Objective:To investigate the clinical characters and prognostic value of PSA flare and bone flare in metastatic castration resistant prostate cancer(mCRPC) patients received Abiterone acetate(AA) therapy.Methods:A retrospective study was conducted for 93 mCRPC patients treated with AA from Jul.2016 to Dec.2020. Mean age was (75.4±8.9)years, median PSA was 58.2 (16.4, 148.6)ng/ml. Patients received at least 6 months of AA treatment. PSA flare was defined as an increase of PSA after AA therapy followed by a decrease. Bone flare was defined as disease progression after 3 months of therapy, typically based on increased lesion intensity or number, and reevaluation 6-9 months later showed improvement in the scan. The clinical characters and prognostic value of the flare phenomenon was evaluated and analyzed respectively.Results:The median follow up time was 16 months(6, 54 months), fourteen patients showed PSA flare at first month after AA treatment, and median time of duration was 2 months(1, 7 months). The serum alkaline phosphatase (ALP) had a similar rising trend along with PSA flare[115.5(98.0, 198.5)U/L vs. 119.0(97.0, 288.8)U/L, P=0.016]. Seven patients showed bone flare and 3 cases co-existed with PSA flare. Multivariate Cox regression analysis indicated bone flare was an independent protective factor for progression free survival(PFS)( HR=0.117, 95% CI 0.015-0.895, P=0.039), PSA flare had no significant influence on PFS ( HR=1.314, 95% CI 0.554-3.121, P=0.536)and overall survival(OS)( HR=1.348, 95% CI 0.393-4.263, P=0.635). Log-rank test showed patients with bone flare had a longer PFS( P=0.016) and OS( P=0.047) compared with patients without bone flare. Conclusions:PSA flare always faded away after 2 months AA therapy and had no influence on PFS and OS. Bone flare maybe an indication for better prognosis.

Objective:To investigate differential expression gene (DEG) in mice with ventilator-induced lung injury (VILI) by bioinformatics analysis, and to verify the key genes by reproducing the VILI mouse model.Methods:① Experiment 1 (bioinformatics analysis): the microarray dataset of GSE9368 and GSE11662 regarding VILI mice and those in the spontaneous breathing control group were downloaded from the gene expression omnibus (GEO) database. DEG obtained by R and Venn map was further used to obtain common DEG. DAVID online database was used to obtain gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, the protein-protein interaction (PPI) analysis of common DEG was carried out by using Search Tool for the Retrieval of Interacting Genes Database (STRING) and the key genes were screened out by using CytoScape software, molecular complex detection (MCODE) analysis plug-in and CytoHubba plug-in with maximum cluster centrality (MCC), maximum neighbor connectivity (MNC) and degree. ② Experiment 2 (related protein verification): VILI mouse model was reproduced by high tidal volume (20 mL/kg) ventilator. Spontaneous breathing control group was set up. Hematoxylin-eosin (HE) staining was performed to assess lung injury and the key genes screened in experiment 1 were verified by immunohistochemical staining.Results:① Experiment 1 results: a total of 114 DEG were screened from GSE9368 dataset, including 99 up-regulated genes and 15 down-regulated genes. A total of 258 DEG were screened from GSE11662 dataset, including 188 up-regulated genes and 70 down-regulated genes. Furthermore, 66 common DEG were obtained, including 61 up-regulated genes and 5 down-regulated genes. GO analysis showed that the common DEG were mainly involved in inflammatory response, immune response, leukocyte and neutrophil chemotaxis. KEGG analysis showed that the common DEG were involved cell adhesion, cytokine receptor interaction and tumor necrosis factor (TNF) signaling pathway. STRING and CytoScape analysis were used to construct gene PPI network diagram and important sub modules. And the CytoHubba plug-in with MCC, MNC and degree algorithms was used to perform topology analysis and then taken an intersection to obtain eight genes including suppressor of cytokine signaling 3 (SOCS3), interleukin-1β (IL-1β), matrix metalloproteinase-9 (MMP-9), integrin Itgam, CXC chemokine ligand 2 (CXCL2), CXC chemokine receptor 2 (CXCR2), Sell and CC chemokine receptor 1 (CCR1). ② Experiment 2 results: a mouse model of high tidal volume VILI was reproduced. Compared with the spontaneous breathing control group, the lung tissue was injured slightly at 0 hour after the end of ventilation, and the lung tissue structure was significantly damaged at 6 hours after the end of ventilation, showing bleeding in alveolar cavity, significant increase and collapse of alveolar wall thickness, and infiltration of inflammatory cells. The top three genes from intersection and topological analysis including IL-1β, SOCS3 and MMP-9 were verified by immunohistochemical staining. The results showed that the expressions of IL-1β, SOCS3 and MMP-9 were gradually increased with time of ventilation, the differences were found at 6 hours as compared with those in the spontaneous breathing control group [IL-1β (integral A value): 8.40±2.67 vs. 5.10±0.94, SOCS3 (integral A value): 9.74±1.80 vs. 5.95±1.31, MMP-9 (integral A value): 11.45±6.20 vs. 5.36±1.28, all P < 0.05]. Conclusion:Bioinformatics analysis based on GSE9368 and GSE11662 data sets found that VILI is mainly related to inflammatory injury, cytokines and immune cell infiltration; IL-1β, SOCS3 and MMP-9 might be biomarkers of VILI.

Objective:To study the preparation technology of ambroxol hydrochloride double-layer tablets and evaluate the drug re-lease in vitro. Methods:The best technology was screened by orthogonal design, and the drug release in vitro was evaluated by UV spectrophotometry. Results:The drug release in vitro was in accordance with the corresponding standard. Conclusion:The preparation technology is simple and the quality is stable for industrial production.

ObjectiveTo investigate the clinical effect of phacoemulsification combined with goniosynechiolysis in treatment of angle-closure glaucoma.Methods163 cases of angle-closure glaucoma with cataract were divided into observed group (86 cases) and control group (87 cases).The observed group was treated with phacoemulsification cataract extraction, artificial crystalline implantation and goniosynechiolysis, while the control group was treated with compound trabeculectomy.The vision, intraocular pressure and chamber depth after surgeries in the two groups were compared.ResultsThe vision in observed group was enhanced distinctly after surgery,which was significantly better than the control group(P ＜0.05).The intraocular pressure in 2 weeks of observed group after surgery was significantly better than the control group(P ＜ 0.01).Moreover the anterior chamber depth and chamber angle of observed group was better than the control group after operation(P ＜ 0.05).ConclusionThe treatment of phacoemulsification combined with goniosynechiolysis for angle-closure glaucoma had good clinical effect.

This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells. The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal (NLS) sequence, and then inserted into the eukaryotic expression vector pDsRed1-C1. The recombinants were transfected into HepG2 cells. The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy. The cell proliferation was measured by MTT, and the cell cycle and apoptosis of HepG2 cells by flow cytometry. The results of restriction analysis, DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid. The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation. Cell cycle analysis showed that the cell ratio of G(0)/G(1) in the wild type group was significantly increased as compared with that in the mutant type group, and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group. It was concluded that the subcellular localization of P21 may contribute to the development of hepatic cancer.

This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells.The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal (NLS) sequence,and then inserted into the eukaryotic expression vector pDsRed1-C1.The recombinants were transfected into HepG2 cells.The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy.The cell proliferation was measured by MTT,and the cell cycle and apoptosis of HepG2 cells by flow cytometry.The results of restriction analysis,DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid.The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation.Cell cycle analysis showed that the cell ratio of G0/G1 in the wild type group was significantly increased as compared with that in the mutant type group,and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group.It was concluded that the subcellular localization of P21 may contribute to the development of hepatic cancer.

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-alpha, IL-1beta and TGF-beta1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-kappaB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-alpha, IL-1beta, IL-6 and COX-2, and inhibit NF-kappaB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-beta1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysac-charide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA 1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-α, IL-1β and TGF-β1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA 1, 2 could significantly reduce the production of pro-inflammatory cyto-kines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibit NF-κB nuclear transloca-tion. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-β1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflam-matory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

OBJECTIVETo explore the expression, characteristics and roles of macrophage colony-stimulating factor receptor (M-CSF-R) in human leukemia cell lines.

METHODSPeripheral blood mononuclear cells (PBMCs) collected from 3 healthy persons, cord blood mononuclear cells (CBMCs) collected from 5 healthy persons and 4 human myelomonocytic leukemia cell lines including J6-1, J6-2, K562 and HL-60 were studied by using ABC immunoperoxidaes assay, indirect immunofluorescene staining, flow cytometry, and Western blot.

RESULTSM-CSF-R was noticed to be localized in the cytoplasm, nucleus and at the membrane in 4 human leukemia cell lines; expression of M-CSF-R was not detected in normal human PBMCs without PHA stimulation. Human PBMCs stimulated by PHA expressed a low level of M-CSF-R. Frequencies of membrane bound M-CSF-R (M-CSF-mR) expression in J6-1, J6-2, K562 and HL-60 were 78.9%, 72.6%, 54.9% and 58.0% respectively. Frequencies of cytoplasm and nucleus associated M-CSF-R (M-CSF-cnR) were 52.3%, 44.3%, 28.0% and 65.3% respectively. One form of M-CSF-R with a molecular weight of 120 000 was detected both in the cytoplasm and nucleus of HL-60 cells. The half-life of M-CSF-cnR in leukemia cells mentioned above was longer than that of corresponding M-CSF-R in stimulated CBMCs, and the half-life of M-CSF-mR in leukemia cells was extended except that of M-CSF-mR in K562 cells. Both anti-M-CSF-R monoclonal antibody and recombinant human M-CSF soluble receptor could cause the growth arrest of HL-60 cell in G(0)/G(1) phase, and could inhibit the formation of colony of HL-60 cell in soft agarose.

CONCLUSIONSExpression of M-CSF-R in leukemia cells is heterogeneous. The accumulation of cellular M-CSF-R results in the low degradation rate of cellular M-CSF-R in leukemia cells, which could be a potential mitotic signal. Signal mediated by M-CSF-R is important and necessary for the growth of HL-60 cell.

Cell Line ; HL-60 Cells ; Humans ; Leukemia ; Leukocytes, Mononuclear ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Receptor, Macrophage Colony-Stimulating Factor ; Tumor Cells, Cultured