discovered that NUDT1 promoted HCC proliferation and inhibited intracellular ROS production. Therefore, si-NUDT1 was designed and synthesized. NUDT1 silencing can inhibit the proliferation of tumor cells and increase the production of intracellular ROS to further improve the efficacy of SDT. Then, si-NUDT1 assembled with PT2 and DSPE-PEG-FA to prepare a novel tumor-targeting nanodrug (PT2-siRNA@PEG-FA) for synergic SDT and gene therapy of HCC.
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Carcinoma Hepatocelular , Cationes , Terapia Genética , Neoplasias Hepáticas , Polímeros , ARN Interferente Pequeño , Tiofenos , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Tiofenos/química , Tiofenos/uso terapéutico , Tiofenos/farmacología , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/tratamiento farmacológico , Polímeros/química , Humanos , ARN Interferente Pequeño/genética , Terapia Genética/métodos , Cationes/química , Terapia por Ultrasonido , Especies Reactivas de Oxígeno/metabolismo , Animales , Ratones , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Hep G2 , Ratones DesnudosRESUMEN
Hepatocellular carcinoma (HCC) is characterized by a high degree of malignancy and mortality. Sorafenib (SOR), a multi-kinase inhibitor, is clinically used in the treatment of HCC. However, SOR suffers from serious side effects and drug resistance. The development of novel therapeutic strategies for HCC therapy is urgently needed. Sonodynamic therapy (SDT) has unique advantages in treating deep tumors due to the merits of deep tissue penetration, low side effects, and the absence of drug resistance. Here, we developed multifunctional nanoparticles (NPs) termed SOR-TCPP@PEG-FA by assembling SOR, tetrakis (4-carboxyphenyl) porphyrin (TCPP), and folic acid (FA)-modified DSPE-PEG. The FA group enhances the tumor targeting capability of these NPs, while TCPP generates ROS under ultrasound (US) irradiation, which are toxic to tumor cells, and SOR with chemotherapeutic effects is released, thus realizing the synergistic SDT and chemotherapy of tumors.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Nanopartículas , Porfirinas , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Sorafenib , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Porfirinas/farmacología , Porfirinas/uso terapéutico , Línea Celular TumoralRESUMEN
Combining phototherapy with other treatments has significantly advanced cancer therapy. Here, we designed and fabricated calcium-enriched carbon nanoparticles (Ca-CNPs) that could effectively deplete glutathione (GSH) and release calcium ions in tumors, thereby enhancing the efficacy of photodynamic therapy (PDT) and the calcium overload effect that leads to mitochondrial dysfunction. Due to the electrostatic interaction, π-π stacking interaction, multiple hydrogen bonds, and microporous structures, indocyanine green (ICG) was loaded onto the surface of Ca-CNPs with a high loading efficiency of 44.7 wt%. The obtained Ca-CNPs@ICG can effectively improve the photostability of ICG while retaining its ability to generate singlet oxygen (1O2) and undergo photothermal conversion (Ca-CNPs@ICG vs. ICG, 45.1% vs. 39.5%). In vitro and in vivo experiments demonstrated that Ca-CNPs@ICG could be used for near-infrared fluorescence imaging-guided synergistic calcium overload, photothermal therapy, and GSH depletion-enhanced PDT. This study sheds light on the improvement of 1O2 utilization efficiency and calcium overload-induced mitochondrial membrane potential imbalance in tumor cells.
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Nanopartículas , Neoplasias , Fotoquimioterapia , Humanos , Verde de Indocianina/farmacología , Verde de Indocianina/química , Calcio , Terapia Fototérmica , Nanopartículas/química , Neoplasias/terapia , Imagen Óptica , Carbono/farmacologíaRESUMEN
Vascular disruption agent (combretastatin A-4 phosphate) and phototherapeutic (IEICO-4F) assembled nanoparticles (IFC NPs) were prepared for the first time. The IFC NPs have a high photo energy utilization efficiency of up to 96.1%, and could significantly inhibit tumor growth by photodynamic and photothermal therapy enhanced tumor vascular disruption.
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Nanopartículas , Neoplasias , Fotoquimioterapia , Humanos , Neoplasias/tratamiento farmacológico , Nanopartículas/uso terapéutico , Línea Celular TumoralRESUMEN
Objective To develop and establish a simple and repeatable method of primary culture of mouse brain microvascular endothelial cells (BMECs) of relatively high purity. Methods Isolated and cultured from the brain tissue of ICR mice aged 2-3 weeks old, the mouse BMECs were gained through physical fragmentation methods such as cutting up and passing through cell sieve, BSA density gradient centrifugation and chemical enzyme digestion. The digestion time of 1 g/L type II collagenase was strictly controlled within 15-20 minutes. The cultured cells were identified by cell morphological observation and immunocytochemical staining to detect factor VII-associated antigen. Results In 24 hours after seeding, the spindle-shaped or polygon-shaped cells began to migrate from the micro vessel segment and gradually gathered and grew in clusters; after 7 days, the fused cells showed a typical single layer and arranged in the shape of paving stone. VII-associated antigen showed positive in the cytoplasm which was brown red. The purity of microvascular endothelial cells was above 95%. Conclusion We have successfully established a simple and repeatable primary culture method for mouse BMECs of relatively high purity.
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Encéfalo , Células Endoteliales , Animales , Células Cultivadas , Ratones , Ratones Endogámicos ICRRESUMEN
OBJECTIVE: To investigate the prevalence of human papillomavirus (HPV) in archival head and neck cancer (HNC) collected from the Tong-Liao area, which is located in east Inner Mongolia, China. METHODS: The presence of HPV in 54 HNCs and 25 benign biopsies was detected and the sequence variation of the E6 gene in HPV-positive samples was analyzed to determine their lineage/sublineage classification. RESULTS: HPV was detected in only 4 out of 54 HNCs and no benign biopsies were found to be HPV-positive. After further p16INK4a immunostaining, only 3 cases of HNC were positive for both HPV and p16INK4a. Phylogenetic analysis of the isolated E6 gene shows that the HPV 16, HPV 31, and HPV 58 isolated in this study belong to lineage A. CONCLUSIONS: The prevalence of HPV in HNC from this area is very low. The lineage/sublineage classification of the 3 HPV types in HNC in this area is consistent with the previous reported data of HPV lineage distribution in cervical cancer within China.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Neoplasias de Cabeza y Cuello/virología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Adulto , Anciano , China/epidemiología , ADN Viral/genética , ADN Viral/aislamiento & purificación , Femenino , Neoplasias de Cabeza y Cuello/epidemiología , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 16/patogenicidad , Papillomavirus Humano 31/aislamiento & purificación , Papillomavirus Humano 31/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/genética , Neoplasias/patología , Neoplasias/virología , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , FilogeniaRESUMEN
INTRODUCTION: Human papillomavirus (HPV) is the main etiologic factor for cervical cancer (CC). To investigate the prevalence of HPV types in archival CC and its precursors collected form Tongliao area, which is located in the east of Inner Mongolian autonomous region, China, and compare the genotype distribution of HPV in cervical lesions between Han Chinese and Mongolian. METHODS: The infections of HPV in a total of 175 cases of formalin-fixed, paraffin-embedded samples, including 71 low-grade squamous intraepithelial lesions (LSIL), 27 high-grade squamous intraepithelial lesions (HSIL), and 77 CC were detected by the combination of consensus primers nested polymerase chain reaction (PCR) and type-specific primers nested PCR. RESULTS: Overall, HPV prevalence was 93.5% in CC, 92.6% in HSIL, and 63.4% in LSIL. Human papillomavirus 16 was the most predominant HPV type in all cervical lesions, detected in 83.1% of CC, 77.8% of HSIL, and 33.8% of LSIL. Human papillomavirus 45 was the second most predominant HPV type in CC (16.9%) and HSIL (11.1%). Human papillomavirus 33 was the second most predominant HPV type in LSIL (8.5%). Human papillomavirus 18, equal with HPV 45, was the second most common type in Mongolian CC (15.6%), whereas in Han Chinese specimens, no HPV 18 was found. CONCLUSIONS: The prevalence of HPV 45 in CC and HSIL in Tongliao area were relatively higher than other regions of China. Comparing the distribution of HPV types in Han Chinese and Mongolian, the prevalence of HPV 18 in CC from Mongolian was significantly higher than that in Han Chinese.
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Carcinoma de Células Escamosas/virología , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Lesiones Precancerosas/virología , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Adulto , Anciano , Carcinoma de Células Escamosas/etnología , Carcinoma de Células Escamosas/genética , Cuello del Útero , China/epidemiología , ADN Viral , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Infecciones por Papillomavirus/etnología , Infecciones por Papillomavirus/genética , Reacción en Cadena de la Polimerasa , Lesiones Precancerosas/etnología , Lesiones Precancerosas/genética , Neoplasias del Cuello Uterino/etnología , Neoplasias del Cuello Uterino/genética , Displasia del Cuello del Útero/etnología , Displasia del Cuello del Útero/genéticaRESUMEN
High-risk human papillomavirus (HPV) is the principal cause of various cancers including cervical cancer, anal cancer, vulvar cancer, and some head and neck cancers. In the viral life cycle, by interacting with both viral and host DNA and proteins, the HPV E2 protein plays a pivotal role in viral transcriptional regulation and DNA replication, and it is also associated with modification of various cellular processes, including host gene transcription, RNA processing, apoptosis, ubiquitination, and intracellular trafficking, to create a convenient environment for a replicative cycle of the virus and contribute to the HPV pathogenesis. Elucidating the roles of E2 protein throughout the viral life cycle will improve our understanding of the viral life cycle and pathogenesis and help us identify novel antiviral agents with therapeutic potential. This article reviews the research progress in the structure, roles, and activity of high-risk HPV E2 protein, particularly that of HPV-16.