Effect of TGF-beta1 on biological characteristics of umbilical cord blood hematopoietic progenitor cells during ex-vivo expansion / 中国实验血液学杂志

To investigate the effects of TGF-beta1 on biological characteristics of hematopoietic progenitor cells (HPC) in umbilical cord blood (UCB) during ex-vivo expansion and feasibility of using it for expansion of UCB HPC, different concentrations of TGF-beta1 were added in the serum-free medium containing a combination of hematopoietic growth factors for expansion of UCB CD133(+) cells and enumeration of nucleated cells (NC), progenitor colonies, immunophenotyping, cell cycle and expression of adhesion molecules of the NC were monitored at every interval. The results showed that total number and expansion of NC from all groups of TGF-beta1 were remarkably less than those in control at each interval. However the content and total numbers as well as expansion of CD34(+), CD133(+), CD34(+)CD38(-) and CD34(+)CD133(+) cells from all groups of TGF-beta1 were more than those in control at each interval during expansion; the plating efficiency and expansion of CFU-GM, CFU-mix and HPP-CFC from NC of TGF-beta1 group were more than those in control at each interval. The contents of cells in G(0)/G(1) phase of NC of TGF-beta1 group at every interval were high. Meanwhile, TGF-beta1 could elevate the expression of some adhesion molecules on NC during expansion such as CD54, CD49d and CD11a, and the contents of CD34(+) cells coexpressing these adhesion molecules in NC of TGF-beta1 group were significantly more than those in control at each interval. In conclusion appropriate dose of TGF-beta1 could accelerate expansion of CD133(+) cells, delay and decrease over-differentiation of HPC, increase the content of HPC in expanded products, upregulate the expression of adhesion molecules on expanded HPC, thus it could promote engraftment of expanded progenitor cells and advantage the ex-vivo expansion of UCB HPC.

Preliminary study on the molecular mechanism of K562 cell apoptosis induced by As2S2 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the apoptotic inducing effect of As(2)S(2) on K562 cells.</p><p><b>METHODS</b>The apoptotic inducing effect of As(2)S(2) on K562 cells was determined by flow cytometry, DNA fragmentation analysis and morphology observation. Expression of protein was determined by Western-blot. RT-PCR was used to evaluate changes in gene expression.</p><p><b>RESULTS</b>Apoptosis of K562 cells was induced by 48 - 72 h exposure to 5 micromol/L As(2)S(2). Apoptosis was induced in (34.4 +/- 3.3)% treated cells by 72 h exposure to 3 micro mol/L As(2)S(2), in (21.8 +/- 3.6)% treated cells by 48 h exposure to 5 micromol/L As(2)S(2) and in (46.0 +/- 5.2)% treated cells by 72 h exposure to As(2)S(2) at the same concentration. With 5 micromol/L As(2)S(2), the protein level of Bcr-Abl and JAK2 decreased, while bax expression was upregulated and c-myc was downregulated both in protein and mRNA level. The activity of caspase 3 in K562 cells was increased by As(2)S(2). As(2)S(2) also induced apoptosis of fresh mononuclear cells derived from chronic myelogenous leukemia (CML) patients.</p><p><b>CONCLUSION</b>As(2)S(2) can induce apoptosis of CML cells. The decline of Bcr-Abl may play an important role. The upregulation of bax, increase of the activity of caspase 3, downregulation of c-myc and decrease of JAK2 may also be involved in the mechanism.</p>

Studies on the dynamics of biological characteristics of CD133+ cells from human umbilical cord blood during short-term culture / 中国实验血液学杂志

This study was to investigate dynamics of biological properties of CD133(+) cells from human umbilical cord blood (UCB) during short-term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133(+) cells. The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133(+) cells were monitored during ex-vivo expansion, and compared with those of CD34(+) cells. The results showed that the contents of CD133(+) and CD34(+) cells in fresh UCB were (1.05 +/- 0.73)% and (1.40 +/- 0.56)% respectively. About 79.62% of CD34(+) cells expressed CD133, and more than 97% of CD133(+) cells were CD133(+)CD34(+), markedly higher than that in CD34(+) fraction (P < 0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin-A between the two fractions. Expansion of CD133(+), CD133(+)CD34(+) and CD34(+)CD38(-) cells at 10 days and those of CFU-mix, HPP-CFC and CD34(+)CD38(-) cells at 6 days from CD133(+) cells group were significantly higher than those from the CD34(+) cell group (P < 0.05). Analysis of immunophenotype showed that CD133(+)CD34(+) cells declined gradually while CD133(-)CD34(+) and CD133(-)CD34(-) cells increased during ex-vivo expansion; basal telomerase activities of fresh UCB CD133(+) and CD34(+) cells were low but significantly exceeded that of CD34(-) fraction (P < 0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133(+) cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34(+) subsets were not affected significantly during expansion. It is concluded that CD133(+) population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34(+) cells, CD133(+) cells have great expansion potential for ex-vivo expansion and is a suitable target cell for ex-vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.

Synergism of As2S2 and STI 571 in inducing apoptosis of K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the synergistic effect of As(2)S(2) and STI 571 on K562 cells and its mechanism.</p><p><b>METHODS</b>The inhibitive effect of As(2)S(2) on the proliferation of K562 cells was determined by cell number count. Cell apoptosis was assessed by flow cytometry, DNA fragmentation and morphology. Protein expression was determined by Western-blot and gene expression by RT-PCR.</p><p><b>RESULTS</b>As(2)S(2) could significantly inhibit the proliferation and induce apoptosis of K562 cells in a dose and time-dependent manner at concentrations from 1 micromol/L to 5 micromol/L for 24 approximately 72 h. 34.4%, 21.8% and 46.0% of the treated-cells displayed apoptosis at 3 micro mol/L for 72 h, 5 micromol/L for 48 h and 5 micromol/L for 72 h, respectively. Compared to treatment with STI571 (0.25 approximately 1.00 micromol/L) or As(2)S(2) (1 approximately 5 micromol/L) alone, treatment of K562 cells with As(2)S(2) and STI571 combination induced more cell apoptosis. (18.4 +/- 1.4)% and (15.8 +/- 1.2)% cells underwent apoptosis at 1 micromol/L STI571 for 48 h and 5 micromol/L As(2)S(2) for 48 h, respectively, and (40.6 +/- 2.0)% cells did in combination treatment (P < 0.05). For U937 cells, the percentages of apoptotic cells were (6.0 +/- 1.1)% at 1 micromol/L STI571 for 48 h, (4.5 +/- 1.2)% at 5 micromol/L As(2)S(2) for 48 h, and (7.3 +/- 1.0)% in combination treatment. As(2)S(2) decreased the bcr-abl fusion protein expression and PTK activity of c-abl and bcr-abl, but not for bcr-abl expression.</p><p><b>CONCLUSION</b>Combination treatment with As(2)S(2) and STI 571 induced more apoptosis of K562 cells. The reduction of PTK activity may be involved in the mechanisms.</p>

New Retinoid SX-116 Induces Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 / 中国实验血液学杂志

In this research, the effect of novel retinoid SX-116 on acute promyelocytic leukemia cell line NB4 was studied in vitro. Cell proliferation, cell morphological characters, flow cytometry, DNA electrophoresis and RT-PCR were observational parameters. The results showed that treated with SX-116 at 10(-6) mol/L, the growth and survival of NB4 cells were markedly inhibited, morphological changes of apoptosis, including membrane blebbing, chromosome condensation and fragmentation of nuclei were observed in NB4 cells after 24 hours exposure of SX-116. Further studies showed "DNA ladder" in genomic DNA electrophoresis, as well as a typical apoptotic peak below G(1) phase presented in flow cytometry. The expression of apoptosis - related gene bcl-2 and p53 were examined. The level of bcl-2 mRNA was downregulated by 6-hour treatment of SX-116, while the gene restored to the normal level by following 12-, 24- and 48-hour exposure. However, p53 mRNA was unchanged during the treatment. The results demonstrated that SX-116 could induce apoptosis of NB4 cells while the mechanism remains to be studied.