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BACKGROUND: Macrovascular invasion (MaVI) occurs in nearly half of hepatocellular carcinoma (HCC) patients at diagnosis or during follow-up, which causes severe disease deterioration, and limits the possibility of surgical approaches. This study aimed to investigate whether computed tomography (CT)-based radiomics analysis could help predict development of MaVI in HCC. METHODS: A cohort of 226 patients diagnosed with HCC was enrolled from 5 hospitals with complete MaVI and prognosis follow-ups. CT-based radiomics signature was built via multi-strategy machine learning methods. Afterwards, MaVI-related clinical factors and radiomics signature were integrated to construct the final prediction model (CRIM, clinical-radiomics integrated model) via random forest modeling. Cox-regression analysis was used to select independent risk factors to predict the time of MaVI development. Kaplan-Meier analysis was conducted to stratify patients according to the time of MaVI development, progression-free survival (PFS), and overall survival (OS) based on the selected risk factors. RESULTS: The radiomics signature showed significant improvement for MaVI prediction compared with conventional clinical/radiological predictors (P < 0.001). CRIM could predict MaVI with satisfactory areas under the curve (AUC) of 0.986 and 0.979 in the training (n = 154) and external validation (n = 72) datasets, respectively. CRIM presented with excellent generalization with AUC of 0.956, 1.000, and 1.000 in each external cohort that accepted disparate CT scanning protocol/manufactory. Peel9_fos_InterquartileRange [hazard ratio (HR) = 1.98; P < 0.001] was selected as the independent risk factor. The cox-regression model successfully stratified patients into the high-risk and low-risk groups regarding the time of MaVI development (P < 0.001), PFS (P < 0.001) and OS (P = 0.002). CONCLUSIONS: The CT-based quantitative radiomics analysis could enable high accuracy prediction of subsequent MaVI development in HCC with prognostic implications.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/cirugía , Humanos , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/cirugía , Pronóstico , Estudios Retrospectivos , Tomografía Computarizada por Rayos X/métodosRESUMEN
Hearing loss is the second most common nonfatal problem affecting the Chinese population. Historical studies have suggested an association between exposure to heavy metals, such as cadmium and lead, and hearing loss. Few studies have investigated this relationship in the general population in China. We conducted a case-control study with 1008 pairs of participants from a cross-sectional epidemiological survey conducted in Zhejiang Province. A self-designed questionnaire was adopted to collect information on demographics, chronic diseases, lifestyles and environmental noise. Pure-tone averages of hearing thresholds at frequencies of 0.5, 1, 2, and 4 kHz were computed. Blood lead and cadmium levels were analyzed with an atomic absorption spectrometer. After adjusting for all other potential confounding factors, compared with the lowest blood cadmium quartile (0.00-0.53 µg/L), blood cadmium quartile 2 (0.54-0.92 µg/L), quartile 3 (0.93-1.62 µg/L) and quartile 4 (1.63-57.81 µg/L) exhibited significantly elevated risks for hearing loss, with odds ratios of 1.932 (95% CI: 1.356-2.751), 2.036 (95% CI: 1.423-2.914) and 1.495 (95% CI: 1.048-2.133), respectively (P-trend<0.001). However, an association of lead with hearing loss was not found. Young age (less than 60 years), male sex and current smoking were associated with increased blood cadmium concentration. Additionally, a positive association between blood cadmium and lead concentrations was found. Therefore, we conclude that exposure to environmental cadmium may be a risk factor for hearing loss among the general population in China.
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Cadmio/toxicidad , Exposición a Riesgos Ambientales/efectos adversos , Pérdida Auditiva , Plomo/toxicidad , Encuestas y Cuestionarios , Adulto , Factores de Edad , Pueblo Asiatico , China/epidemiología , Femenino , Pérdida Auditiva/sangre , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Factores SexualesRESUMEN
BACKGROUND: Biopsy Gleason score (GS) is crucial for prostate cancer (PCa) treatment decision-making. Upgrading in GS from biopsy to radical prostatectomy (RP) puts a proportion of patients at risk of undertreatment. PURPOSE: To develop and validate a radiomics model based on multiparametric magnetic resonance imaging (mp-MRI) to predict PCa upgrading. STUDY TYPE: Retrospective, radiomics. POPULATION: A total of 166 RP-confirmed PCa patients (training cohort, n = 116; validation cohort, n = 50) were included. FIELD STRENGTH/SEQUENCE: 3.0T/T2 -weighted (T2 W), apparent diffusion coefficient (ADC), and dynamic contrast enhancement (DCE) sequences. ASSESSMENT: PI-RADSv2 score for each tumor was recorded. Radiomic features were extracted from T2 W, ADC, and DCE sequences and Mutual Information Maximization criterion was used to identify the optimal features on each sequence. Multivariate logistic regression analysis was used to develop predictive models and a radiomics nomogram and their performance was evaluated. STATISTICAL TESTS: Student's t or chi-square were used to assess the differences in clinicopathologic data between the training and validation cohorts. Receiver operating characteristic (ROC) curve analysis was performed and the area under the curve (AUC) was calculated. RESULTS: In PI-RADSv2 assessment, 67 lesions scored 5, 70 lesions scored 4, and 29 lesions scored 3. For each sequence, 4404 features were extracted and the top 20 best features were selected. The radiomics model incorporating signatures from the three sequences achieved better performance than any single sequence (AUC: radiomics model 0.868, T2 W 0.700, ADC 0.759, DCE 0.726). The combined mode incorporating radiomics signature, clinical stage, and time from biopsy to RP outperformed the clinical model and radiomics model (AUC: combined model 0.910, clinical model 0.646, radiomics model 0.868). The nomogram showed good performance (AUC 0.910) and calibration (P-values: training cohort 0.624, validation cohort 0.294). DATA CONCLUSION: Radiomics based on mp-MRI has potential to predict upgrading of PCa from biopsy to RP. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 5 J. Magn. Reson. Imaging 2020;52:1239-1248.
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Prostatectomía , Neoplasias de la Próstata , Biomarcadores , Biopsia , Humanos , Imagen por Resonancia Magnética , Masculino , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/cirugía , Estudios RetrospectivosRESUMEN
OBJECTIVE: To observe the in vivo effect of Danlou Tablet (, DLT) on myocardial ischemia and reperfusion (I/R) injury. METHODS: DLT effects were evaluated in mouse heart preparation using 30-min coronary occlusion followed by 24-h reperfusion and compared among sham group (n=6), I/R group (n=8), IPC group (ischemia preconditioning, n=6) and DLT group (I/R with DLT pretreatment for 3 days, 750 mgâ¢kg-1â¢day-1, n=8). The effects of DLT were characterized in infarction size (IS) compared with risk region (RR) and left ventricle using the Evans blue/triphenyltetrazolium chloride double dye staining method in vivo. Furthermore, the dose-dependent effect of DLT on I/R injury was evaluated by double staining method. Five different concentrations of DLT (0.625, 1.25, 2.5, 5 and 10 gâ¢kg-1â¢day-1) were chosen in this study, and dose-response curve of DLT was obtained on these data. RESULTS: The ratio of IS to left ventricle was significantly smaller in the DLT and IPC groups than the I/R group (P<0.05 or P<0.01), the ratio of IS to RR was also reduced in the DLT and IPC groups (P<0.01), while there were no differences in RR among the four groups (P>0.05). Experiments showed incidence of arrhythmias was reduced in the DLT group (P<0.01). Furthermore, DLT produced a dose-dependent inhibitory effect with a half maximal inhibitory concentration of 1.225 gâ¢kg-1â¢day-1. CONCLUSIONS: Our research concluded that DLT was effective in reducing I/R injury in mice, and provided experimental supports for the clinical use of DLT.
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Cardiotónicos/uso terapéutico , Medicamentos Herbarios Chinos/uso terapéutico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Animales , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Temperatura Corporal/efectos de los fármacos , Cardiotónicos/farmacología , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Masculino , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Factores de Riesgo , ComprimidosRESUMEN
OBJECTIVE: To evaluate the therapeutic effect of nasal coblation plasma surgery for the treatment of persistent allergic rhinitis (PAR). METHODS: One hundred patients with mite-sensitized moderate to severe PAR who underwent nasal coblation plasma surgery (inferior turbinoplasty plus nasal agger ablation) were enrolled in this study. There were 68 male and 32 female patients aged 16 to 62 years (mean, 36.3 years). The visual analogue scale (VAS) for global rhinitis symptoms, nasal provocation test (NPT), anterior rhinomanometry, and T&T olfactometry were used to assess the short-term outcomes, preoperatively and postoperatively at the end of three months after surgical procedure. SPSS19.0 software was applied for statistical analysis. RESULTS: At three months after treatment, the total nasal symptom VAS scores significantly decreased from 7.0 ± 2.0 to 2.5 ± 1.5 (X(-) ± s; t = 18.00, P = 0.0001). All patients were allergic to house dust mites with positive NPT before treatment. At three months from the coblation intervention, 88.0% of the patients changed from positive NPT to negative, while 12.0% remained as positive. There was a significant reduction in total nasal resistance, which diminished from 0.772 ± 0.224 to 0.221 ± 0.112 kPa·s·L(-1) after treatment (t = 22.00, P = 0.0001). Preoperative olfactory tests showed hyposmia in 31.0% of the patients, with 22 cases for slight and 9 cases for moderate disorder. Three months after treatment, 13.0% were diagnosed as hyposmia, with 7 cases for slight and 6 cases for moderate disorder (χ(2) = 10.44, P = 0.005). CONCLUSION: Nasal coblation plasma surgery provides favorable short-term outcomes in terms of remarkable improvement in nasal symptoms, hyperreactivity of nasal mucosa, nasal flow and olfactory function in patients with moderate to severe PAR, but long-term effect needed further observation.
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Ablación por Catéter/métodos , Rinitis Alérgica Perenne/cirugía , Adolescente , Adulto , Femenino , Humanos , Hipotermia Inducida , Masculino , Persona de Mediana Edad , Procedimientos Quírurgicos Nasales , Rinitis Alérgica Perenne/fisiopatología , Rinomanometría , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: To investigate the effect of transfusion of apoptotic and necrotic thymocytes prior to sepsis on the survival rate of mice. METHODS: BALB/c mice are divided into 3 groups and received intravenous injection of PBS (control), apoptotic thymocytes, or necrotic thymocytes. Three days later, cecal ligation and puncture (CLP) were performed to induce sepsis in these mice, and their survival and organ damage were observed. RESULTS: The survival rates of mice in PBS group was 44.6% at the end of first week after CLP, and obvious lung and kidney damages were observed. A significant increase in the survival rate was found in apoptotic cell transfusion group (69.6%, P=0.012), with also lessened lung and kidney damages. The survival rate of mice in necrotic cell transfusion group was only 31.6% at 2 weeks, significantly lower than that in PBS group (P=0.035), and the lung and kidney damage was even more obvious. CONCLUSION: Transfusion of apoptotic thymocytes 3 days before induction of sepsis can reduce organ damage and improve the survival rate of mice, while necrotic cell transfusion produces the opposite effect.
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Sepsis/mortalidad , Sepsis/terapia , Timo/citología , Animales , Apoptosis , Modelos Animales de Enfermedad , Transfusión de Linfocitos , Ratones , Ratones Endogámicos BALB C , Necrosis , Tasa de SupervivenciaRESUMEN
Genome-wide hypomethylation has been confirmed in patients with primary immune thrombocytopenia (ITP). Proteins containing methylcytosine-binding domain (MBD) are involved in promoter methylation as transcriptional repressors and promote the gene-silencing effect of DNA methylation. The purpose of this study was to investigate the methylation pattern of T cells and the relationship between genomic methylation and the expression of MBD2 and MBD4 in ITP patients. DNA deoxymethylcytosine content of CD4(+) cells from peripheral blood mononuclear cells was measured by enzyme-linked immunoassay. Real-time polymerase chain reaction was performed to quantify the transcription levels of MBD2 and MBD4 in peripheral blood mononuclear cells and CD4(+) cells. DNA dmC content in CD4(+) cells of ITP patients was significantly lower than in the controls (p = 0.001). The mRNA level of MBD2 and MBD4 in CD4(+) cells of ITP patients was statistically lower than those of the controls (p < 0.001). Positive correlations between methylation indexes and expression of each enzyme were observed in the control group (r(2) = 0.718, p = 0.004 for MBD2; r(2) = 0.608, p = 0.015 for MBD4). However, inverse correlations were found in ITP patients (r(2) = 0.604, p = 0.008 for MBD2; r(2) = 0.498, p = 0.027 for MBD4). Our results indicate that decreased expression of MBD2 and MBD4 might involve in the pathogenesis of ITP.
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Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Púrpura Trombocitopénica Idiopática/genética , ARN/análisis , Enfermedad Aguda , Adulto , Linfocitos T CD4-Positivos/patología , Enfermedad Crónica , Metilación de ADN , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Epigénesis Genética , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Púrpura Trombocitopénica Idiopática/fisiopatologíaRESUMEN
Mesenchymal stem cells (MSCs), which are poorly immunogenic and have potent immunosuppressive activities, have emerged as a promising candidate for cellular therapeutics for the treatment of disorders caused by abnormal immune responses. In this study we investigated whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) could ameliorate colitis in a trinitrobenzene sulfonic acid (TNBS)-induced colitis model. TNBS-treated colitic mice were infused with hUC-MSCs or vehicle control. The mice were sacrificed on day 1, 3, and 5 after infusion, and their clinical and pathological conditions were evaluated by body weight, colon length, and histological analysis. The expression levels of proinflammatory cytokine proteins in colon were examined by ELISA. The homing of hUC-MSCs was studied by live in vivo imaging and immunofluorescent microscopy. hUC-MSCs were found to migrate to the inflamed colon and effectively treated the colitic mice with improved clinical and pathological signs. The levels of IL-17 and IL-23 as well as IFN-γ and IL-6 were significantly lower in the colon tissues of the hUC-MSC-treated mice in comparison with the vehicle-treated mice. Coculture experiments showed that hUC-MSCs not only could inhibit IFN-γ expression but also significantly inhibit IL-17 production by lamina propria mononuclear cells (LPMCs) or splenocytes of the colitic mice or by those isolated from normal animals and stimulated with IL-23. Systemically infused hUC-MSCs could home to the inflamed colon and effectively ameliorate colitis. In addition to the known suppressive effects on Th1-type immune responses, hUC-MSC-mediated modulation of IL-23/IL-17 regulated inflammatory reactions also plays an important role in the amelioration of colitis.
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Colitis/patología , Colitis/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Animales , Movimiento Celular , Proliferación Celular , Separación Celular , Supervivencia Celular , Técnicas de Cocultivo , Colon/patología , Citocinas/biosíntesis , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Membrana Mucosa/patología , Bazo/metabolismo , Bazo/patología , Ácido TrinitrobencenosulfónicoAsunto(s)
Quiste Broncogénico , Enfermedades de la Laringe , Humanos , Masculino , Persona de Mediana EdadRESUMEN
This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration. By a series of DNA manipulation, a hybrid system of two adenovirus vectors was constructed. One vector contains loxP-flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recolonization. The other vector carries Cre and phiC31 gene. Vectors only expressing Cre or phiC31 were used as controls. 293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000, and the expression of target genes was identified by fluorescence microscopy and RT-PCR. The results showed that after being identified by PCR, restriction analysis and sequencing, an adeno-integrase hybrid system was successfully constructed. The system expressed RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro. It is concluded that the adeno-integrase hybrid system is successfully constructed, which lays a good foundation for further investigation of its therapeutic application.
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Vectores Genéticos , Hemofilia B/genética , Integrasas/genética , Adenoviridae/genética , Línea Celular , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Hemofilia B/terapia , Humanos , TransfecciónRESUMEN
OBJECTIVE: To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase. METHODS: The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. RESULTS: The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. CONCLUSION: Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.
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Factor IX , Hemofilia B , Animales , Factor IX/genética , Expresión Génica , Terapia Genética , Vectores Genéticos , Genómica , Hemofilia B/terapia , Humanos , Hidrodinámica , RatonesRESUMEN
OBJECTIVE: To compare the responses to sepsis between C57BL/6 and BALB/c mice. METHODS: Thirty C57BL/6 mice and 30 BALB/c mice were randomized into sham-operated group and sepsis group (n=15). Sepsis model was established by cecal ligation puncture (CLP) in the mice, and 6 h after the operation, 5 mice from each group were selected randomly for cytokine detection including IL-1beta, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN-gamma and TNF-alpha by Bio-plex. The other 10 mice in each group were used for survival analysis. RESULTS: The survival rates of BALB/c and C57BL/6 mice were both 100% in one week after the sham operation, but lowered to 10% and 50% in one week after CLP, respectively. The survival rate of C57BL/6 mice was significantly lower than that of BALB/c mice (P<0.05). After CLP, C57BL/6 mice showed significantly greater IL-4, TNF-alpha and IL-10 production than the sham-operated mice, but the concentrations of the 8 cytokines in BALB/c mice after CLP showed no significant increment. CONCLUSION: Compared with BALB/c mice, C57BL/6 strain mouse is more sensitive to sepsis.
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Citocinas/sangre , Modelos Animales de Enfermedad , Sepsis/sangre , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Distribución Aleatoria , Especificidad de la EspecieRESUMEN
In order to evaluate whether mesenchymal stem cells (MSCs) from non-hematopoietic tissues are able to regulate megakaryocytopoiesis, we identified human MSCs from adult bone marrow (ABM), fetal pancreas (FPan) and umbilical cord (UC), and their abilities to support megakaryocyte (MK) differentiation from CD34(+) hematopoietic progenitor cells (HPCs) were comparatively studied. First, MSCs were isolated from ABM, FPan and UC then their growth kinetics, molecular characterization and mesodermal differentiation capacity were determined. ABM-MSCs, FPan-MSCs and UC-MSCs were irradiated and cocultured with human umbilical cord blood (UCB) CD34(+) cells, and the expansion efficiency of MK progenitor cells and MK formation were analysed and compared. Finally, SCF, IL-6 and GM-CSF expression by the three types of MSCs were also examined. Our results showed that FPan-MSCs and UC-MSCs shared most of the characteristic of ABM-MSCs, including morphology, immunophenotype, adipogenic and osteogenic differentiation potentials. Compared with ABM-MSCs, fetal MSCs had higher proliferative capacity. After 7 days' coculture, the maximal production of CD34(+)/CD41a(+) cells was obtained in a group of CD34(+) HPCs + ABM-MSCs. Furthermore, this group produced more MK colonies than other groups (p < 0.05). Surface antigen and ploidy analysis morphological observation demonstrated that a proportion of expanded cells in each group differentiated into mature MKs. ABM-MSCs, FPan-MSCs and UC-MSCs were revealed to express SCF, IL-6 and GM-CSF at mRNA level. We conclude that FPan-MSCs and UC-MSCs have the ability to promote megakaryocytopoiesis, while ABM-MSCs expand more MK progenitor cells from CD34(+) HPCs than MSCs from non-hematopoietic tissues and CD34(+) cells alone.
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Células de la Médula Ósea/citología , Feto/citología , Células Progenitoras de Megacariocitos/citología , Células Madre Mesenquimatosas/citología , Páncreas/citología , Cordón Umbilical/citología , Adulto , Diferenciación Celular , Citometría de Flujo , Humanos , Páncreas/embriología , Valores de ReferenciaRESUMEN
Combined deficiency of factor V and VIII (F5F8D) is a rare, autosomal recessive disorder caused by mutations of either lman1 or mcfd2. To identify mutations of these two genes in a Chinese F5F8D family, the samples of peripheral blood were collected from the proband and her parents. Coagulation tests were carried out, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg) and coagulate activity of FV, FVIII (FV:C, FVIII:C). The genomic DNA was extracted, then all the exons and intron/exon boundaries of these two genes were amplified by polymerase chain reaction (PCR). The products were finally analyzed by direct sequencing. The results showed that the proband's APTT, PT, TT, Fg, FV:C and FVIII:C were 82.2 sec, 19.6 sec, 18.6 sec, 2.9 g/L, 7.1% and 18.7% respectively, while those parameters of the parents were all within the normal range. Two pathogenic mutations were identified in lman1 gene of the proband: one was the heterozygous c.912_913insA in exon 8 resulting in a frameshift of p.Glu305fsX20; the other was the heterozygous c.1366C > T in exon 11 resulting in p.Arg456X. The proband's father and mother were heterozygous for c.1366C > T and c.912_913insA respectively. It is concluded that F5F8D of the proband is caused by a novel compound heterozygous mutation of the lman1 gene, which has never been reported.
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Deficiencia del Factor V/genética , Hemofilia A/genética , Lectinas de Unión a Manosa/genética , Proteínas de la Membrana/genética , Mutación , Niño , Exones , Factor V/genética , Deficiencia del Factor V/etiología , Factor VIII/genética , Femenino , Hemofilia A/etiología , Heterocigoto , Humanos , LinajeRESUMEN
The present study was aimed to isolate and identify human mesenchymal stem cells from adult bone marrow (BM-MSC) and umbilical cord (UC-MSC), and to compare their ability to support in vitro long-term hematopoiesis. MSC from bone marrow and umbilical cord were isolated by using density gradient centrifugation or enzyme digestion. MSC were further purified by adherent culture. Immunophenotype, adipogenic and osteogenic differentiation potential of BM-MSC and UC-MSC were detected. The hematopoietic supporting capacity of BM-MSC and UC-MSC was assessed by LTC-IC assay. Nonadherent cells in each group were collected for phenotypic analysis at 3, 5 and 7th week of culture. The results showed that BM-MSC and UC-MSC in culture shared a similar spindle-shaped morphology and adhered to the tissue culture substrate. They were both positive for CD90, CD105, CD73, CD29, CD54, CD166, HLA-ABC, and negative for HLA-DR, CD34 and CD45. BM-MSC and UC-MSC could differentiate into adipocytes or osteoblasts confirmed by oil red O staining and von Kossa staining, separately. LTC-IC assay showed that at 5th week of culture, the difference of the CFC yields between UC-MSC group and BM-MSC group was not statistically significant (p>0.05). At 6, 7, 9th week of culture, the CFC yields in the UC-MSC group were lower than those of BM-MSC (p<0.05). The phenotypic analysis of nonadherent cells at 3, 5, 7th week of culture indicated that along with prolongation of time, the percentages of CD34+ cells and CD117+ cells in each group decreased markedly, and the percentages of CD33+ cells, CD13+ cells and CD11b+ cells increased gradually. It is concluded that MSC from human adult bone marrow and umbilical cord can be successfully isolated and identified. UC-MSC are able to support long-term hematopoiesis in vitro, but its hematopoietic supportive capacity is weaker than those of BM-MSC.
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Células de la Médula Ósea/citología , Hematopoyesis , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Adulto , Técnicas de Cultivo de Célula , Separación Celular , Células Cultivadas , Sistema Hematopoyético , HumanosRESUMEN
Inherited afibrinogenemia is a rare autosomal recessive bleeding disease characterized by complete absence of fibrinogen in blood. To identify the genotype in a Chinese family with inherited afibrinogenemia, the samples of peripheral blood were collected from 6 members of 3 generations. The activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (Fg, clauss) were tested. Fg was also analyzed by using immunoturbidimetry method. DNAs of six members were extracted by using a DNA extract kit. All the exons and exon-intron boundaries of the three fibrinogen genes were amplified by using PCR and analyzed by direct sequencing. The results showed that the parents of proband were 3 degree consanguinity. A homozygous c.934_935insA in FGA was found in proband which results in the change of protein p.Ser312fsX42. The parents, grandmother, maternal grandmother and father's sister were all detected with heterozygous mutation which was same as that in proband. In conclusion homozygous c.934_935insA in FGA is a cause of inherited afibrinogenemia and a novel mutation being reported.
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Afibrinogenemia/genética , Fibrinógeno/genética , Mutación del Sistema de Lectura , Heterocigoto , Afibrinogenemia/etiología , Niño , Exones , Femenino , Humanos , Masculino , LinajeRESUMEN
This study was aimed to design and screen short hairpin RNA (shRNA) molecules targeting multidrug resistance gene (mdr1), as well as to investigate the effects of shRNA expression vector on K562/A02 cells. Mdr1-shRNA expression vector was transfected into K562/A02 cells by lipofectamine 2000, and G418 was added to screen and establish the stable expression cell strain. The expressions of mdr1 mRNA and protein were detected by real-time RT-PCR and Western blot respectively. The sensitivity of cells to chemodrugs after interference were tested by CCK8 assay. The function of p-glycoprotein was determined by Rhodamine 123 efflux experiment. The results showed that all of 4 mdr1-shRNA expression vectors could significantly knockdown the expression of p-glycoprotein as compared with control vector, moreover, the vector targeting 508 - 526 sites of mdr1 gene was the best one. It is concluded that the mdr1-shRNA expression vector gained by screening can significantly knockdown the expression of mdr1 gene and reverse leukemia drug resistance, paving the way for the application of RNAi in the following animal experiments.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , ARN Interferente Pequeño/genética , Subfamilia B de Transportador de Casetes de Unión a ATP , Secuencia de Bases , Técnicas de Silenciamiento del Gen , Genes MDR , Vectores Genéticos , Humanos , Células K562 , Leucemia/genética , Interferencia de ARN , ARN Mensajero/genética , TransfecciónRESUMEN
The present study was purposed to evaluate the safety of mesenchymal stem cell (MSC)-based therapy impacting on atherosclerosis. Allogeneic MSCs were obtained from rabbit bone marrow aspirates and expanded in vitro. New Zealand white rabbits were divided into three groups: 24 rabbits with hypercholesterolemia receiving intravenous injection of either 5 x 10(7) MSCs (n = 12) or saline (n = 12) after 5 weeks on a high lipid diet and additional rabbits (n = 6) fed with standard rabbit diet were served as controls. Body weight and blood lipids were measured at weeks 0, 5, 9 and 13 during the study. All rabbits were sacrificed at week 13. Atherosclerotic lesion size and vasa vasorum were evaluated by using pathological analysis and immunocytochemical technique. The results showed that the aortic sinus lesion size significantly increased in rabbits infused with MSCs as compared with controls receiving saline (23.35 +/- 3.51% and 11.39 +/- 3.08% respectively). The lesion size in whole aortas of MSC-treated rabbits was 76.64 +/- 12.70% versus 57.61 +/- 9.00% in saline-treated animals (p < 0.05). Moreover, vasa vasorum networks in MSC-treated aortas were more numerous and had increased capillary density. It is concluded that the allogeneic MSC transfusion may result in an increase in atherosclerotic lesion size. In cell therapy with MSCs or cell populations containing MSCs a strategy to attenuate the high potential of MSCs involved in atherogenesis of atherosclerosis should be taken in account.
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Trasplante de Células Madre Mesenquimatosas/efectos adversos , Placa Aterosclerótica/etiología , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , ConejosRESUMEN
The family of T-cell immunoglobulin- and mucin-domain-containing molecules (TIMs) has an important role in immune regulation. TIM-3 is a transmembrane protein preferentially expressed on terminally differentiated Th1 cells and plays a role in T-helper (Th)-1-mediated autoimmune disease. Idiopathic thrombocytopenic purpura (ITP) is an acquired organ-specific autoimmune disease with a polarization of Th1. The purpose of this study was to investigate whether the -1516G>T, -574T>G, 4259G>T single-nucleotide polymorphisms within the TIM-3 gene contribute to the genetic susceptibility to ITP. Genotyping of TIM-3 -1516G>T, -574T>G, and 4259G>T was performed in 187 patients with ITP and 123 healthy individuals by polymerase chain reaction-restriction fragment length polymorphism assay. No significant differences existed in genotype and allele distributions between the patients with ITP and the controls in all three sites. There was strong linkage disequilibrium (LD; r(2) = 0.633) between -574T>G and 4259G>T, whereas -1516G>T was not in LD with -574T>G (r(2) = 0.007) or with 4259G>T (r(2) = 0.002). The -1516G>T, -574T>G, and 4259G>T of TIM-3 gene polymorphisms might not play an important role as a genetic risk factor in the pathophysiology of ITP.