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OBJECTIVE: To explore the application value of the second-generation metagenomic next-generation sequencing (mNGS) in the detection of pathogens in patients with pulmonary infection. METHODS: We conducted a retrospective analysis of 65 pulmonary infection cases treated at our institution and the Fifth People's Hospital of Shanghai between January 2021 and May 2023. All subjects were subjected to mNGS, targeted next-generation sequencing (tNGS), and conventional microbiological culture. A comparative analysis was performed to evaluate the diversity and quantity of pathogens identified by these methodologies and to appraise their respective diagnostic capabilities in pulmonary infection diagnostics. RESULTS: The mNGS successfully identified etiological agents in 60 of the 65 cases, compared to tNGS, which yielded positive results in 42 cases, and conventional laboratory cultures, which detected pathogens in 24 cases. At the bacterial genus level, mNGS discerned 9 genera, 11 species, and 92 isolates of pathogenic bacteria, whereas tNGS identified 8 genera, 8 species, and 71 isolates. Conventional methods were less sensitive, detecting only 6 genera, 7 species, and 33 isolates. In terms of fungal detection, mNGS identified 4 fungal species, tNGS detected 4 isolates of the Candida genus, and conventional methods identified 2 isolates of the same genus. Viral detection at the species level revealed 10 species and 46 isolates by mNGS, whereas tNGS detected only 3 species and 7 isolates. The area under the receiver operating characteristic curve (AUC) with 95% confidence intervals for diagnosing pulmonary infections was 0.818 (0.671 to 0.966) for mNGS, 0.668 (0.475 to 0.860) for tNGS, and 0.721 (0.545 to 0.897) for conventional culture.The mNGS demonstrates superior diagnostic efficacy and pathogen detection breadth in critically ill patients with respiratory infections, offering a significant advantage by reducing the time to diagnosis. The enhanced sensitivity and comprehensive pathogen profiling of mNGS underscore its potential as a leading diagnostic tool in clinical microbiology.
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Introduction: The study was designed to explore how cinobufagin (CB) regulates the development of non-small cell lung cancer (NSCLC) cells through lipid rafts. Material and methods: The effects of CB at gradient concentrations (0, 0.5, 1 and 2 µM) on NSCLC cell viability, apoptosis, reactive oxygen species (ROS) level, phosphorylation of Akt, and apoptosis- and lipid raft-related protein expression were assessed by MTT assay, flow cytometry and Western blot. Cholesterol and sphingomyelin were labeled with BODIPY to evaluate the effect of CB (2 µM) on them. Sucrose density gradient centrifugation was used to extract lipid rafts. The effect of CB on the expression and distribution of caveolin-1 was determined by immunofluorescence, quantitative reverse transcription polymerase chain reaction and Western blot. After overexpression of caveolin-1, the above experiments were performed again to observe whether the regulatory effect of CB was reversed. Results: CB inhibited NSCLC cell viability while promoting apoptosis and ROS level. CB redistributed the lipid content on the membrane surface and reduced the content of caveolin-1 in the cell membrane. In addition, CB repressed the activation of AKT. However, caveolin-1 overexpression reversed the effects of CB on apoptosis, AKT activation and lipid raft. Conclusions: CB regulates the activity of Akt in lipid rafts by inhibiting caveolin-1 expression to promote NSCLC cell apoptosis.
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In vertebroplasty and kyphoplasty, bioinert poly(methyl methacrylate) (PMMA) bone cement is a conventional filler employed for quick stabilization of osteoporotic vertebral compression fractures (OVCFs). However, because of the poor osteointegration, excessive stiffness, and high curing temperature of PMMA, the implant loosens, the adjacent vertebrae refracture, and thermal necrosis of the surrounding tissue occurs frequently. This investigation addressed these issues by incorporating the small intestinal submucosa (SIS) into PMMA (SIS-PMMA). In vitro analyses revealed that this new SIS-PMMA bone cement had improved porous structure, as well as reduced compressive modulus and polymerization temperature compared with the original PMMA. Furthermore, the handling properties of SIS-PMMA bone cement were not significantly different from PMMA. The in vitro effect of PMMA and SIS-PMMA was investigated on MC3T3-E1 cells via the Transwell insert model to mimic the clinical condition or directly by culturing cells on the bone cement samples. The results indicated that SIS addition substantially enhanced the proliferation and osteogenic differentiation of MC3T3-E1 cells. Additionally, the bone cement's biomechanical properties were also assessed in a decalcified goat vertebrae model with a compression fracture, which indicated the SIS-PMMA had markedly increased compressive strength than PMMA. Furthermore, it was proved that the novel bone cement had good biosafety and efficacy based on the International Standards and guidelines. After 12 weeks of implantation, SIS-PMMA indicated significantly more osteointegration and new bone formation ability than PMMA. In addition, vertebral bodies with cement were also extracted for the uniaxial compression test, and it was revealed that compared with the PMMA-implanted vertebrae, the SIS-PMMA-implanted vertebrae had greatly enhanced maximum strength. Overall, these findings indicate the potential of SIS to induce efficient fixation between the modified cement surface and the host bone, thereby providing evidence that the SIS-PMMA bone cement is a promising filler for clinical vertebral augmentation.
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Fracturas por Compresión , Fracturas de la Columna Vertebral , Humanos , Cementos para Huesos/farmacología , Cementos para Huesos/química , Polimetil Metacrilato/farmacología , Polimetil Metacrilato/química , Osteogénesis , Fracturas de la Columna Vertebral/cirugía , Columna VertebralRESUMEN
Chronic granulomatous disease (CGD) is a rare X-linked recessive primary immunodeficiency disease (PID). Herein, a human induced pluripotent stem cell (iPSC) line was generated from the peripheral blood mononuclear cells (PBMCs) of a CGD patient with a mutation (c.785_786delTT) in the CYBB gene. These iPSCs showed the expression of pluripotency markers, the ability to differentiate into three germ layers. They offer a promising technique for studying the pathogenesis and conducting drug screening for CGD patients.
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Enfermedad Granulomatosa Crónica , Células Madre Pluripotentes Inducidas , Humanos , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/metabolismo , Enfermedad Granulomatosa Crónica/patología , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Diferenciación Celular , Mutación/genéticaRESUMEN
BACKGROUND: Omicron had swept the mainland China between December 2022 and January 2023, while SARS-CoV-2 still continued to evolve. To fully prepare for the next wave, it's urgent to evaluate the humoral immune response post BA.5/BF.7 breakthrough infection against predominant sub-lineages among existing vaccination strategies and the elders. METHOD: This study enrolled a longitudinal young-adult cohort from 2/3-dose vaccination to 1 month after breakthrough infection, and an elder cohort at 1 month after breakthrough infection. Seral samples were collected and tested for humoral immune response to SARS-CoV-2 subvariants including WT, BA.2, BA.5, BF.7, BQ.1.1, CH.1.1, XBB.1.5. RESULTS: BA.5/BF.7 breakthrough infection induced higher neutralization activity than solely vaccination in all SARS-CoV-2 strains, while the latest Omicron subvariants, BQ.1.1, CH.1.1, XBB.1.5, exhibited the strongest neutralization evasion ability. There was a negative correlation between age and humoral immune response in WT, BA.5, BQ.1.1, and XBB.1.5. Compared to non-vaccination groups, breakthrough infection in two-dose vaccination groups had significantly higher neutralizing antibody against WT, BA.2, BA.5, BF.7 but not to BQ.1.1, CH.1.1, XBB.1.5 while booster dose against the prototype prior-breakthrough would not further significantly enhance individual's humoral responses against the latest Omicron subvariants. CONCLUSIONS: Newer variants manifest increasing immune evasion from neutralization and repeated prototype-based booster vaccines may not further enhance neutralizing antibody against emerging new variants. Older adults have lower levels of neutralizing antibody. Future vaccination strategies should aim to enhance effective neutralization to contemporary variants.
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Infección Irruptiva , COVID-19 , Humanos , Anciano , COVID-19/prevención & control , SARS-CoV-2 , Envejecimiento , Anticuerpos NeutralizantesRESUMEN
BACH2-related immunodeficiency and autoimmunity (BRIDA) is an inborn error of immunity, newly reported in 2017, presenting with symptoms of immunoglobulin deficiency and ongoing colitis. Studies using a mouse model have demonstrated that BACH2 deficiency predisposes individuals to systemic lupus erythematosus (SLE); however, no BACH2 deficiency has been reported in SLE patients. Here we describe a patient with BRIDA presenting with early-onset SLE, juvenile dermatomyositis, and IgA deficiency. Whole exome sequencing analysis of the patient and her parents revealed a novel heterozygous point mutation in BACH2, c.G1727T, resulting in substitution of a highly conserved arginine with leucine (R576L), which is predicted to be deleterious, in the patient and her father. Reduced BACH2 expression and deficient transcriptional repression of the BACH2 target, BLIMP1, were detected in PBMCs or lymphoblastoid cell lines of our patient. Notably, extreme reduction of memory B cells was detected in the patient's father, although he had no obvious symptoms. SLE symptoms and recurrent fever were relieved by treatment with prednisone combined with tofacitinib. Thus, we present the second report of BRIDA and demonstrate that BACH2 may be a monogenic cause of SLE.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Síndromes de Inmunodeficiencia , Lupus Eritematoso Sistémico , Femenino , Humanos , Masculino , Autoinmunidad , Mutación de Línea Germinal , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genéticaRESUMEN
Background: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare disorder of the immune regulatory system caused by forkhead box P3 (FOXP3) mutations. Abnormal numbers or functions of regulatory T (Treg) cells account for the various autoimmune symptoms. We aimed to explore the molecular genetics and phenotypic spectra of patients with atypical IPEX syndrome in China. Methods: We analyzed the molecular, clinical and immune phenotype characteristics of five Chinese patients with FOXP3 mutations. Results: We summarized the molecular and phenotypic features of five patients with FOXP3 mutations, including two novel mutations. Four of the five patients displayed atypical phenotypes, and one developed immune-related peripheral neuropathy. Three of the five patients showed normal frequencies of Treg cells, but the proportions of subsets of Treg cells, CD4+ T cells and B cells were out of balance. Conclusions: Our report broadens the understanding of the clinical features of atypical IPEX syndrome. Our detailed analyses of the immunological characteristics of these patients enhance the understanding of the possible mechanisms underlying the clinical manifestations.
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Factores de Transcripción Forkhead , Poliendocrinopatías Autoinmunes , Diabetes Mellitus Tipo 1/congénito , Diabetes Mellitus Tipo 1/genética , Diarrea/etiología , Diarrea/genética , Factores de Transcripción Forkhead/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Enfermedades del Sistema Inmune/congénito , Enfermedades del Sistema Inmune/genética , Enfermedades Intestinales/congénito , Enfermedades Intestinales/genética , Fenotipo , Poliendocrinopatías Autoinmunes/congénito , Poliendocrinopatías Autoinmunes/genética , SíndromeRESUMEN
Activated phosphoinositide 3-kinase δ syndrome (APDS) is a rare autosomal dominant primary immunodeficiency disease (PID) which was caused by the acquired mutation of PIK3CD gene. In this study, we generated a human induced pluripotent stem cell (hiPSC) line CHCMUi001-A from the peripheral blood mononuclear cells (PBMCs) of a APDS patient, who has a heterozygous mutation (c.3061 G > A) in the PIK3CD gene. This iPSC line presented a normal karyotype and exhibited characteristics of pluripotent stem cells. This iPSC line can be very useful for not only studying disease mechanisms but also developing new potential clinical treatments for APDS patients.
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Células Madre Pluripotentes Inducidas , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares , Mutación , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , SíndromeRESUMEN
Cell wound healing assay is an important experimental technique for the detection of cell migration in vitro. At present, scratch on monolayer cells using a pipette tip is commonly used. However, it is difficult to guarantee the scratch with same width, and only the migration distance of a certain part is calculated. Therefore, the experimental method needs to be optimized. ATRA was used to treat hepa1-6 mouse hepatoma cells. Circular wound with diameter of 0.2 cm were formed by a circular gap forming device. The whole cell wound region could be captured under the microscope to observe cell migration. There are almost no crawling cells in the wound region. The migration capacity of hepa1-6 cells was evaluated by calculating the healing area. ATRA could significantly inhibit the migration of hepa1-6 cells. Compared with linear wound, the standard deviation of wound healing rate in the circular cell wound method is smaller. The circular cell wound method can ensure the dynamic observation of the same wound region, and calculate the healing area at the two-dimensional level with small error and high repetition rate. It is reliable and easy to operate, can be widely used in laboratory.
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Cicatrización de Heridas , Ratones , Animales , Ratones Endogámicos , Movimiento Celular , Línea CelularRESUMEN
OBJECTIVES: To investigate the associations among expression of programmed cell death ligand 1 (PD-L1), eleven mutated genes, and clinicopathological characteristics in 273 patients with non-small cell lung cancer (NSCLC). METHODS: We retrospectively examined tumor PD-L1 expression in 247 surgically resected primary and 26 advanced NSCLC patients by immunohistochemistry using SP263 antibody assay. Gene mutations of EGFR, TP53, KRAS, PIK3CA, ERBB2, MET, RET, ALK, BRAF, ROS1, and APC were examined by NGS sequence. Data analysis was carried out using SPSS 22.0. The associations among PD-L1 expression, eleven mutated genes and clinicopathological characteristics were assessed by univariate and multivariate analysis. RESULTS: Among the total 273 patients, 68 (24.9%) patients were positive for PD-L1 expression. Data showed that mutated rate of EGFR gene was the highest with 63.0% (172/273), followed by TP53 (11.7%, 32/273) and KRAS (5.5%, 15/273). The female, non-smoker, and patients with adenocarcinoma (ADC) were more likely to have EGFR mutations. Multivariate logistic regression showed that PD-L1 expression was significantly associated with Non-ADC, lymphatic invasion, EGFR wild type and TP53 mutation (p = 0.041, <0.001, 0.004 and 0.014, respectively). Moreover, PD-L1 expression in adenocarcinoma was associated with lymphatic invasion, mutation of TP53 and KRAS gene (p = 0.012, <0.025 and 0.041, respectively). CONCLUSIONS: Mutations of EGFR, KRAS and TP53 should be routinely detected in clinical practice to better guide the immunotherapy for NSCLC patients. Future investigations are warranted to illustrate the potential mechanisms between driver mutations and PD-L1 expression for guiding immunotherapy in patients with NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Adulto , Anciano , Anciano de 80 o más Años , Antígeno B7-H1/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Femenino , Humanos , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas/genética , Estudios Retrospectivos , Adulto JovenRESUMEN
Gene editing is an advanced technique based on artificial nucleases and can precisely modify genome sequences. It has shown great application prospects in the field of medicine and has provided a new precision therapy for diseases. Primary immunodeficiency disease is a group of diseases caused by single gene mutation and characterized by recurrent and refractory infections, with an extremely high mortality rate. The application of gene editing has brought hope for curing these diseases. This article reviews the development of gene editing technology and briefly introduces the research and application of gene editing technology in primary immunodeficiency disease.
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Edición Génica , Enfermedades de Inmunodeficiencia Primaria , HumanosRESUMEN
As a potential chemo-therapeutic agent, all-trans retinoic acid (ATRA) can significantly reverse epithelial-mesenchymal transition (EMT) of hepal-6 hepatocarcinoma cell line in vitro, but the mechanism is unclear. The expression profile of microRNA-200 (miR-200) families is different in hepatocellular carcinoma. In this study, we found that ATRA treatment could up-regulate the expression of miR-200a-3p, 200c-3p, and 141-3p, which were involved in ATRA regulated proliferation and apoptosis of hepal-6 cell, but not colony formation. Meanwhile, miR-200a-3p, 200c-3p, and 141-3p could recovery ATRA inhibited migration and invasion abilities of hepal-6 cells at various levels. miR-200a-3p and 200c-3p prevented ATRA from inducing the differentiation and hepatic functions of hepal-6 cells. Antagomir specific for miR-200a-3p and 200c-3p down-regulated the expression of CK18, but only miR-200a-3p antagomir played prominent role in regulating the expression of these mesenchymal markers, N-Cadherin, Snail and Twist. The transcriptional activities of 8 transcription factors were up-regulated and 35 transcription factors were down-regulated by ATRA. Compared with ATRA group, inhibition of miR-200a-3p, 200c-3p, and 141-3p significantly strengthened the expression of Fra1/Jun (AP1), Ets1/PEA3, Brn3, and Zeb1/AREB6 at varying degrees. Therefore, this result suggested that ATRA may suppress EMT through down-regulating miR-200a-3p, 200c-3p and 141-3p related transcription factors. miR-200 and their downstream genes might be the potentially specific targets for the treatment of hepatocarcinoma.
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Carcinoma Hepatocelular , Hepatitis B Crónica , Neoplasias Hepáticas , Antivirales/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/epidemiología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/epidemiología , Humanos , Incidencia , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/epidemiología , Nucleósidos/uso terapéutico , Nucleótidos/uso terapéuticoRESUMEN
Hepatic stem cell transplantation has been demonstrated as an effective alternative therapy for the end-stage liver failure patients. Therefore, the functional detection of hepatic stem cell is essentially required. The present study confirmed that adenovirus BMP9 (Ad-BMP9) could increase the ALB-Gluc activity of HP14-19 hepatic progenitor cells, the expression of specific hepatic markers ALB, TAT, UGT1A were up-regulated while the hepatic stem cell markers DLK, AFP were down-regulated, and the number of positive Periodic acid-Schiff (PAS) stained cells were significantly higher than those in control group. However, the indocyanine green (ICG) uptake failed to be detectable in induced hepatocytes, which was inconsistent. By using another cell line LC14d, we found out that positive ICG uptake cells were located in the area of low cell density, while positive PAS stained cells were mainly concentrated in the area where cells were overlapped, indicating that different cell confluence might affect the outcomes of ICG uptake and PAS staining. A manual wound healing of Ad-BMP9 induced HP14-19 cells was made, the crawling cells were stained positive for ICG but not for PAS. Therefore, our finding may provide evidence for better application of PAS staining and ICG uptake assay in functional detection of mature hepatocytes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00453-8.
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[This corrects the article on p. 5131 in vol. 12, PMID: 33042410.].
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Our previous studies reported that urine-derived stem cells (USCs) possess a strong self-renewal ability and multidirectional differentiation potential and thus are an ideal candidate cell source for hepatocellular transplantation. USC transplantation may repair the pathological changes of chronic liver injury to a certain extent, and hypoxia pretreatment may improve the recovery efficiency of USCs. Therefore, the present study aimed to investigate the possible mechanism of the improved recovery efficiency of hypoxia-pretreated USCs. A chronic liver injury model was established by intraperitoneal injection of carbon tetrachloride into nude mice. USCs were transplanted via caudal vein injection. Hematoxylin and eosin staining and Masson's staining were performed to determine the pathology of the liver. Immunofluorescence and frozen section biopsy were performed to determine differentiation and cell fusion in vivo. Cell coculture was used to detect cell fusion in vitro. The proliferative ability of USCs was evaluated using cell viability and colony formation assays, and the migratory functions of USCs were evaluated using wound healing and transwell assays. The degeneration of hepatocytes and the level of fibrosis in the hypoxia transplantation group were improved compared with the normoxia transplantation group. It was found that exogenous USCs may be differentiated into functional hepatocytes or fused with hepatocytes in vivo. C-X-C motif chemokine (CXC) ligand 12 (CXCL12) expression levels in liver tissue of the chronic liver injury model were upregulated compared with those in the control group. The expression of CXC receptor 4 (CXCR4) in hypoxia-pretreated USCs was also significantly upregulated. The results suggested that USCs fused with different types of liver cells and that hypoxia treatment promoted the fusion rate in vitro by upregulating CXCR4 signaling. Furthermore, hypoxia pretreatment promoted cell proliferation, migration, and cell fusion by inducing CXCR4 signaling, leading to USC-elicited liver tissue recovery following injury in vivo.
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Movimiento Celular/fisiología , Proliferación Celular/fisiología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/terapia , Receptores CXCR4/metabolismo , Trasplante de Células Madre/métodos , Células Madre/citología , Acondicionamiento Pretrasplante/métodos , Orina/citología , Animales , Hipoxia de la Célula , Línea Celular , Células Cultivadas , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Células Hep G2 , Humanos , Masculino , Ratones Desnudos , Interferencia de ARN , Receptores CXCR4/genética , Regulación hacia ArribaRESUMEN
Hepatocellular carcinoma is the fourth leading cause of cancer-related deaths due to its high rate of recurrence and metastasis. All-trans-retinoic acid (ATRA) can inhibit the malignant behaviors of hepatocarcinoma cells. Autophagy is reportedly involved in the migration and metastasis of various cancer cells. This study aimed to investigate the effect of autophagy on the function of ATRA on hepatocarcinoma cells, and to explore its possible underlying mechanism. Hepatocarcinoma cell lines, Hepa1-6 and HepG2, were treated with ATRA and autophagy inhibitors, including 3-methyladenine (3-MA) and Bafilomycin (Baf). Transmission electron microscopy, laser scanning, western blot, and real-time PCR demonstrated that ATRA induces autophagy in hepatocarcinoma cells. Trypan blue staining, a wound healing assay, and a transwell assay showed that 3-MA and Baf reverses the inhibitory functions of ATRA on the proliferation, migration, and invasion of hepatocarcinoma cells. Flow cytometry, Hoechst staining, periodic acid-Schiff staining, and indocyanine green uptake validated that 3-MA and Baf reverses the function of ATRA on apoptosis and the differentiation of hepatocarcinoma cells. Real-time PCR, western blot, and an immunofluorescence assay demonstrated that the reversal of the epithelial-mesenchymal transition (EMT) process by ATRA is weakened when autophagy is inhibited. Additionally, we confirmed that Bcl-2 is associated with the induction of ATRA-induced autophagy instead of the PI3K/Akt/mTOR pathway. These findings suggest that ATRA induces autophagy and autophagic cell death through the Bcl-2/Beclin1 pathway. Furthermore, ATRA-induced autophagy is involved in the inhibitory effect of ATRA on the malignant behaviors of hepatocarcinoma cells by reversing the EMT process.
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Urine-derived stem cells (USCs) are autologous stem cells that exhibit self-renewal ability and multi-lineage differentiation potential. These characteristics make USCs an ideal cell source for hepatocellular transplantation. Here, we investigated the biological characteristics of USCs and their potential use for the treatment of chronic liver injury. We characterized the cell-surface marker profile of USCs by flow cytometry and determined the osteogenic, adipogenic, and hepatic differentiation capacities of USCs using histology. We established a chronic liver-injury model by intraperitoneally injecting carbon tetrachloride into nude mice. USCs were then transplanted via tail vein injection. To determine liver function and histopathology following chronic liver injury, we calculated the liver index, measured serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and performed histological staining. USCs were small, adherent cells expressing mesenchymal but not hematopoietic stem-cell markers. Some induced USCs underwent osteogenic and adipogenic differentiation. When co-cultured with hepatic progenitor cells, about 10% of USCs underwent hepatic differentiation. The ALT and AST levels of the USC-transplanted group were lower than that of the chronic liver-injury model group, and there were no significant differences between the two USC-transplanted groups. However, hepatocyte degeneration and liver fibrosis substantially improved in the hypoxia-pretreated USC-transplanted group compared with the normoxia USC-transplanted group. Taken together, USCs display desirable proliferation and differentiation characteristics, and USC transplantation partially improves abnormal liver function and pathology associated with chronic liver injury. Furthermore, hypoxia pretreatment promotes cell proliferation, migration, and colony formation by inducing autophagy, leading to USC-elicited liver tissue recovery following injury in vivo.
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Hepatic progenitor cells (HPCs) are potential seed cells for hepatocyte transplantation treatment of liver diseases. ATRA can induce the differentiation and mature function of hepatic progenitor cells, but the mechanism is still poorly understood. Here, by using microRNA array to analyze the expression profiles of microRNA (miR), we found that miR-200 family molecules in HPCs were upregulated after ATRA treatment, especially miR-200a-3p, 200c-3p, and 141-3p. ATRA induction could downregulate the expression of hepatic stem markers Oct4 and AFP, and improve the expression of hepatic markers ALB, CK18, and TAT, and the activity of ALB-GLuc, as well as indocyanine green uptake and glycogen storage function of HPCs. These above effects of ATRA on HPC differentiation were almost inhibited by blocking of miR-200a-3p, but not miR-200c-3p and 141-3p using antagomir. Cell autophagy is associated with ATRA regulation in HPCs, compared with control group, the expression of LC3 and Beclin1 increased in ATRA-treated HPCs, and orange and red fluorescent spot, which represents autophagy flow, also enhanced after ATRA treatment. However, ATRA-induced cell autophagy level was inhibited in antagomir-200a-3p+ATRA-treated cells. Therefore, the present study indicates that antagomir-200a-3p is related to ATRA-induced hepatic differentiation of HPCs through regulating cell autophagy, supporting the possible use of ATRA as a key inducer in HPC-based therapy of liver diseases.
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Hepatocitos/efectos de los fármacos , Hepatopatías/genética , MicroARNs/genética , Tretinoina/farmacología , Animales , Antagomirs/genética , Autofagia/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Hepatopatías/metabolismo , Ratones , Células Madre/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Tretinoina/metabolismoRESUMEN
A large number of functional hepatocytes is required for bioartificial liver therapy. Simian virus 40 Tantigen (SV40T) has been previously reported to improve the immortalized proliferation of primary hepatocytes to generate a sufficient number of cells; however, these longterm immortalized hepatocytes may induce further malignant transformation in vivo. In the present study, the SV40T immortalization gene and two suicide genes, herpes simplex virus thymidine kinase (HSVtk) and cytosine deaminase (CD), were transducted into primary hepatocytes to construct a novel type of Cre/LoxPmediated reversible immortalized hepatocyte line. Polymerase chain reaction analysis and western blotting confirmed that the SV40T, HSVtk and CD genes were successfully inserted into hepatic progenitor cells and their expression was controlled by Cre/LoxP recombination. Total removal of SV40T could be achieved via the ganciclovir (GCV)/HSVtk suicide system. Cells maintained their biosafety in vivo with CD gene expression and 5fluorocytosine (5FC) induced cell death. Following transplantation into the carbon tetrachloride (CCl4) model group, the majority of cells had survived after 14 days postimplantation and a number of the cells had transported into the liver parenchyma. When compared with the CCl4 model group, the transplanted cells repaired the liver biochemical index and pathological structure markedly. Thus, the present study reports a novel reversible immortalized hepatocyte with double suicide genes, which exhibited the cellular phenotype and recovery function of normal liver cells. This method maximally guaranteed the biological safety of immortalized hepatocytes for in vivo application, providing a reliable, safe and ideal cell material for the artificial liver technique.