Effect of Nucleolin on Lymphoma Proliferation by Regulating Thymidine Kinase 1 / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1).@*METHODS@#Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay.@*RESULTS@#The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle.@*CONCLUSION@#The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.

Expression and clinical significance of TRAIL and c-FLIP in peripheral blood mononuclear cell of patients with rheumatoid arthritis / 中国免疫学杂志

Objective:To analyze the expression of TNF-related apoptosis-inducing factor TRAIL,c-FLIP and caspase-8 in peripheral blood mononuclear cells of patients with rheumatoid arthritis at different period,in order to provide a experimental basis that treating and looking for a more effective way and method.Methods:mRNA expression of TRAIL,c-FLIP and caspase-8 were detected by Real-time PCR from peripheral blood mononuclear cells;TRAIL,c-FLIP and caspase-8 were checked by Western blot from peripheral blood mononuclear cells.Results:The mRNA expression level of TRAIL,c-FLIP and caspase-8 from PBMC at different groups of RA:TRAIL mRNA in group M was 3.26±0.78 which was much higher than healthy controls(1.30±0.20) (P=0.028),and those of group L and group H (1.56±0.37 and 1.83±0.26 respectively) was slightly higher than controls(P<0.05).C-FLIP mRNA in low activity group (L),middle activity group (M) and high activity group (H) were 1.27±0.28,1.32±0.34 and 1.93±0.40 re-spectively,which was higher than that of healthy controls (1.08 ± 0.12) (P=0.035,0.034,0.030).The relative level caspase-8 mRNA in group L,group M,group H were(2.77 ±0.97),(4.52 ± 0.85),and(2.13 ± 0.44)which was higher than healthy controls (1.04±0.13) (P=0.023,0.012,0.032).The protein level of TRAIL,c-FLIP and caspase-8 in PBMC from different groups of RA patients:The expression of TRAIL in group M was significantly increased than group L,H and control(P<0.05) with no difference in group L.c-FLIP protein in all protein expressed in group M quantity highest,significantly higher than other groups.Caspase-8 expression in the group M and H was significantly enhanced than control (P=0.003,0.001 ) with no difference in group L (P> 0.05). Conclusion:During the different active stages of RA,in peripheral blood mononuclear cells,the expression of TRAIL,c-FLIP and caspase-8 are increased overall trend,possible can provide certain experimental reference for clinical therapy.

Investigation on the mechanisms of p15INK4B gene demethylation by valproate in Molt-4 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the antitumour effects of sodium valproate (VPA) on the proliferation, differentiation and cell cycle of Molt-4 cell and to investigate its demethylation mechanisms.</p><p><b>METHODS</b>After Molt-4 cells trated with VPA at different concentrations, cell viability and growth curve were assessed by MTT assay. Cell cycle changes were analyzed by flow cytometry. The expression level of p15, DNA methyltransferase 1 (DNMT-1), DNMT3A and 3B mRNA were detected by RT-PCR and the methylation level was detected by hn-MSPCR.</p><p><b>RESULTS</b>VPA significantly inhibited the proliferation of Molt-4 cells. After 48 h culture with 5.0 mmol/L VPA, the percentages of Molt-4 cells in G(0)/G(1) phase was (66.87 ± 3.31)% and in S phase was (8.47 ± 2.56)%, while in control group, the cells in G(0)/G(1) phase increased and in S phase decreased significantly. The p15 gene in Molt-4 cells failed to express due to its hypermethylation. The expression level of p15 gene mRNA increased significantly after exposure to VPA for 48 h. As compared with control group, the expression of DNMT-1 was down-regulated in a dose-dependent manner. The expression level of DNMT3B decreased at 10.0 mmol/L concentration.</p><p><b>CONCLUSION</b>VPA has a demethylation effect on p15 INK4B gene by inhibiting the DNMT-1 and DNMT3B gene activities to recover p15 gene activity, which arrests Molt-4 cell in G(0)/G(1) phase.</p>