Expression Changes of Hepcidin and Ferroportin 1 in Murine Model of Iron Overload / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the changes of hepcidin and ferropotin 1 expression in murine model of iron overload.</p><p><b>METHODS</b>The murine model of iron overload was established, C57BL/6 mice were injected with iron dextran intraperitoneally (10 mg) every 3 days for 4 weeks. Blood routine, serum ferritin and pathological sections were tested at the appointed time-point respectively (before iron injection, 2 weeks and 4 weeks after treatment of iron injection). The serum hepcidin was assayed by enzyme-linked immunosorbent method. The expression of ferroportin 1 in bone marrow cells was detected by RT-PCR and Western blot, respectively. The labile iron pool of bone marrow cells was measured by flow cytometry.</p><p><b>RESULTS</b>The absolute number and percentage of reticulocytes in the iron-overloaded mice were significantly decreased along with the increase of iron injection times (r=-0.938, r=-0.947), while no significant change was found in the number of white blood cells, hemoglobin level and platelet count. The level of serum ferritin was increased along with increase of iron injection time (r=0.894). Iron overload was found in pathological sections of different organs. Furthermore, serum hepcidin was increased along with increase of iron injection time (r=0.957). RT-PCR and Western blot analyses showed that the expressions of ferroportin 1 at mRNA and protein level were increased in the murine model of iron overload (P<0.05). Labile iron pool in bone marrow cells was also found to be increased in the murine model of iron overload(P<0.05).</p><p><b>CONCLUSION</b>The expressions of hepcidin and ferroportin 1 are increase in a murine model of iron overload, which may be contributed to the suppression effect on erythropoiesis in bone marrow.</p>

Influence of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice / 中国实验血液学杂志

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.

Effects of CD4(+)CD25(+) regulatory T cells on allogeneic hematopoietic stem cell transplantation in sensitized mice / 中国实验血液学杂志

The aim of this study was to investigate the effects of CD4(+)CD25(+) regulatory T cells (Treg) on allogeneic hematopoietic stem cell transplantation (HSCT) in sensitized mice so as to provide experimental evidence for clinical treatment of allogeneic HSCT rejection in sensitized recipients. The BALB/c mice were divided into 5 groups: group A - mice were sensitized with injection of splenocytes; group B - mice were sensitized with splenocytes and treated with >5×10(5) Treg on day 7 before transplantation; group C - mice were sensitized with splenocytes and treated with 5×10(5) Treg on day 13 and 7 before transplantation; group D - mice were not sensitized, but treated with equal volume of PBS as control; group E - blank control. Each group had 15 mice. On day 0 of transplantation, mice in each group were irradiated lethally with 8 Gy by linear accelerator, and the bone marrow cells of C57BL/6 labeled by fluorescence staining were intravenously injected via the tail vein. The fluorescent cells in peripheral blood and organ tissue were detected by flow cytometry on different time points for homing assessment. Survival rates and hematopoietic reconstitution were also recorded and monitored. The results showed that on 12 and 24 hours after transplantation, as compared with the sensitized group, the number of fluorescence homing cells in different tissue of the applied Treg groups increased significantly and the differences were statistically significant (P < 0.05). The mice in sensitized group and blank control group all died on the 6-13 day, whereas the median survival time of mice in applied Treg once and twice were 15 days and 16 days respectively. Comparing with sensitized group, the difference was statistically significant (P < 0.001), but there was no significant difference between these two groups applied regulatory T cell (P > 0.05). It is concluded that applying Treg can induce immune tolerance of sensitized recipient to allogeneic HSCT and inhibit immune destruction and prolong the survival time, but can not induce full immune tolerance and at last sensitized mice died of rejection of hematopoietic stem cells.

Separation and amplification of CD4(+)CD25(+) regulatory T cells from sensitized mice / 中国实验血液学杂志

The aim of this study was to separate and amplify CD4(+)CD25(+)Treg cells from splenocytes of sensitized mice. The percentage of CD4(+)CD25(+)Treg cells was detected by flow cytometry in sensitized and normal control mice. CD4(+)T, CD4(+)CD25(+)Treg and CD4(+)CD25(-) T cells were isolated from mouse splenocytes by MACS. CD4(+)CD25(+)Treg cells were expanded in vitro cultures in addition of CD3/CD28 MACSiBead and IL-2. The activity of cells was detected with 0.4 trypan blue staining. The purity of cells after sorting, the main surface marker and the level of Foxp3 were detected by flow cytometry. The results showed that CD4(+)CD25(+)Treg cell proportion was higher in sensitized mice than normal control mice (P < 0.05). The average purity of CD4(+)CD25(+)Treg cells was 87. The activity of these cells was more than 97, and the expression of Foxp3 in these cells was high. The amplification multiples achieved 42 times after 2 weeks in vitro. The percentage of CD4(+)CD25(+) regulatory T cells was 85.32, and the expression of Foxp3 decreased from (76.92 ± 1.72) to (75.33 ± 2.11) (P > 0.05). It is concluded that the sorting of CD4(+)CD25(+)Treg cells is isolated successfully by MACS without affecting the vitality of target cells. The amplification of CD4(+)CD25(+)Treg cells is successful in vitro. Expression of surface markers and Foxp3 gene does not obviously change after amplification, so that to establish a practical method to recover and enlarge the amount of CD4(+)CD25(+)Treg cells in good condition.

Effect of thalassemia panel reactive antibody on proliferation and apoptosis of cord blood CD34(+) cells / 中国实验血液学杂志

The study was purposed to explore the effect of panel reactive antibody (PRA) serum from patients with β-thalassemia on proliferation and apoptosis of the CD34(+)cells from cord blood and its mechanism. CD34(+) cells of umbilical cord blood were incubated with different sera and complement respectively. After incubation, the samples were centrifuged and the supernatants were collected for lactate dehydrogenase (LDH) detection, and the CD34(+) cells were harvested and measured for the apoptosis by flow cytometry with Annexin V/PI. The intracellular DNA synthesis were also quantified by [(3)H]TdR incorporation using liquid scintillation counter. The results showed that concentration of LDH in PRA positive groups was higher as compared with control group, and the DNA synthesis of CD34(+) cells in PRA positive groups were inhibited. There were no differences in the percentage of cell apoptosis and necrosis among different groups. It is concluded that thalassemic serum PRA impairs the cell membrane, inhibits the DNA synthesis, which can be increased by addition of the complement, but PRA had no significant effect on apoptosis of CD34(+) cells.

Effects of mesenchymal stem cells on lymphocyte proliferation of sensitized mice in vitro and their action manner / 中国实验血液学杂志

This study was purposed to investigate the effects of mesenchymal stem cells (MSC) on lymphocyte proliferation of sensitized mice in vitro and their action manner so as to more understand the possible mechanisms of bone marrow-derived MSC action on the engraftment of hematopoietic stem/progenitor cells in sensitized mice. Bone marrow-derived MSC were cultured by adherent culture method, the MSC or culture supernatant were used as immune cells or immunologic factor, the spleen lymphocytes of sensitized mice were used as effector cells. The phytohemagglutinin (PHA) was used to stimulate the lymphocyte proliferation, the MTT method was used to detect the mixed lymphocyte reaction. The results showed that bone marrow-derived MSC could inhibit the lymphocyte proliferation of sensitized mice, the MSC cultured supernatant also exhibit this effect. Moreover, the inhibitory effect of MSC supernatent increased along with the increase of cell ratio and concentration, while ratio of these two kind of cells was 1:1, the inhibitory effect was the highest (p < 0.05). It is concluded that the MSC can suppress lymphocyte proliferation of sensitized mice in vitro through cell-cell direct contact or cell-cell indirect interaction.

Experimental study on rejection of allogeneic donor bone marrow cells in sensitized recipients / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a murine model of sensitization, and investigate the effect and mechanism of sensitization on allogeneic donor bone marrow cells (BMCs).</p><p><b>METHODS</b>Sensitized BALB/c mice were established by transfusions of allogeneic splenocytes. The donor reactive antibodies were detected by binding and complement-dependent cytotoxicity assays. After irradiation, 1 × 10(7) BMCs of C57BL/6 donor mice were injected into non-sensitized or sensitized BALB/c recipient mice. The distribution pattern of donor BMCs in peripheral blood, spleen and bone marrow of recipient mice were analyzed at different time points (2 h, 12 h and 48 h) post transplantation. Hematopoietic recovery post transplantation was assessed, and survival was monitored. Moreover, sera and splenocytes derived from non-sensitized or sensitized recipients were incubated with allogeneic BMCs in vitro, and the cytotoxic indexes were calculated in the immune experiments.</p><p><b>RESULTS</b>The binding and complement-dependent cytotoxicity assays showed that a high level of donor reactive antibodies was presented in sensitized sera. Compared with the non-sensitized recipients, the homing assay showed significantly decreased distributions of allogeneic donor BMCs in peripheral blood, spleen and femur of sensitized recipients. Non-sensitized recipients survived long term after irradiation, while all the sensitized recipients died within 12-15 days. Fourteen days post transplantation, the white blood cells and BMCs of non-sensitized recipients were (3240 ± 300) × 10(6)/L and (396 ± 27) × 10(6)/femur, respectively; while the white blood cells and BMCs of sensitized recipients were (320 ± 80) × 10(6)/L and (6 ± 2) × 10(6)/femur, respectively; the differences were statistically significant between this two groups (P < 0.05). Seven days post transplantation, the percentage of donor cells in bone marrow of non-sensitized and sensitized recipients was (48.07 ± 4.70)% and (0.77 ± 0.11)%, respectively, and the differences were statistically significant (P < 0.05). Furthermore, the white blood cells and BMCs following transplantation decreased along with time in sensitized recipients. The immune experiments of complement-dependent cytotoxicity reaction, cytotoxic T lymphocytes reaction and antibody-dependent cellular cytotoxicity showed the cytotoxic indexes were higher in sensitized group than the non-sensitized group.</p><p><b>CONCLUSION</b>A sensitized model was established by transfusions of allogeneic spleen cells. Allogeneic donor BMCs were rejected in sensitized recipients, and its mechanism might be through immune impairment pathways.</p>

Transfusion of hematopoietic stem/progenitor cells into marrow cavity in sensitized mouse model / 中国实验血液学杂志

The study was aimed to investigate the strategy of transfusion of allogeneic hematopoietic stem/progenitor cells (HS/PC) into marrow cavity of mouse model in sensitized transplantation. A sensitized BALB/c mouse model was established by repeated transfusion of allogeneic spleen cells. The normal BALB/c mice were used as non-sensitized controls. The non-sensitized or sensitized recipients were transplanted by transfusion of allogeneic HS/PCs into bone marrow cavity. The survival rate and hematopoietic recovery were monitored. Moreover, non-sensitized and sensitized sera were obtained and incubated with allogeneic HS/PC respectively, the percentage of dead cells was calculated using complement-dependent cytotoxicity (CDC) tests. The results showed that non-sensitized recipients got long-term survival after the transfusion of HS/PC into marrow cavity, and the hematopoietic recovery increased along with time. However, among the sensitized recipients, one mouse died of anesthetic accident, the other 9 mice (9/10) died within 2 weeks after the transfusion of HS/PC in marrow cavity, and the hematopoietic recovery declined along with time. Histopathologic analysis demonstrated that the sensitized recipients died of bone marrow failure. The results of CDC tests showed that the percentage of dead cells in non-sensitized and sensitized group was 7.80 ± 1.93% and 50.80 ± 3.12%, respectively, and the differences were statistically significant (p < 0.05), indicating sensitized sera were capable of impairing allogeneic HS/PC. It is concluded that the strategy of the marrow cavity transfusion of HS/PC can not enhance engraftment of allogeneic donor cells in sensitized recipients.

Therapeutic effect of focal adhesion kinase gene silence on leukemia / 中国实验血液学杂志

This study was aimed to investigate the effects of focal adhesion kinase (FAK) gene silence on leukemia cell growth, leukemogenesis and efficacy of chemotherapy drug. Vector containing lentiviral-FAK-shRNA was constructed and transfected into BCR/ABL-BaF3 leukemic cells, the cell growth and apoptosis were detected in vitro. The effect of FAK shRNA on leukemogenesis was studied in a murine model with leukemia. The apoptosis of leukemia cells and survival of leukemic mice treated by FAK shRNA combined with drug STI571 were monitored. The results showed that FAK gene expression was knocked down by lentiviral-FAK-shRNA. FAK gene silencing inhibited leukemia cell growth in vitro. The apoptosis test results showed that the percentages of Annexin V(+) cells in vector control group and FAK shRNA group were (3.46 ± 0.56)% and (7.3 ± 0.79)%, respectively, and the difference was statistically significant (p < 0.05). The mice in vector control group died at day 21 to 27, while the mice in FAK shRNA group died between day 52 and 60, and the difference was statistically significant (p < 0.05). Moreover, FAK gene silence combined with drug STI571 could enhance the apoptosis of leukemia cells and prolong survival time of leukemic mice. It is concluded that FAK gene silence inhibits leukemogenesis and promotes efficacy of chemotherapy drug on leukemia cells, indicating FAK gene silence may be considered as a new therapeutic strategy for leukemia.

Effect of the serum panel reactive antibody on proliferation and differentiation of cord blood CD34+ cells in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>The low rate of engraftment in children with beta-thalassemia has seriously restricted the popularity of the hematopoietic stem cell transplantation (HSCT). Panel reactive antibody (PRA) has been regarded as one of the important factors for the success of kidney transplantation. Poly-transfusion before transplantation is associated with the production of PRA. Also PRA is produced in the children with beta-thalassemia major who need poly-transfusion for life. PRA might be one of factors inducing the low rate of engraftment in children with beta-thalassemia. This study focused on observing the effect of PRA on the proliferation, differentiation, apoptosis and necrosis of cord blood CD34(+) cells in vitro by incubating the cord blood CD34(+) cells with serum containing PRA.</p><p><b>METHOD</b>Seven samples of cord blood were collected and the HLA typing for every sample was done. Seven sera positive for PRA and seven negative sera were selected respectively. Mononuclear cells (MNCs) were obtained by Ficoll-Hypaque density gradient centrifugation. CD34(+) cells were isolated from MNCs by positive selection using an immunomagnetic separation (CD34(+) progenitor cell isolation kit and auto-MACS). The CD34(+) cells of umbilical cord blood were incubated with the serum and complement in the following groups: A (absence of serum), B (presence of PRA positive serum), C (presence of PRA positive serum and complement), D (presence of complement), and E (presence of PRA negative serum). After incubation the samples were centrifuged and the supernatant was collected for LDH detection. At the same time the CD34(+) cells were harvested for assessing the expression of Annexin V and CD95 of the CD34(+) cells by flow cytometry and also for the detection of the DNA synthesis by (3)H-TaR incorporation. Meanwhile the cells were inoculated into the methylcellulose cultural system. The proliferation and hematopoietic potential of the CD34(+) cell of cord blood by the colony formation assay were detected on the day 10.</p><p><b>RESULT</b>The concentration of LDH in group A was (20.71 +/- 2.81) U/L, which was significantly lower than that in group B (64.28 +/- 5.12) U/L and group C (84.29 +/- 4.99) U/L. The concentration of LDH in group B was significantly lower than that in group C, while there were no significant differences in the concentration of LDH among groups A, D and E (P > 0.05). The cpm in group A was (22 629 +/- 3288), which was significantly higher than that in group B (4598 +/- 2178) and group C (1626 +/- 1192). And the cpm in group B was significantly higher than that in group C. There were no significant differences in the cpm among groups A, D and E (P > 0.05). On day 10 of culture, the total colonies, granulocyte-macrophage colony forming unit (CFU-GM), mixed colony forming unit (CFU-GEMM) and erythroid burst colony forming unit (BFU-E) in group A were significantly higher than that in group B and C. The total colonies, CFU-GM and CFU-GEMM in group B were significantly higher than those in group C. No significant differences were found in the total colonies, CFU-GM, CFU-GEMM and BFU-E among groups A, D and E (P > 0.05). There were no statistically significant differences in the CD95 and Annexin V expression among all the groups (P > 0.05).</p><p><b>CONCLUSION</b>PRA could impair the membrane, decrease the DNA synthesis, and inhibit the colony formation of CD34(+) cord blood cells, which could be strengthened by the presence of the complement at the given concentration in our study. PRA had no significant influence on the apoptosis of CD34(+) cells in vitro.</p>

Experimental study on the effect of sensitized sera on engraftment of hematopoietic stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of sensitized sera on engraftment of hematopoietic stem cells (HSCs) and its mechanism.</p><p><b>METHODS</b>Bone marrow cells (BMCs) from C57BL/6 mice were incubated with sensitized sera (group I) or normal sera (group II), and then washed and transplanted into irradiated BALB/c mice. The survival analysis and engraftment evaluation of the recipients were observed. The incubated BMCs were bound with goat anti mouse IgG for and labeled with Annexin V for apoptosis detection.</p><p><b>RESULTS</b>Seven out of ten recipient mice in group I died of bone marrow failure at day 10 after transplantation, while all of those in group II were long-term survived. Engraftment assay showed recipients blood count and BMCs progressively decreased along with time passing in group I; in addition, the chimeric percentage of donor cells progressively decreased as well. The percentage of BMCs binding with goat anti mouse IgG in group I or group II were (90.3 +/- 5.1)% and (5.2 +/- 2.4)%, respectively, and the difference was statistically significant (P < 0.01). However, no significant difference was found in the apoptosis detection between the two groups (P > 0.05).</p><p><b>CONCLUSION</b>The engraftment capacity of HSCs is significantly impaired by sensitized sera, the antibodies in sensitized sera may bind to antigens expressed on HSCs but do not induce apoptosis.</p>

Establishment of sensitized animal models and their sensitization effects on engraftment of hematopoietic stem cells / 中国实验血液学杂志

This study was aimed to establish sensitized animal models, explore the changes of immune function in these sensitized recipients, and investigate effects of sensitization on the engraftment of hematopoietic stem cells (HSCs). Different doses of spleen cells (1x10(5), 1x10(6) and 1x10(6)x2 at intervals of 7 days) from C57BL/6 were infused into BALB/c, the immunity function of sensitized models was tested by complement-dependent cytotoxicity method, mixed lymphocyte reaction and ELISA. After irradiation with gamma-ray of 60Co in dose 8 Gy, sensitized mice were transplanted 1x10(7) C57BL/6 bone marrow cells via tail vein or intra-bone marrow, and survival rate was detected daily. The results showed that different levels of donor reactive antibody were induced in all sensitized models. Comparing with normal mice, profound proliferation of spleen cells were found in groups of injected 1x10(6) and 1x10(6), continuous injections at intervals of 7 days. Sensitized model received bone marrow cells of C57BL/6 via tail vein died on day 10 to 14 after transplantation, and sensitized model mice received bone marrow cells of 1x10(6)x2 at intervals of 7 days via intra-bone marrow also died within two weeks after transplantation. It is concluded that different sensitized mouse models are established by different doses of allogeneic spleen cells infusion, the changes of immune function in sensitized mice are correlative with sensitization. Donor HSCs are rejected in sensitized models, and the engraftment can not be improved by intra-bone marrow injection.