Objective:To further improve the understanding of paroxysmal nocturnal hemoglobinuria (PNH), we retrospectively analyzed and summarized the clinical characteristics, treatment status, and survival status of patients with PNH in Zhejiang Province.Methods:This study included 289 patients with PNH who visited 20 hospitals in Zhejiang Province. Their clinical characteristics, comorbidity, laboratory test results, and medications were analyzed and summarized.Results:Among the 289 patients with PNH, 148 males and 141 females, with a median onset age of 45 (16-87) years and a peak onset age of 20-49 years (57.8% ). The median lactic dehydrogenase (LDH) level was 1 142 (604-1 925) U/L. Classified by type, 70.9% (166/234) were classical, 24.4% (57/234) were PNH/bone marrow failure (BMF), and 4.7% (11/234) were subclinical. The main clinical manifestations included fatigue or weakness (80.8%, 235/289), dizziness (73.4%, 212/289), darkened urine color (66.2%, 179/272), and jaundice (46.2%, 126/270). Common comorbidities were hemoglobinuria (58.7% ), renal dysfunction (17.6% ), and thrombosis (15.0% ). Moreover, 82.3% of the patients received glucocorticoid therapy, 70.9% required blood transfusion, 30.7% used immunosuppressive agents, 13.8% received anticoagulant therapy, and 6.3% received allogeneic hematopoietic stem cell transplantation. The 10-year overall survival (OS) rate was 84.4% (95% CI 78.0% -91.3% ) . Conclusion:Patients with PNH are more common in young and middle-aged people, with a similar incidence rate between men and women. Common clinical manifestations include fatigue, hemoglobinuria, jaundice, renal dysfunction, and recurrent thrombosis. The 10-year OS of this group is similar to reports from other centers in China.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.

Objective: To explore the situation of nephrology nursing research in more than a decade, and to provide reference for the development of nursing scientific research in China. Methods: A total of 1 927 papers of nephrology nursing, which ranged from January 1, 2008 to November 8, 2018, were retrieved from the Web of Science core collection database and visualized by CiteSpace V and ECharts.js. Results: The cited frequency of international nephrology nursing research increased year by year, and the number of papers growed tendency in fluctuation. The United States and Australia were in the leading position on nephrology nursing research, and China ranked sixth. Research hotspots focused mainly on the keywords such as chronic kidney disease, hemodialysis, peritoneal dialysis, quality of life, and disease management. Co-occurrence cluster analysis revealed that peritoneal dialysis and home hemodialysis were the frontiers. Conclusions International nephrology nursing research has developed steadily. Chinese specialists should track the research trends of international core institutions in real time, explore research hotspots and frontiers, and strive for prompting nursing research to be in line with the international.

OBJECTIVE:To optimize the formulation of Budesonide sustained-release tablet. METHODS:Using the cumula-tive releases in 2,4,8 h as investigation indexes,central composite design-response surface method was used to optimize the amount of hydroxypropylcellulose L(HPC-L),amount of soybean phosphatides,and filler(fixed total 200 mg)lactose- micro-crystalline cellulose mass ratio in the formulation of Budesonide sustained-release tablet,and the verification test was conducted. The release behaviors of prepared sustained-release tablet and original preparation in pH 7.2,7.0,6.8 phosphate buffer were com-pared. RESULTS:The optimal formulation was as follow as budesonide of 9 mg,HPC-L of 46.49 mg,soybean phosphatides of 9.23 mg,filler lactose-microcrystalline cellulose mass ratio of 1:2.9;the cumulative releases in 2,4,8 h were 21.9%,50.1%, 99.5%,the relative errors with predicted values (22.0%,50.0%,98.5%) were 0.45%,0.20%,1.02%(n=3),respectively. Compared with cumulative release of original preparation,the f2 was higher than 50. CONCLUSIONS:Budesonide sustained-re-lease tablet is successfully prepared,which shows similar release behavior to original preparations.

Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.

Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1)and to explore its intracellular location and functions.Methods By using Hela cDNA as the template,the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid.The recombinant plasmid was transfected into 293 cells using lipofectamine 2000.The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence,respectively.Scratch-repair experiment was used to detect the cancer cells’ migration ability.Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells.The recombinant plasmid was successfully expressed in eukaryote cells.CAP1 was located in the cytoplasm.The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration.Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells.CAP1 protein was located in the cytoplasm.The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1.

OBJECTIVETo investigate the reversal effects of different concentrations of DNA methylation inhibitor, 5-aza-2-deoxycytidine, on the hypermethylation of maternally expressed gene 3 (MEG3) gene promoter, and then the inhibitory effect of restoration of MEG3 expression on the proliferation of ovarian cancer cells.

METHODSHuman ovarian cancer OVCAR3 cells were treated with various concentration of 5-aza-2-deoxycytidine (0, 1, 5, 10, 20 µmol/L, respectively) for 6 days. Then the methylation status of MEG3 promoter was detected by methylation specific PCR (MSP). The alteration of MEG3 gene expression was detected by RT-PCR. Cell proliferation was determined by MTT assay and EdU incorporation assay.

RESULTSAfter treated with 5-aza-2-deoxycytidine, the methylation status of MEG3 in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 0.79 ± 0.00, 0.67 ± 0.00, 0.65 ± 0.03 and 0.61 ± 0.01 folds, respectively (P < 0.05 for all). The relative expressions of MEG3 mRNA in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 2.04 ± 0.16, 2.44 ± 0.17, 3.19 ± 0.34 and 5.34 ± 0.39, respectively (P < 0.05 for all). In contrast to the negative control, the inhibition rates of the OVCAR3 cell growth were increased significantly when treated with 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine in 2, 4 and 6 days. There were (40.78 ± 0.80)%, (35.65 ± 0.33)%, (31.81 ± 0.66)%, (27.33 ± 1.27)% and (17.75 ± 1.85)% of EdU-positive cells in the 0, 1, 5, 10 and 20 µmol/L 5-aza-2-deoxycytidine groups (P < 0.01 for all).

CONCLUSIONSMaternally expressed gene 3 promoter hypermethylation is reversed by 5-aza-2-deoxycytidine in ovarian cancer cells. The downregulation of MEG3 gene might be resulted from the methylation, and the re-expression of MEG3 partly contribute to the growth inhibition of epithelial ovarian cancer cells.

Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Methylation ; Female ; Humans ; Neoplasms, Glandular and Epithelial ; Ovarian Neoplasms ; Promoter Regions, Genetic ; RNA, Messenger

Objective To evaluate the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) in ovarian cancer cell lines, and to investigate the biological effects of down-regulated MALAT-1 on OVCAR3 cells.Methods qRT-PCR analysis was used to examine the expression level of MALAT-1 gene in ovarian cancer cells, including ES-2, A2780, SKOV3 and OVCAR3 cell lines.For functional research, four shRNA oligos specially targeting MALAT-1 and a empty vector were designed and constructed into pGPU6/GFP/Neo, then transfected into OVCAR3 cells.qRT-PCR was used to confirm the effective suppression of MALAT-1.Changes of proliferation and adhesion of cells were analyzed by CCK-8 and adhesion assays.Wound-healing, transwell migration and invasion assays were used to examine migration and invasion of MALAT-l-silencing cells in vitro.Results The expression of MALAT-1 gene in OVCAR3 cells was high, and qRT-PCR results confirmed successfully the knockdown of MALAT-1 after transient transfection.After successful suppression of MALAT-1, the proliferation, wound-healing and adhesion ability in vitro were inhibited to some degree.In transwell migration assay, the number of migration cells in MALAT-1-silencing group was 52.17±4.48, which is much less than that in the negative and control groups (286.50± 12.23 and 295.67±6.96, respectively).In invasion assay, the number of invasion cells passing the transwell membrane in MALAT-1-silencing group (37.33±2.40) was also decreased significantly, compared to that in the negative and control groups (239.00±15.72 and 222.67±20.85, P ＜ 0.05).Conclusions shRNA-mediated silence of MALAT-1 can effectively inhibit the proliferation, adhesion, migration and invasion abilities of ovarian cancer cell line OVCAR3 in vitro, indicating MALAT-1 is expected to be a target gene for the treatment of ovarian cancer.

Objective To study the relationship between the methylation status of CPG islands in MEG3 gene promoter region of epithelial ovarian cancer and its clinical and pathological features. Methods The promoter methylation status was evaluated by MSP (methylation-specific polymerase chain reaction ) in 47 cases of ovarian cancer tissue and 15 cases of normal control. Results The methylation ratio (42.6%) of the MEG3 genes in the ovarian cancer was statistically significantly higher (P = 0.035 ) than that (13.3%) in the normal control. The methyation rate of the group with an age > 60 years old was slightly higher than that of the group with an age≤60 years old, without statistically significant (P > 0.05), so was observed in ovarian cancers of stage Ⅰ andⅡ than that in stage Ⅲ and Ⅳ. There were also no significant differences in MEG3 gene methylation positive rate neither in different pathological grading nor in various ovarian cancer tissues (P > 0.05). Conclusion Abnormal methylation in MEG3 gene may be associated with epithelial ovarian cancer , but no relation to its clinical pathology.

Objective To investigate the inhibitory effects of RNA interference (RNAi) on the expression of matrix metalloproteinase-2 (MMP-2) gene and growth, adhesion,invasiveness and migration of ovarian cancer cells. Methods One specific target sequence of MMP-2 gone and one non-specific sequence (NC group) were chosen,the medium DMEM as blank group.After transfection of ovarian cancer OVCAR-3 cells, the RT-PCR and Western blot were used to detect mRNA and protein expression of MMP-2 gene, the growth ability was detected by MTT assay, the abilities of adhesion was detected by cell adhesion assay, the invasion and migration were detected by Matrigel invasion assay and wound healing assay. Results By contrast to the NC group,the mRNA expression was decreased by 73.8 ％,78.8 ％ and 78.4 ％(P＜ 0.05) in 24 h,48 h and 72 h after transfection and protein expression was decreased by 72.6 ％,81.2 ％ and 76.4 ％(P＜ 0.05) respectively at the same time. The 48 h group had the most efficient inhibitory effect. Cell growth curve revealed that cell growth was not significantly inhibited (P＞ 0.05). Adhesion was significantly reduced,the inhibitory rate was 55.0 ％ at 60 min and 44.8 ％ at 90 min (P＜ 0.05),respectively. Invasion and migration were significantly reduced as well,the inhibitory rate on invasion and migration were 29.7 ％ and 35.8 ％(P＜0.05), respectively. Conclusion siRNA mediated MMP-2 down-regulation in ovarian OVCAR-3 cells can inhibits its adhesion,invasion and migration,but do not significantly affect its growth,suggesting a important target to ovarian cancer gene-therapies.