Analysis of the Physiological Activities and Functions in Vitro of Apheresis Platelets during Storage / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the changes of physiological activities and functions in vitro of apheresis platelets during storage.</p><p><b>METHOD</b>17 units of apheresis platelets were randomly chosen and stored at 20 °C to 24 °C with agitation. Platelet counting (Plt), mean platelet volume (MPV), blood gases, pH value, glucose (Glu) concentration, lactate (LA) concentration, LDH concentration, thromboelastogram (TEG), hypotonic shock response (HSR), CD62p expression rate and anew expression rate were measured on days 0, 1, 3, 5 after platelet storage. Changes of physiological activities and functions in vitro were systematically evaluated by above-mentioned indexes.</p><p><b>RESULTS</b>During storage, Plt, MPV and HSR were not significantly changed; but pH value, blood gases, Glu, LA, LDH, HSR, expression rate of CD62p and anew expression rate were significant differenty. Among thromboelastogram indexes, R value increased obviously with prolongation of storage time; K value and αAngle were not significantly changed; MA was not significantly changed on day 1 and 3, but was slightly increase on day 5.</p><p><b>CONCLUSION</b>The physiological activities and functions in vitro of apheresis platelets are kept well during storage. For clinical transfusion of apheresis platelet during storage, clinical effect of transfusione is not influenced.</p>

MIP-1α promotes the migration ability of Jurkat cell through human brain microvascular endothelial cell monolayer / 中国实验血液学杂志

This study was purposed to explore the mechanism of central nervous system (CNS) leukemia resulting from brain metastasis of human acute T-cell leukemia (T-ALL) cells and the role of MIP-1α in migration of Jurkat cells through human brain microvascular endothelial cells (HBMEC). The real-time PCR, siRNA test, transendothelial migration test, endothelial permeability assay and cell adhesion assay were used to detect MIP-1α expression, penetration and migration ability as well as adhesion capability respectively. The results showed that the MIP-1α expression in Jurkat cells was higher than that in normal T cells and CCRF-HSB2, CCRF-CEM , SUP-T1 cells. The MIP-1α secreted from Jurkat cells enhanced the ability of Jurkat cells to penetrate through HBMEC, the ability of Jurkat cells treated by MIP-1α siRNA to adhere to HBMEC and to migrate trans endothelial cells decreased. It is concluded that the MIP-1α secreted from Jurkat cells participates in process of penetrating the Jurkat cells through HBMEC monolayer.

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.

Comparative analysis of collecting mononuclear cells from peripheral blood by using Fenwal CS-3000 plus, haemonetics MCS plus and COBE spectra separators / 中国实验血液学杂志

This study was aimed to compare the collection efficiency of mononuclear cells (MNC) from peripheral blood as well as the changes of blood-related indices in patient by using 3 cell separators. MNC were collected from 94 tumor patients by using Fenwal CS-3000plus, Haemonetics MCSplus and COBE spectra separators. Routine blood test was performed before and after MNC collection to detect the potential effects of cell separators on blood-related indices in the patients. MNC count was performed. The percentages of CD3(+), CD4(+) and CD8(+) in peripheral blood cells were determined. The results showed that the MNC counts were (3.08 ± 0.79)×10(9), (3.21 ± 1.12)×10(9), and (3.22 ± 1.84)×10(9) per bag by CS-3000plus, MCSplus and COBE spectra, respectively. And the corresponding decrease of platelet percentage was (6.86 ± 5.70)%, (8.05 ± 5.14)% and (5.89 ± 4.48)%, respectively. The CD3, CD4 and CD8 ratios in peripheral blood of patients before and after treatment were significantly statistical different (P < 0.001). It is concluded that the MNC collection can be performed successfully with CS-3000plus, MCSplus and COBE spectra, and their collections can meet the needs in clinic.

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.

Correlation between reactive oxygen species of cryopreserved peripheral blood mononuclear cells and expression of homing adhesion molecules on peripheral blood hematopoietic stem/progenitor cells / 中国实验血液学杂志

This study was purposed to detect the level of reactive oxygen species (ROS) in cryopreserved peripheral blood mononuclear cells (PBMNC) and the expression of homing molecules on peripheral blood hematopoietic stem/progenitor cells, and to analyze the correlation between ROS level and the expression of homing molecules. The survival percentage of PBMNC was determined by trypan blue exclusion rate. The expression of antigens on peripheral blood hematopoietic stem/progenitor cells, such as CD34, CD133 and some related homing molecules (VLA4, CXCR4 and CD44) were detected by flow cytometry. The correlation of the ROS levels with the expression of homing molecules was evaluated by the correlation coefficient. The results showed that the survival rate of PBMNC was gradually reduced along with prolongation of cryopreserved time, and there was a significant difference in the survival percentage of cryopreserved PBMNC between experimental group frozen for 3, 6, 9, 12 months and control group (P < 0.05). The expression of marked antigen CD34 and CD133, and homing adhesive molecules of CD44, VLA4 and CXCR4 declined with the extension of frozen time, and there was significant statistical difference (P < 0.05) between experimental group cryopreserved for a year and control group. The level of ROS in PBMNC gradually increased along with the prolongation of cryopreserved time, and there was significant statistical difference (P < 0.05) between experimental group freezing-stored for 6, 9 and 12 months and control group. The correlation coefficients of ROS level and expression percentage CD34(+) VLA4(+), CD34(+) CXCR4(+), CD34(+) CD44(+) PBMNC were -0.50, -0.457, -0.465, respectively. It is concluded that ROS level in PBMNC increases significantly with the prolonged cryopreservation time, especially for more than 6 months. There is a significant negative correlation between the ROS level of PBMNC and the expression rate of homing molecules. Higher level of ROS may be one of the reasons for the reduction of homing capability in peripheral blood stem/progenitor cells after cryopreservation.

Effect of different oxygen concentrations on biological properties of bone marrow hematopoietic stem cells of mice / 中国实验血液学杂志

This study purposed to investigate the effects of different oxygen concentrations and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and their possible mechanisms through simulating oxygen environment to which the peripheral blood HSC are subjected in peripheral blood HSCT. The proliferation ability, cell cycle, directed differentiation ability, ROS level and hematopoietic reconstitution ability of Lin(-)c-kit(+)Sca-1(+) BMHSC were detected by using in vitro amplification test, directional differentiation test, cell cycle analysis, ROS assay and transplantation of Lin(-)c-kit(+)Sca-1(+) HSC from sublethally irradiated mice respectively. The results showed that oxygen concentrations lower than normal oxygen concentration, especially in hypoxic oxygen environment, could reduce ROS generation and amplify more primitive CD34(+)AC133(+) HSC and active CD34(+) HSC, and maintain more stem cells in the G(0)/G(1) phase, which is more helpful to the growth of CFU-S and viability of mice. At the same time, BMHSC exposed to normal oxygen level or inconstant and greatly changed oxygen concentrations could produce a high level of ROS, and the above-mentioned features and functional indicators are relatively low. It is concluded that ROS levels of HSC in BMHSCT are closely related with the oxygen concentration surrounding the cells and its stability. Low oxygen concentration and antioxidant intervention are helpful to transplantation of BMHSC.

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.