Effect of RYBP Gene Silencing on Sensitivity of HL-60 Cells to Chemotherapeutic Drugs / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of RYBP gene on sensitivity of HL-60 cells to chemotherapy drugs by using RNA interference.</p><p><b>METHODS</b>Plasmid expressing RYBP specific shRNA was constructed and then was used to establish the RYBP knockdown stable HL-60 cell line. Q-PCR and Western blot were used to confirm the efficacy of RYBP gene silencing at mRNA and protein level respectively; then the DNA ladder and Annexin V labeled flow cytometry were used to detect cell apoptosis; CCK-8 was used detect the sensitivity of HL-60 cells to the chemotherapeutic drug cytarabine or daunorubicin.</p><p><b>RESULTS</b>The lentiviral-RYBP-shRNA vector was succesfully and effectively inhibit the expression of RYBP at mRNA and protein in HL-60 cells. It was found that without chemotherapy drug treatment the apoptosis rate of RYBP shRNA group was lower than that of the empty vector control group (NC group). When treated with cytarabine, the apoptosis rate and inhibitive rate of RYBP shRNA group were lower than those of NC group. Besides, when treated with daunorubicin, the apoptosis rate of RYBP shRNA group was lower than that of NC group, while the inhibitive rate had no significant difference.</p><p><b>CONCLUSIONS</b>RYBP gene silencing can inhibitive the apoptosis of HL-60 cells and significantly reduce the sensitivity to cytarabine, but this gene silencing can't affect the sensitivity to daunorubicin.</p>

Expression of histone demethylase lysine specific demethylase 1 in acute leukemia and its clinical significance / 中国实验血液学杂志

The aim of this study was to investigate the expression of histone demethylase lysine specific demethylase1 (LSD1) in patients with acute leukemia (AL) and its clinical significance. LSD1 protein expression level was detected by semi-quantitative Western blot in HL-60 and SHI-1 leukemia cell line, in bone marrow mononuclear cells of acute AL patients with different condition [new diagnosis, complete remission (CR) and relapse] and in patients with non malignant hematopathy (control). Clinical data of AL patient followed up was collected. The relationship of LSD1 expression level with clinical prognosis was analyzed. The results showed that in HL-60 and SHI-1 leukemia cell line, LSD1 expression was strong positive, relative amount (LSD1/β-actin gray level rate) was 4.647 ± 3.840 and 1.628 ± 0.185 (n = 4) respectively. In 72 AL patients, LSD1 expression levels were quite different. LSD1 positive rate was 56.9% (41/72), average relative amount was 1.053 ± 1.976. In 17 controls, LSD1 positive rate was 0%, relative amount was 0.004 ± 0.012. The LSD1 positive rate in newly diagnosed AML or ALL group (90.4%, 77.8%) and refractory/relapse AML or ALL group (100%, 100%) was higher than that in AML or ALL CR group (4.7%, 0%) (p = 0.000), relative amount of LSD1 showed no statistically difference between newly diagnosed AML and ALL groups (1.177 ± 1.646, 1.275 ± 1.845) or refractory/relapse group (2.050 ± 2.470, 4.107 ± 3.676) and CR group (0.029 ± 0.033, 0.019 ± 0.024) (p > 0.05). In all AL patients, LSD1 positive rate in newly diagnosed (84.6%) and refractory/relapse groups (100%) was higher than that in CR group (3.8%). LSD1 relative amount in newly diagnosed group (1.274 ± 1.760), refractory/relapse group (3.359 ± 3.319) and CR group (0.027 ± 0.031) was higher than that in control group (p < 0.01), and in refractory/relapse group was higher than that in newly diagnosed group and CR group (p < 0.01), in newly diagnosed group was higher than that in CR group (p < 0.01). It is concluded that overexpression of LSD1 is correlated with refractory or relapse in AL. LSD1 expression level can reflect disease status of AL patients and may be a predictive biomarker for unfavourable prognosis of AL.

Effect of different cytokine combinations on the expression of CD49d and CXCR4 and ex vivo expansion of umbilical cord blood mononuclear cells / 中国实验血液学杂志

This study was purposed to explore the effect of different cytokine combinations on the expansion of the mononuclear cells drived from umbilical cord blood (CB) ex vivo and expression of CXCR4 and CD49d on CD34+ cells after expansion. Human fresh CB mononuclear cells were cultured in serum-free and stroma-free medium containing different combinations of cytokine for 7 days. At day o and 7, the total cells were counted, CD34+ cells and CD34+CXCR4+, CD34+CD49d+ cells were assayed by flow cytometry, and CFU were determined. According to the different combinations of cytokine, experiments were divided into four groups: control, SF group (SCF + FL), SFT group (SCF + FL + TPO) and SFT6 group (SCF + FL + TPO + IL-6). The results showed that the SF (SF group) combination supported only low expansion of total cells, CD34+ cells and CFU. The addition of TPO in SF group restored UCB stem/progenitors expansion to a higher level than that in SF group, while there was no difference between groups SFT and SFT6 (P > 0.05). The cytokine combinations in groups SF, SFT and SFT6 all could upregulate the expression levels of CD49d and CXCR4 on expanded cord blood CD34+ cells, but there were no significant differences between groups SF, SFT and SFT6 (P > 0.05). It is concluded that SCF + FL has no strong synergistic effects on primitive hematopoietic cells. TPO plays an important role in enhancing expansion of umbilical cord blood hematopoietic cells, while IL-6 only shows a neutral effect on it. SCF + FL + TPO combination not only promotes progenitor cells expansion but also upregulates the expression of CD49d and CXCR4 on CD34+ cells from cord blood.

In vitro expansion of cord blood mononuclear cells supported by fetal bone marrow stromal cells and cytokines / 中国实验血液学杂志

This study was aimed to explore the role of human fetal bone marrow stromal cells (FBMSC) in combination with exogenous cytokines in supporting the in vitro expansion of cord blood mononuclear cells and the expression of CXCR4(+) and CD49d(+) in CD34(+) cells. Mononuclear cells (MNC) separated from cord blood (CB) were cultured in a serum-free support culture system with FBMSC or exogenous cytokines or both of them. On day 0, 6, 10 and 14, total cells were counted, CD34(+), CD34(+)CXCR4(+) and CD34(+)CD49d(+) cells were quantitated by FACS, and hematopoietic progenitor cells were assessed by semisolid culture assay. The results showed that after culturing for 14 days, CD34(+) cells, CD34(+)CXCR4(+) cells, CD34(+) CD49d(+) cells and colony forming unit (CFU) were significantly increased (P < 0.05). Compared with other groups, expansion multiple of CD34(+), CD34(+)CXCR4(+), CD34(+)CD49d(+) cells and CFU were higher than that in FBMSC and cytokine group (P < 0.05). It is concluded that the culture system used in this study can not only support the expansion of CB MNCs but also increase the number of hematopoietic stem and progenitor cells which has chemokine and adhesion capacity. This culture system may be a feasible way for in vitro culture of cord blood cells.