Objective To investigate the effect of human hepatocellular carcinoma HepG2 cell-derived Exosome on the differentiation of mesenchymal stem cells(MSC)into cancer-associated myofibroblasts(CAF)and the impacts of CAF on liver cancer cell proliferation,migration,and invasion. Methods The protein expression of HepG2 cell-derived Exosome was detected by Western blotting. MSCs were separated from human adipose tissue and cultured with HepG2 cell-derived Exosome(100 ng/nl)to initiate differentiation. The expressions of mesenchymal markers and several interleukins were also detected by Western blotting. HepG2 cells were co-cultured with the conditioned media(CM),in which HepG2 Exosome induced the differentiation of MSC into CAF. The expressions of epithelial and mesenchymal markers were detected by real-time polymerase chain reaction(PCR)and Western blotting. Cell proliferation was assessed using MTS assay. Transwell chambers were used in the in vitro migration and invasion assay. Results HepG2 cell-derived particles expressed CD63,70 kilodalton heat shock proteins,and 90 kilodalton heat shock proteins. With the treatment of HepG2 cell-derived Exosome,the expressions of mesenchymal marker α-smooth muscle actin,fibroblast activation protein α,interleukin(IL)-6,IL-8,and IL-1β were up-regulated,while vascular endothelial growth factor had no significant change. The conditioned media which HepG2 Exosome induced MSC differentiation CAF(CAF-CM)could significantly promote HepG2 cells proliferation(1.075±0.104),compared to BSA control(0.874±0.066,P=0.023)and MSC-CM(0.649±0.034,P=0.0005). CAF-CM could significantly enhance cell migration [(42.5±9.1) cells vs.(18.5±3.1) cells,P=0.001] and invasion [(29.0±3.5) cells vs.(13.1±3.7) cells,P=0.009] compared to its control group. Moreover the conditioned medium which HepG2 Exosome induced MSC to differentiate into CAF could also promote the expressions of mesenchyme-related genes Smad interacting protein 1(P=0.040),β-catenin(P=0.038),fibronectin(P=0.029),and Vimentin(P=0.013)and inhibit the expression of epithelial related genes zonula ocdudens-1(P=0.010).Conclusions Exosome extracted from HepG2 cells can induce human adipose-derived MSC to differentiate into cancer-associated myofibroblasts. CAF-like cells can promote the migration of the liver cancer cell line HepG2.

Animals ; Animals, Newborn ; Cerebral Cortex ; drug effects ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Hypoxia-Ischemia, Brain ; metabolism ; Progesterone ; pharmacology ; Rats ; Rats, Sprague-Dawley

Objective To clone ,construction ,express and purify Tp47 of Treponema pallidum (Tp) ,and assess the immunoreac‐tivity by Western‐Blot .Methods Tp47 was amplified by polymerase chain reaction ,and then cloned to the vector pGEX‐6P‐1 .The correct sequence of the recombinant plasmids pGEX‐6P1‐Tp47 was transformed into Escherichia coli BL21 (DE3) and induced .The expression product was analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis and Western‐Blot .The expression protein was purified .Serum of different clinical stages of syphilis was used as the antibody to detect the immunoreactivity of the protein by Western‐Blot .Results A fusion protein with molecular weight about 71 × 103 was attained .Western‐Blot proved that the recombinant protein can react with Tp IgG positive sera .And the specificities and sensitivities of the diagnostic reagent detected by sera were 100% .Conclusion The recombinant protein Tp47 was expressed and purified with good antigen activity ,which could provide the basis of theory and practice for the development of early diagnostic kit applying to detect Tp infection .

Phorbol ester-induced release of growth hormone (GH) and prolactin (PRL) from human somatotrophic tumors was examined in vitro. 12-O-tetradecanoyl-phorbol-13- acetate (TPA)strongly stimulated GH and PRL secretion and showed an additive effect on GH secretion if used in combination with GH releasing hormone (GHRH). In contrast, staurosporine exerted a variable inhibitory effect on GH release. There was no correlation between such effects and gsp mutations.The findings suggested that TPA doesn't act directly through cAMP signal transduction system.

Phorbol ester-induced release of growth hormone (GH) and prolactin (PRL) from human somatotrophic tumors was examined in vitro. 12-O-tetradecanoyl-phorbol-13- acetate (TPA)strongly stimulated GH and PRL secretion and showed an additive effect on GH secretion if used in combination with GH releasing hormone (GHRH). In contrast, staurosporine exerted a variable inhibitory effect on GH release. There was no correlation between such effects and gsp mutations.The findings suggested that TPA doesn't act directly through cAMP signal transduction system.

Chronic myeloid leukemia (CML) is characterized by the accumulation of active BCR-ABL protein.Imatinib is the first-line treatment of CML;however,many patients are resistant to this drug.In this study,we aimed to compare the differences in expression patterns and functions of time-series genes in imatinib-resistant CML cells under different drug treatments.GSE24946 was downloaded from the GEO database,which included 17 samples of K562-r cells with (n=12) or without drug administration (n=5).Three drug treatment groups were considered for this study:arsenic trioxide (ATO),AMN107,and ATO+AMN107.Each group had one sample at each time point (3,12,24,and 48 h).Time-series genes with a ratio of standard deviation/average (coefficient of variation) ＞0.15 were screened,and their expression patterns were revealed based on Short Time-series Expression Miner (STEM).Then,the functional enrichment analysis of time-series genes in each group was performed using DAVID,and the genes enriched in the top ten functional categories were extracted to detect their expression patterns.Different time-series genes were identified in the three groups,and most of them were enriched in the ribosome and oxidative phosphorylation pathways.Time-series genes in the three treatment groups had different expression patterns and functions.Time-series genes in the ATO group (e.g.CCNA2 and DAB2)were significantly associated with cell adhesion,those in the AMN107 group were related to cellular carbohydrate metabolic process,while those in the ATO+AMN107 group (e.g.AP2M1) were significantly related to cell proliferation and antigen processing.In imatinib-resistant CML cells,ATO could influence genes related to cell adhesion,AMN107 might affect genes involved in cellular carbohydrate metabolism,and the combination therapy might regulate genes involved in cell proliferation.

Background Corneal neovascularization (CNV) is an important cause of visual impairment and graft rejection after allograft corneal transplantation in inflammatory corneal diseases. The mechanisms and therapy relating to CNV are intensely investigated at all times. Objective This study was to evaluate the effect of eicosapentaenoic acid (EPA) on CNV induced by alkali cauterization and its mechanism. Methods The animal models of corneal neovasculation were induced in the right eyes in 72 Sprayue-Dawley rats by putting a piece of 3 mmfilter paper with 1 mol/L NaOH at the center of the cornea for 30 seconds. The rats were then divided randomly into the 0.02 mg EPA treatment group (24 rats) ,0.03 mg EPA treatment group (24 rats) ,model group (24 rats) and normal group (6 rats). EPA of 0.04 ml with doses of 0.02 mg or 0. 03 mg or saline solution of 0. 04 ml was injected subconjunctivally in model rats and immediately after cauterization. The presence of CNV and corneal edema were observed daily by slit lamp biomicroscope. 1,4,7 and 14 days after operation, corneal histopathological examination was performed by hematoxylin and eosin staining. The vascular endothelial cells were stained with CD34 by immunohistochemistry,and the expression of IL-1α,IL-6 mRNA and the nuclear factor-κBp65 ( NF-κBp65 ) proteins was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The use of animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by Hebei Province( version 1998 ). Results Under the slit lamp, CNV grew slowly from days 2-4 with obvious corneal edema and defect of epithelium. Larger CNV area and less edema were seen from days 7-10. Maximal vessel growth was observed 14 days after injury with thinner vessels in the model group. Histological examination showed that part of the corneal epithelium was damaged;serious corneal edema, more inflammatory cells and a lot of CNV in the stroma were presented in the model group. However, repairing of the corneal epithelium without CNV ,light corneal edema and less inflammatory cells were found in both the 0. 02 mg EPA and 0. 03 mg EPA treatment groups 7 days after alkali cauterization. The relative area of CNV in the 0. 02 mg EPA treatment group was ( 15.80±6.43 )% and ( 11.06±2. 14)% ,and that in the 0. 03 mg EPA treatment group was (16. 10±7.41 )% and (11.06±2. 51 )%, showing significant reduction in comparison with the model group [ (84. 74±7.77)% and (89.63±7.50) % ] 7 days and 14 days after operation ( P＜0. 05 ). Stronger expression of CD34 in the vascular endothelial cells of the cornea stroma was observed in the model group and an absence of CD34 was observed in the EPA-treated groups on the 7th day. RT-PCR revealed that the expression of IL-1α mRNA and IL-6 mRNA was lower in the EPA treatment groups than the model group ( P＜0. 05 ), and Western blot analysis showed that the expression of NF-κB/p65 in the corneas in the EPA treatment groups was significantly lower than that in the model group on the 4th day after operation (P＜0.05).Conclusion Topical application of EPA suppresses CNV induced by alkali burn possibly by inhibiting the expression of NF-κB,IL-1α and IL-6.

Abstract: Objective　To analyze the serotype distribution, drug resistance rate and drug resistance gene carrying of Streptococcus pneumoniae isolates in hospitalized patients, and evaluate the coverage of the vaccine to the serotype of Streptococcus pneumoniae in this area, so as to provide reference for the rational use of antibiotics in clinic. Methods　A total of 150 strains of non-repetitive Streptococcus pneumoniae isolated from inpatients from January 2015 to December 2019 were collected for serotyping and antimicrobial sensitivity test. The carrying rates of pbp2b, ermB and tetM were detected by PCR. Results　The PCR classification rate of 150 strains of Streptococcus pneumoniae was 93.1%, and the classification rate of capsular swelling test was 100%, and a total of 19 serotypes were divided, mainly 19F and 6B. Children's serotypes were predominantly 19F, 6B, and 15A; adult serotypes were predominantly 19F, 14, and 23F. The coverage rates of the PCV7, PCV10, PCV13 and PPV23 vaccines were 36.8%, 42.1%, 57.9% and 68.4%, respectively. Strains with serotypes of 19F, 6B, 3, and 23F had higher rates of resistance to antimicrobials. The sensitivity of Streptococcus pneumoniae to penicillin was greater than 96.0%. Antimicrobials with significant differences in resistance rates between invasive and non-invasive strains were penicillin, moxifloxacin, and levofloxacin. The percentage of strains carrying both ermB and tetM resistance genes was 96.0%, and the concordance rate between pbp2b, ermB and tetM resistance genes and the resistance phenotype was >98.0%. A total of 10 multi-resistance combinations were detected, with a multi-resistance rate of 62.6%, and the multi-drug resistance pattern of Streptococcus pneumoniae was mainly concentrated in the 19F and 6B serotypes. Conclusion　There are significant age differences in the serotypes of Streptococcus pneumoniae in this area. The vaccine currently used has low coverage in this region and therefore offer limited protection to the population. The drug resistance rates of Streptococcus pneumoniae varied significantly among serotypes. Erythromycin and tetracycline are not recommended for clinical treatment of Streptococcus pneumoniae. Penicillin can still be used as the first choice for clinical treatment of Streptococcus pneumoniae infection.

Objective To explore whether the expression level of heparanase (HPA) and its coagulation proteins on leukemic blast membrane could determine the hemostatic balance on the surface of leukemia cells.Methods Forty patients of leukemia were studied,and 20 patients with iron dificient anemia as the control group.Expression of tissue factor (TF),heparanase (HPA),tissue factor pathway inhibitor (TFPI),and urokinase plasminogen activator receptor (UPAR) on leukemic blast surfaces were analyzed by flowcytometry.Results The expression of TF,UPAR,and HPA in AML,ALL,CML,CLL and CRAL groups were significantly higher compared with the control group (t =.3.289,3.507,2.701,P ＜0.05; t =2.498,0.802,3.090,P ＜0.05; t =2.642,3.308,2.696,P ＜0.05; t =3.417,3.434,2.382,P ＜0.05; t =2.193,2.272,2.263,P ＜0.05).There were no significantly differences between the leukemic cell expression of TFPI and the control group (P ＞0.05).Expression of TF,UPAR,HPA in AML patients were significantly higher than ALL,CML and CLL groups (t =2.463,2.179,2.276,P ＜0.05; t =2.637,2.402,2.095,P ＜0.05; t =2.548,2.425,2.412,P ＜0.05).The levels of TF,UPAR and HPA in M3,M4 and M5 patients were higher than that of M1,M2 groups (P ＜0.05).There were no significantly differences among M3,M4 and M5 (P ＞0.05).Conclusions These results suggest that TF,UPAR and HPA are predominately expressed on leukemic blast surface,particularly in M3and M4,5 subtypes.The expression of coagulation proteins on blast membrane might determine the hemostatic balance on the surface of leukemia cells.

The chemical shift of fluoxetine hydrochloride appears at delta 14.15 in 19F NMR analysis. The delta moved upfield slightly from 14.158 to 14.145 when the concentration of solution became diluted from 2.00 to 0.05 mmol x L(-1). Spiking test was suggested to confirm the existence of the compound for qualitative analysis. 19F NMR detection sensitivity test illustrated that a concentration of 17 mg in 1 L water could be detected while the sample was scanned 500 times with optimum parameters. In quantitative analysis, standard curve of concentration versus fluorine signal intensity was proposed to determine the amount of fluoxetine. Long capillary tube containing trifluoroacetic acid was used as internal standard for the integration measurements and straight line was obtained with good fitting. Direct additions of trifluoroethanol to fluoxetine solutions gave a poorer standard curve.
Fluorine ; chemistry ; Fluoxetine ; analysis ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Trifluoroacetic Acid ; analysis