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Objective To explore the role of E2A﹣HLF fusion gene in the prognosis evaluation of B﹣cell acute lymphoblastic leukemia (B﹣ALL), and to improve the accuracy of stratified treatment. Methods The clinical characteristics, treatment effect and survival time of two B﹣ALL patients with E2A﹣HLF fusion gene who were admitted to the Second Hospital of Shanxi Medical University were retrospectively analyzed, and the related literature was reviewed. Results The first patient was an 8 years old girl, developed with fever, abdominal pain, and slightly increased white blood cells (WBC), and also accompanied by hypercalcemia. Another patient was a 27 years old man, developed with jaw pain and anemia, WBC was normal. Precursor B﹣ALL (pre﹣B﹣ALL) was identified by flow cytometry (FCM) in the two cases. E2A﹣HLF fusion gene was screened out at first diagnosis for the girl, but found after relapse for the man. Both patients early received intensive treatment with high﹣dose methotrexate after the first complete remission, but relapsed after 3 and 6 months respectively. The girl did not receive allogeneic hematopoietic stem cell transplantation (allo﹣HSCT) after relapse and died of severe infection. The man received allo﹣HSCT complete remission, and maintained complete remission within 5 months after HSCT, E2A﹣HLF fusion gene was also negative, but eventually died of multiple transplantation﹣related complications. Conclusions E2A﹣HLF fusion gene occurs mostly in the pre﹣B﹣ALL patients with hypercalcemia, and it shows extremely poor prognosis with a high multidrug resistance rate and high early recurrence rate. The allo﹣HSCT can increase the leukemia﹣free survival rate, and the relapse and overall survival may be improved when these patients received allo﹣HSCT in the first complete remission.
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This study aims to knock out the goat β-lactoglobulin (BLG) gene using CRISPR-Cas9 system and knock in human lactoferrin (hLF) at the BLG locus, and further study the effect of RAD51 stimulatory compound (RS-1) on homologous recombination efficiency. First, we designed an sgRNA targeting the first exon of goat BLG gene and constructed a co-expression vector pCas9-sgBLG. This sgRNA vector was then transfected into goat ear fibroblasts (GEFs), and the target region was examined by T7EN1 assay and sequencing. Second, we constructed a targeting vector pBHA-hLF-NIE including NEO and EGFP genes based on BLG gene locus. This targeting vector together with pCas9-sgBLG expression vector was co-transfected into GEFs. Transfected cells were then treated with 0, 5, 10 and 20 μmol/L RS-1 for 72 h to analyse the EGFP expression efficiency. Next, we used 800 μg/mL G418 to screen G418-resistent cell clones, and studied hLF site-specific knock-in cell clones by PCR and sequencing. The editing efficiency of sgBLG was between 25% and 31%. The EGFP expression efficiency indicated that the gene knock-in efficiency was improved by RS-1 in a dose-dependent manner, which could reach 3.5-fold compared to the control group. The percentage of positive cells with hLF knock-in was increased to 32.61% when 10 μmol/L RS-1 was used. However, when the concentration of RS-1 increased to 20 μmol/L, the percentage of positive cells decreased to 22.22% and resulted in an increase of senescent cell clone number. These results suggested that hLF knock-in and BLG knock-out in GEFs were achieved by using CRISPR/Cas9 system, and optimum concentration of RS-1 could improve knock-in efficiency, which provides a reference for efficiently obtaining gene knock-in cells using CRISPR/Cas9 in the future.
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Objective To study the effect of S1P on HLF cell fibrosis and its mechanism. Methods (1) The expression of ECM in HLF cells was analyzed by using Western Blot after treatment by S1P(1 μmol/L), FTY720-P(5μmol/L),ponesimod(5μmol/L)and SEW2871(5μmol/L)24 h;(2)The HLF cells were pre-treated using selective S1PR antagonist W146(1 μmol/L),JTE-013(0.2 μmol/L),and TY-52156(1.25 μmol/L)1 h before incubation by S1P and S1PR agonists 24 h and then the expression of ECM was analyzed;(3)The HLF cells were pre-incubated using JTE-013(0.2μmol/L)and TY-52156(1.25μmol/L)for 1 h and then the expression of ECM was analyzedafter being treated by S1P and S1PR agonists 24 h. Results (1)S1P and selective S1P receptor agonist increased the expression of ECM to various extents;(2)The S1P1R antagonist W146 did not affectthe expression of ECM induced by S1P and S1PR agonists and S1P2R antagonist JTE-013 and S1P3R antagonist TY-52156 both decreased the expression of ECM induced by S1P and S1PR agonists;(3)The expression of ECM induced by S1P and S1PR agonists further decreased using both JTE-013 and TY-52156 but not using ponesimod. Conclusion S1P2R and S1P3R are activated under the influence of S1P so as to increase the synthesis of ECM and promote fibrosis gene expression in HLF cells.
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Objective: To investigate a new method for altering the secondary metabolism of inactive actinomycete strain by magnetic nanoparticle-aided microwave mutagenesis to obtain antitumor mutants. Methods: HLF-43, a marine-derived actinomycete strain without antitumor activity with the inhibition rate of 3.9% at the 1000 μg/ml sample concentration on K562 cells, was used as the initial strain in the present study. The spore suspension of HLF-43 was exposed to the microwave radiation (the frequency 2450 MHz, the power 464 W)under the presence of Fe3O4 magnetic nanoparticles, then spread on neomycin-containing plates and the neomycin-resistant (neo) mutants were selected during incubation at 28°C for 4-14 days. The initial strain HLF-43 and its neo mutants were fermented to obtain fermentation samples and the antitumor neo mutants were obtained by the test of antitumor activity for these samples at 100 μg/ml. The antitumor activity was assayed by the MTT method using K562 cells. Results: A new method has been created for altering the secondary metabolism of actinomycete strain by magnetic nanoparticle-aided microwave mutagenesis. By the method coupled with the neo mutant selection, a total of 80 neo mutants were obtained from the inactive-initial strain HLF-43 and 28 of them were antitumor mutants with the inhibition rates over 20% at the 100 μg/ml sample concentration on the K562 cells. The antitumor mutants could be obtained at quite high frequency of 35% (28/80). Conclusion: Compared with the microwave mutagenesis without the aid of Fe3O4 magnetic nanoparticles, the Fe3O4 magnetic nanoparticle-aided microwave mutagenesis is more favorable to obtain the neo mutants resistant to higher concentration of neomycin and to obtain the antitumor neo mutants at higher frequency.
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To study the inhibitory effects of laminarin (acidic polysaccharide) J201A on the proliferation of human lung fibroblasts (HLF) and its related mechanism of action. Methods: Effect of J201A on the proliferation of HLF was evaluated by MTT assay, and the effects on cell cycle and synthesis of proteins of HLF were assessed by flow cytometry. The existence of J201A receptors on HLF was confirmed by fluorescent staining assay. Results:J201 A inhibited the synthesis of proteins and the proliferation of HLF at G0/G1 stage of cell cycle and J201A receptors existed on HLF. Conclusion:J201A exerted its antifibrotic activity by inhibiting HLF at G0/G1 stage of cell cycle and the synthesis of proteins.