Role of antioxidant in protecting the biological function of hematopoietic stem cells / 中国实验血液学杂志

In peripheral blood hematopoietic stem cell transplantation (PBHSCT) , the mobilization and circulating of bone marrow hematopoietic stem cells in blood with higher oxygen concentration all increase reactive oxygen species(ROS) production, which has negative effect on the biological function of BMHSC. In order to investigate the protective effect of antioxidant on hematopoietic stem cells (HSC), the ascorbic acid 2-phosphate (AA2P), an ascorbic acid derivative of vitamin C, was added in HSC culturing by imitating oxygen conditions which BMHSC experienced in peripheral blood stem cell transplantation. The protective effect of above-mentioned culture methods on the biologic functions of BMHSC was evaluated by vitro amplification assay, committed division assay, reactive oxygen species (ROS) measurement, CD34(+) HSC engraftment. The results showed that the ROS level in HSC from in vitro cultures was much higher than that freshly separated BMHSC, and the amplified AC133(+)CD34(+) HSC, BFU-E, CFU-GM, CFU-GEMM colonies, migration rate and severe combined immunodeficiency (SCID)-repopulating cells (SRC) were all much more than HSC cultured without AA2P. It is concluded that antioxidant intervention may be an effective methods for protecting the biological function of PBHSC and improving the therapeutic effect of PBHSCT.

Influence of reactive oxygen species on mouse bone marrow hematopoietic stem cell apoptosis / 中国实验血液学杂志

Objective of this study was to investigate the mechanism of the biological function damage resulting from increased ROS in peripheral blood stem cells during peripheral blood stem cell transplantation. Bone marrow hematopoietic stem cells (BMHSC) were cultured at the oxygen concentration imitated according to the bone marrow oxygen concentration (5% O2) including mean venous oxygen concentration (12% O2), mean arterial oxygen concentration (20% O2). The ROS level in BMHSC was detected by using fluorescent probe, the percentage of BM-HSC in cell cycle was determined by flow cytometry, the apoptosis rate was assayed by Annexin V/PI double staining, the expression levels of ATM gene and P21 protein were measured by PCR and Western respectively. The results showed that as compared with control group (5% O2), the ROS levels were lower, the percentage of cells in G1, S,G2/M phase increased (P < 0.01), the apoptosis rate of cells obviously increased (P < 0.01), the expression level of ATM gene obviously decreased (P < 0.01), while the expression level of P21 protein significantly was enhanced (P < 0.01) in 12% O2, 20% O2 and 5%-12%-20% O2 groups. It is concluded that ROS results in the apoptosis of BMHSC through inhibiting the expression of ATM gene and activating P21 protein.

Effect of various oxygen concentrations on biological function of human bone marrow hematopoietic stem/progenitor cells / 中国实验血液学杂志

Hypoxia in bone marrow is suitable for the perfect preservation of biological functions of bone marrow hematopoietic stem cells (BM HSC). It is deserved to study whether the biological functions of BM HSC are influenced when being exposed to environment of oxygen at various concentration during amplification of BM HSCs in normal oxygen condition in vitro and process of peripheral blood hematopoietic stem cell transplantation (PBSCT). This study was purposed to investigate the effects of various oxygen concentrations on biological functions of human BM HSCs. The BM HSCs were amplified in vitro, the amplification level of CD34(+) HSCs and CD34(+)AC133(+) HSCs were detected by flow cytometry, the apoptosis and cell cycle distribution of CD34(+) HSCs amplified in various oxygen concentrations were assayed by flow cytometry with Annexin V/PI double staining as well as PI and Ki-67 antibody, respectively, the differentiation of amplified CD34(+) HSCs in vitro was determined by direction differentiation assay, the migration ability of amplified CD34(+)AC133(+) HSCs was measured by migration test. The results indicated that the oxygen environment below normal oxygen, especially hypoxia, could amplify more primitive CD34(+)AC133(+) HSCs and CD34(+) HSCs with activity, arrest more HSCs in G₀/G₁ phase, promote the generation of BFU-E, CFU-GM, CFU-GEMM, and better preserve the migration ability of HSCs. While the above functional indicators of BM HSCs were poor when HSCs exposed to normoxia, oxygen-unstable and oxygen-severe changeable environments. It is concluded that the biological functions of BM HSCs in PBSCT are related with oxygen concentration and its stability, the culture of BM HSCs in lower oxygen environment may be more beneficial for PBSCT.

Effects of protein kinase C activity on CD44 expression and subcellular distribution in human erythrocytes / 中国实验血液学杂志

The study was aimed to investigate the effects of protein kinase C (PKC) on standard type CD44 expression and subcellular distribution in human erythrocytes. PKC activity was detected by the incorporation of [gamma-(32)P]-ATP into exogenous substrate, phosphorylation of CD44 was determined by autoradiograph, distribution of CD44 was observed by indirect immunofluorescence, and expression of CD44 was analyzed by flow cytometry. The results showed that PKC activity reached the maximal level at 30 minutes after treatment with phorbol-myristate-acetate (PMA), and the peak of CD44 phosphorylation and CD44 expression appeared at the same time, which all increased significantly as compared with control group (p < 0.001). PKC activation resulted in CD44 aggregation on membrane and colocalization of PKC and CD44. Calphostin C could inhibit the above reaction resulted from PKC activation. It is concluded that PKC activation can up-regulate CD44 expression by phosphorylation, and result in the coherent migration and colocalization of CD44 and PKC in human erythrocytes.