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Objective:To investigate the mid-term efficacy of prostatic artery embolization (PAE) for the treatment of lower urinary tract symptoms (LUTS), urinary retention (UR) or hematuria secondary to benign prostatic hyperplasia (BPH).Methods:This was a retrospective study conducted from February 2014 to December 2018 in 140 patients who underwent PAE for LUTS, UR or hematuria secondary to BPH, including 85 patients with LUTS (60 patients with LUTS and 25 LUTS combined with hematuria), 52 patients with UR (50 patients with UR and 2 UR combined with hematuria) and 3 patients with hematuria. All patients were followed up for 24 months. Clinical success rates were evaluated. Friedman test was performed to compare the differences in International Prostate Symptom Score (IPSS), quality of life (QoL) score, and prostatic volume (PV) between baseline and follow-up time points (3, 6, 12 and 24 months). A post hoc test was performed by the Bonferroni method.Results:Significant differences in IPSS, QoL score and PV between baseline and follow-up time points were observed in 85 patients with LUTS ( P<0.001 for all), and clinical success rates at 3, 6, 12, 24 months after PAE were 95.3% (81/85), 91.8% (78/85), 87.1%(74/85), 83.5%(71/85). The success rate of extubation in patients with UR within 1 month after PAE was 98.1% (51/52). The average interval from PAE to catheter-independence was (6.8±3.7) days, and clinical success rates were 94.1% (48/51), 92.2% (47/51), 88.2% (45/51), 84.3% (43/51), respectively. The interval from PAE to the resolution of hematuria was (3.4±2.5) days, and clinical success rates were 90.0%(27/30), 90.0%(27/30), 83.3%(25/30), 80.0%(24/30), respectively. Conclusions:PAE was an effective treatment option for symptoms secondary to BPH in mid-term follow up.
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Objective To explore the effect and mechanism of necroptosis related proteins in middle cerebral artery occlusion (MCAO) induced brain ischemia/reperfusion injury in mice.Methods C57BL/6 mice were used to establish the brain ischemia/reperfusion injury model induced by MCAO.MCAO mice were treated with z-VAD.fmk (zVAD,1.1 g/kg),GSK'872 (0.7 g/kg) and combined intervention of zVAD and GSK'872,and neurological defect was evaluated by mNSS while brain infarct volume was measured by TTC staining.Western blot and immunofluorescence assay were used to detect protein expression and location of RIP1,RIP3 and MLKL,respectively.Results Neurological defect and brain infarction were caused by MCAO.Compared with MCAO group,zVAD,GSK'872 and the combined intervention alleviated neurological defect and reduced brain infarct volume significantly (P＜0.05 or P＜0.01).The protein levels of RIP3 and RIP1 MLKL were increased in mice of MCAO group,while GSK'872 and the combined intervention obviously downregulated the aforementioned protein expression [RIP1 (GSK'872:0.64± 0.02 vs MCAO:1.28±0.02,P＜0.01);RIP3 (GSK'872:1.08±0.02 vs MCAO:1.45±0.02,P＜0.01);MLKL (GSK'872:0.54±0.01 vs MCAO:1.00±0.01,P＜0.01)].However,zVAD only slightly reduced protein expression of MLKL (P＜0.05) but didn't change the protein expression of RIP1 and RIP3 (P＞0.05).Conclusion RIP1,RIP3 and MLKL are involved in the execution of necroptosis and contribute to the pathological progress of brain ischemia/reperfusion injury.
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BACKGROUND:Oligodendrocytes are mostly differentiated from oligodendrocyte precursor cel s. A suitable medium and cel seeding density have a significant impact on the process of the isolation of oligodendrocyte precursor cel s to obtain oligodendrocytes. OBJECTIVE:To explore the optimization of oligodendrocyte culture conditions. METHODS:Oligodendrocyte precursor cel s isolated from the newborn rats 48 hours after birth were cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×104 cel s/cm2, 4×104 cel s/cm2, 8×104 cel s/cm2, 16×104 cel s/cm2, 32×104 cel s/cm2, and 64×104 cel s/cm2, respectively. Oligodendrocyte precursor cel s were induced to differentiate into oligodendrocytes at 72 hours after cel adhesion. Morphology of differentiated oligodendrocyte precursor cel s were observed under a light microscope, and the differentiation results were identified by immunofluorescence staining after 7-day induced differentiation. RESULTS AND CONCLUSION:Morphology of oligodendrocyte precursor cel s were recognized when cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×104 cel s/cm2, 4×104 cel s/cm2, and 8×104 cel s/cm2, respectively. Immunofluorescence staining showed that myelin basic protein-positive cel s were found after 7-day induced differentiation, and the positive cel number were 16.40±3.30, 49.95±2.33, and 76.95±4.86 in DMEM/F12 medium, and 12.65±2.53, 32.10±1.17, and 54.05±1.56 in DMEM/high glucose medium (P<0.05). These findings indicate that DMEM/F12 medium is more suitable for culturing oligodendrocyte precursor cel s compared with DMEM/high glucose medium to some extent. The number of differentiated oligodendrocytes was gradual y increased with the enhanced seeding density of oligodendrocyte precursor cel s, and the seeding densities from 4×104 to 8×104 cel s/cm2 were appropriate for the observation of cel morphology.
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Aim To explore the relationship between Mre11/Rad50/Nbs1 ( MRN ) complex focus formation and DNA double-strand breaks( DSBs) caused by cinob-ufagin in human hepatocellular carcinoma HepG2 cells. Methods The Na+,K+-ATPaseα1 subunit expression level in liver cancer tissues was detected by immunohis-tochemistry. After HepG2 cells were treated with 5μmol·L-1 cinobufagin for 6, 12 and 24 h, the drug-in-duced DSBs were assessed by single cell gel electro-phroesis ( SCGE ) , the gene transcription and protein levels of Mrel1, Nbs1, Rad50 and p53 were evaluated by Real time-PCR and Western blot. The cell cycle in parallel was analyzed by flow cytometry. Results The Na+, K+-ATPase α1 subunit expression level in liver cancer tissues was significantly increased compared with the tissue adjacent to carcinoma ( P <0. 05 ) . The 5μmol · L-1 cinobufagin could induce the DSBs in a time-dependent manner (P <0. 05), and it could up-regulate the gene expression levels of Mre11, Nbs1, Rad50 and p53 in HepG2 cells ( P<0. 05 ) . The pro-portions of HepG2 cells in S phase were ( 21. 32 ± 4. 21) % in the control group, and (33. 25 ± 5. 72) %, (56. 72 ± 6. 29) % and (67. 32 ± 9. 42) % in HepG2 cells treated with 5 μmol · L-1 cinobufagin for 6, 12 and 24 h, respectively. The proportions of cells in S phase in cinobufagin groups were significantly increased compared with the control group ( P<0. 05 ) . Conclu-sion Cinobufagin could induce the cell cycle arrest in liver cancer HepG2 cells by activation of Mre11/Rad50/Nbs1 Complex.
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Objective To investigate the signaling pathway and the key signal molecules of protein kinase (RIPK)3 in SH-SY5Y cells. Methods SH-SY5Y cells were transfected with RIPK3 expression plasmid vector to upregulate intracellular RIPK3, while the SH-SY5Y cells were transfected with empty vector plasmid, which was considered as control group. Western blot assay was used to check the expression of exogenous RIPK3 in cells. The proliferation rate of SH-SY5Y cells was determined by MTT assay at designated time to detect exogenous RIPK3 activity. Whole transcriptome sequencing (RNAseq) was used to detect the transcription of genes. Whole-transcriptomic gene transcription was measured by following Ingenuity Pathway Analysis (IPA) to obtain downstream signaling pathways and the key molecule, which were partly confirmed by following droplet digital PCR (ddPCR). Results Exogenous RIPK3 showed biological activity in SH-SY5Y, which inhibited the proliferation of cells. IPA showed that znic finger protein 36 (ZFP36) was significantly up-regulated as compared with that of the control group. The tran?scription levels of ZFP36 downstream genes such as tumor necrosis factor (TNF), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF) and mRNA-decapping enzyme 2 (DCP2) were affected at the same time. Conclusion Within the limitations of this study, it seems that RIPK3 is notable for the development, inflammation and tumorigenesis of the nervous system as an independent regulator of ZFP36 gene and downstream effectors.
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Objective To investigate the metabolism characteristics and the potetial biomarker candidates of osteonecro?sis of the femoral head (ONFH) using metabolomic technology. Methods The femoral head specimens from 23 ONFH patients (25 necrotic femoral heads) and 18 normal femoral heads from femoral neck fracture patients were collected for histopathological examination to confirm the diagnosis of all samples. All the metabolites of bone trabecula were extracted for ultra-high perfor?mance liquid chromatography-MS/MS analyzed. The measured variables was pretreat, and PCA (principal component analysis), PLS?DA (partial least squares?discriminant analysis) and OPLS?DA (orthogonal?partial least squares?discriminant analysis) models were employed to confirm the difference between these two groups after UPLC?MS/MS (ultra?high performance liquid chromatogra?phy?mass spectrometry/mass spectrometry) analysis. At last, the differential variables were screened out by PLS?DA and variate analysis (Kruskal?Wallis H test). The changed metabolites were confirmed by MS and MS/MS aligned in HMDB (human metabolo?mic database) and Massbank. The changed metabolites with the most obviously changed peak abundance, D?arginine, L?proline and L?glutamine, were picked out as the potential diagnostic biomarkers. After binary logistic regression analysis, the combined biomarkers candidates were further analyzed by receiver operating characteristic (ROC) curve to evaluate the significance of the combined biomarkers. Results Significant distinction of metabolites expression mode can be seen in PCA, PLS?DA and OPLS?DA models scoring plots between ONFH and control groups. Twelve changed metabolites in ONFH bone trabeculas were con?firmed by multi?variate statistical analysis and variate statistical analysis. Compared with the femoral neck fracture patients, the in?creased metabolites included D?arginine, L?proline, L?glutamine, creatine, uracil, uridine, LysoPC(20∶4(5Z, 8Z, 11Z, 14Z)), Ly?soPC(16∶0), PC(20∶1(11Z)/18∶3(6Z, 9Z, 12Z)) and PE(P?16∶0e/0∶0). The decreased metabolites were reticulataxanthin and β?cryptoxanthin. According to the change fold of peak abundance and variable weight projection in PLS?DA, the most obviously dif? ferential metabolites were picked out as the biomarker candidates of ONFH. The potential biomarkers candidates were identified as D?arginine, L?proline and L?glutamine. The area under the curve of D?arginine, L?proline and L?glutamine ROC were 0.873, 0.712 and 0.862. The area under the curve of ROC was 0.946 after combining D?arginine, L?proline, L?glutamine using binary lo?gistic regression analysis. Conclusion PCA, PLS?DA and OPLS?DA models were used to find out the differential variables in the metabolites of bone trabeculas in ONFH and femoral neck fracture patients. Twelve metabolites were identified by MS/MS, and 3 obviously changed metabolites, D?arginine, L?proline, L?glutamine, were indicated as biomarker candidates. These 3 obviously changed metabolites showed a good diagnostic significance.
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Objective To investigate the effects of synuclein-γ (SNCG) gene silencing on the proliferation and apoptosis of glioma U87-MG cells.Methods Five small hairpin RNA templates targeting SNCG and a negative control were synthesized and cloned into the lentiviral vector system and all the constructs were sequenced.Then the recombinant lentiviral vectors were used to infect U87-MG cells.The lentiviruses which can effectively inhibit protein expression levels of SNCG were selected by RT-PCR for further study.Colony formation and flow cytometry assay were used to investigate the effects of SNCG downregulation by RNA interference on the clony formation,proliferation,and apoptosis of U87-MG cells,respectively.Results The lentiviral vectors carrying 5 shRNAs targeting the SNCG gene were successfully constructed,and SNCG siRNA3 and siRNA5 showed higher interfering efficiency than other vectors.In comparison with the group of negative control,SNCG siRNA3 and siRNA5 were observed to significantly inhibit SNCG expression at the mRNA levels (the relative mRNA levels:siRNA3 (0.17± 0.01)％,siRNA5 (0.13±0.01)％ vs (1.00±0.10)％,P＜0.05).Also,SNCG suppression mediated by RNAi significantly inhibited the clone formation (colony number:siRNA3 (66± 12),siRNA5 (1 ± 1) vs (80± 5),P＜0.05),and the proliferation (ratio of cells in S phase:siRNA3 (41.2±0.7) ％,siRNA5 (39.9±0.5) ％ vs (47.6±2.2) ％,P ＜0.05),but promoted the apoptosis (cell apoptosis:siRNA3 (22.9± 0.4) ％,siRNA5 (28.6± 0.9) ％ vs (1.1 ± 0.1) ％,P＜0.01) of transfected U87-MG cells.Conclusion SNCG suppression at the mRNA level mediated by RNAi can inhibit the proliferation and the clony formation,but induce the apoptosis of glioma U87-MG cells in vitro,suggesting that SNCG suppression mediated by an RNAi strategy may become a novel approach for treating human gliomas.
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As the major pathway mediating specific protein degradation in eukaryotes, ubiquitin-proteasome system (UPS) is involved in various physiological and pathological processes such as cell cycle regulation, immune response, signal transduction and DNA-repair. Deubiquitinases (DUB) maintain the balance of UPS and related physiological processes via reversibly removing ubiquitin from the covalently modified protein substrates, which have been implicated in various disease processes in case of their imbalance expression. Because DUB plays critical regulating roles in the UPS pathway, they may be also the ideal drug targets for severe and intractable human diseases, such as cancer and neurodegenerative disease. With the rapid development of proteomic technology, systematical investigation of specific substrates and interacting proteins of varied DUB via mass spectrometry approach may shed light on these DUB's biological function and regulating roles in the physiological and pathogenic states. In this review, we briefly introduce the characteristics of DUB and summarize the recent application and progresses of proteomics in DUB research.
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Humans , Mass Spectrometry , Proteasome Endopeptidase Complex , Metabolism , Proteomics , Signal Transduction , Ubiquitin , Metabolism , Ubiquitin-Specific Proteases , MetabolismABSTRACT
The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6J mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues' labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥ 95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.
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Animals , Mice , Amino Acids , Chemistry , Diet , Isotope Labeling , Lysine , Chemistry , Mice, Inbred C57BL , Proteins , Chemistry , Proteomics , MethodsABSTRACT
Aim To investigate the effect of ouabain and cinobufogenin on cell proliferation,apoptosis and cell cycle on HepG2,and explore their molecular mechanism.Methods The anti-proliferative effect on HepG2 cells was determined by MTT assay.The HepG2 cells were stained with Hoechst 33342,and its morphological changes were observed under fluorescence microscope;The cell cycle was measured by flow cytometry.The Cyclin A1,CDK 2,PCNA and p21~(CIP1) expression levels of HepG2 cells treated with ouabain and cinobufogenin were dectected in mRNA and protein by Real time PCR and Western blot.Results Ouabain and cinobufogenin could inhibit cell proliferation on HepG2 cells,and the inhibitory effects were in time and dose dependent manners.The HepG2 cells treated with ouabain and cinobufogenin showed the typical morphological features of apoptosis.Cell cycle analysis showed that the S phase of HepG2 cells treated with ouabain and cinobufogenin increased significantly compared with the control group.Real-time quantitative PCR and Western blot results showed that ouabain and cinobufogenin could down-regulate Cyclin A1,CDK 2,and PCNA expressions(P<0.05)and up-regulate p21~(CIP1) expression(P<0.05).Conclusion Nα~+,K~+-ATPase inhibitor has the anti-proliferative effect on HepG2 cells and induce apoptosis and S phase arrest.These effects might be related with proteins associated with cell cycle closely.
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AIM: To find one kind of peptide that will conjugate with ouabain and inhibit its biological function, and provide a new treatment strategy for primary hypertension. METHODS: In this study, ouabain was used as a target to screen ouabain conjugated peptide (OCP) from Ph. D. -7 phage display peptide library. After 3 rounds of bio-panning, the products were identified by ELISA and DNA electrophoresis analysis and sequencing. Peptide was synthesized and analyzed the activity by radioligand binding assay. The inhibitory ratio of cell proliferation was measured by MTT and the cell morphology changing was measured by Hoechst 33342/PI staining. The expression of Na~+-K~+-ATPase α1-subunit and β1-subunit were detected by RT-PCR and immunocytochemistry. The levels of the free intracellular Na~+ in EAhy926 cells were measured by laser confocal microscope. RESULTS: The ouabain conjugated peptide was found out, and it was occupied in 0.64(9/14). The analysis of protein showed that the obtained peptides had no homology with Na~+-K~+-ATPase. The amino acid sequence of synthesized peptide was Arg-Cys-Met-Thr-Ser-Arg-Ser. There was binding activity between OCP and ~3H-ouabain. The MTT assay showed that OCP could reverse the inhibition action of ouabain on vascular endothelial EAhy926 cells in a dose and time-dependent manner. The number of apoptotic cells had significantly decreased detected by Hoechst 33342/PI staining. The results of RT-PCR and immunocytochemistry showed that OCP could inhibit the up-regulated expression of Na~+-K~+-ATPase α1-subunit and down-regulated expression of Na~+-K~+-ATPase β1-subunit induced by ouabain in EAhy926 cells. CONCLUSION: The OCP could reverse the growth inhibition and death induction of ouabain in EAhy926 cells, which would provide the basis for studying the interaction between ouabain and Na~+-K~+-ATPase and explore novel anti-ouabain agents.
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2000 fold). In bystander effect experiment, the death rate of mixed cells was still over 50% even when they contained only 20% TK+ target cells. Fourteen days after nude mice bearing tumors were treated i.p. with GCV, the mean weight and volume of transfected tumors were less than those of untransfected tumors (P