[Reactive oxygen species mediate the injury and deficient hematopoietic supportive capacity of umbilical cord derived mesenchymal stem cells induced by iron overload].

OBJECTIVE: To explore the effects of iron overload on umbilical cord derived mesenchymal stem cells (UC-MSC) and elucidate the involvement of reactive oxygen species (ROS) in this process. METHODS: The iron overload model of MSC was established by in vitro addition of ferric ammonium citrate (FAC) into culture medium. Cell proliferation and apoptosis were determined by Annexin V/PI double staining and population doubling time (DT) respectively. Co-culture system was used to assess the hematopoietic support capacity of UC-MSC in different groups. Thereafter the ROS level was detected with fluorescent probe 2', 7'-dichlorofluorescin diacetate (DCFH-DA). And the ROS related signaling factors of p-p38MAPK, p38 MAPK, P53 were measured by Western blot. RESULTS: (1) The DT of UC-MSC in iron overload group was significantly longer than that of control ((24.43 ± 2.72) h vs (16.03 ± 2.31) h, P < 0.05). But the difference was insignificant after two passages (P > 0.05). (2) Apoptosis in iron overload group was higher than that of control (12.75% ± 0.32% vs 3.63% ± 0.80%, P < 0.05). (3) The colony forming capacity of mononuclear cell (MNC) co-cultured with UC-MSC of iron overload group for 1/2 weeks significantly decreased. (4) The ROS level of UC-MSC with iron overload was higher than that of control in time and concentration-dependent fashions and it peaked at 400 µmol/L of FAC for 12 h (1499 ± 86 vs 548 ± 97, P < 0.05). (5) The expressions of p-p38MAPK and P53 increased in response to FAC compared with control. But such an effect was partially inhibited after the use of antioxidants. CONCLUSIONS: Iron overload may impair the proliferation, survival and hematopoiesis supportive function of UC-MSC by enhancing the generation of ROS. And ROS stimulates the signaling pathways of p-p38MAPK and P53.

[A preliminary study about the apoptostic mechanism of RNA targeting basic fibroblast growth factor in glioma U251 cells].

OBJECTIVE: To preliminarily investigate the mechanism of small interfering RNA (siRNA) induced apoptosis in glioma U251 cells by silencing basic fibroblast growth factor (bFGF). METHODS: U251 cells were divided into the normal control group, the mock group and experiment group, the mock and experiment group were transfected with mock vector (Ad-null) and the recombinant adenovirus carrying bFGF-siRNA (Ad-bFGF-siRNA) respectively at a multiplicity of infection (MOI) of 100. After 72 hours, the expression of related proteins was revealed by the method of Western blot. Mitochondrial transmembrane potential (ΔΨm) was measured with flow cytometry and confocal microscopy, Groups were compared using single factor analysis of variance (One-way ANOVA). RESULTS: After U251 cells were transfected with bFGF-siRNA, the results of Western blot showed that after 72 hours of transfection the bFGF protein in the experiment group decreased obviously, meanwhile Cytochrome C, Caspase-3 and Bax showed increased expression while in the Bcl-xl and Bcl-2 proteins decreased expression. The proportion of high mitochondrial membrane potential of cells by flow cytometry, the experimental group was 74.4% ± 4.7% decreased significantly compared with the control group 92.1% ± 2.5%, the mock group 90.9% ± 1.8% (F = 28.805, P < 0.05); laser scanning confocal microscopy results showed that the red fluorescence and green fluorescence ratio of the experimental group was 0.83 ± 0.12 decreased significantly compared with 1.36 ± 0.40 of the control group and 1.32 ± 0.35 of the mock group(F = 7.920, P < 0.05). CONCLUSION: siRNA targeting bFGF induced U251 cell apoptosis may be achieved through the mitochondrial pathway.

[Clinical significance of leukemia stem/progenitor cell related gene expression in acute leukemia].

This study was aimed to detect the expression of leukemia stem/progenitor cell (LSPC) related genes (ABCB1, BMI-1, HOXB4) in the patients with acute leukemia, and to explore its clinical significance in acute leukemia. Bone marrow samples were collected from de novo acute leukemia patients (41 cases), patients with complete remission (CR, 16 cases) and the patients with non-malignant hematologic diseases (10 cases) respectively. And the expressions of ABCB1, BMI-1, HOXB4 genes were detected by comparative real-time quantitative PCR (RQ-PCR) with SYBR Green assay. The results showed that the expressions of ABCB1, BMI-1, HOXB4 were not detected in the patients with non-malignant hematologic diseases, but were higher (relative expressive level: 4.26 ± 2.26, 3.72 ± 1.91, 3.74 ± 2.38) in de novo acute leukemia patients and lower (relative expressive level: 2.14 ± 1.47, 2.07 ± 0.99, 1.47 ± 0.89) in the acute leukemia patients with CR (p < 0.05). The expressions of LSPC related genes were lower (relative expressive level: 1.77 ± 1.29, 2.09 ± 1.26, 1.78 ± 1.49) in the patients acquired CR/partial remission (PR) than those in the patients not acquired CR/PR (relative expressive level: 7.23 ± 1.78, 3.96 ± 0.92, 4.48 ± 2.57) (p < 0.01). Univariate analysis revealed that there were more cases with the expression of LSPC immunophenotype (CD34(+)CD38(-)CD96(+) and CD34(+)CD38(-)CD123(+)) and more hyperleukocytosis cases in patients with any higher expression of LSPC related gene (p < 0.05). Analysis of multiple parameters discovered larger significance (p < 0.01). It is concluded that there is a good relationship between LSPC related genes (ABCB1, BMI-1, HOXB4) and LSPC immunophenotype. The expression of LSPC-related genes is higher in de novo acute leukemia patients, and lower in patients acquired CR/PR. The patients with higher expressed LSPC-related genes display worse response to chemotherapy, lower CR/PR rate and higher leukocytosis, the analysis of multiple parameters may be a good method for assessing the therapeutic efficacy/prognosis of acute leukemia.

[Effect of Buyang Huanwut decoction on apoptosis of splenocytes in rats with sepsis].

OBJECTIVE: To observe the change in number of spleen T lymphocyte, B lymphocyte, apoptotic splenocytes and the expression of Bcl-2 and Bax protein in rats with sepsis; to study the effect of Buyang Huanwu decoction (BYHWD) on the immune function in rats with sepsis. METHODS: One hundred and eighty healthy male Wistar rats were randomly divided into four groups: sham operation group (n=30), model group (n=50), BYHWD treatment group (n=50) and BYHWD prevention group (n=50) . Sepsis model was reproduced by cecal ligation and puncture (CLP). The rats of BYHWD treatment group and BYHWD prevention group were given BYHWD (1 g/ml) 15 ml/kg at 30 minutes after surgery, and 3 days before surgery, once a day, respectively. The rats were sacrificed and their spleens were harvested at 6, 12, 24, 48, 96 hours after reproduction of the model. Morphological changes in spleen, the expression of T lymphocyte, B lymphocyte, apoptotic cells, Bcl-2 and Bax were determined in all the groups. RESULTS: By light microscopy, it was found that white pulp became atrophic and splenic nodules destroyed after CLP. The pathology was most obvious in model group, followed by BYHWD treatment group, and least obvious in prevention group. CD4(+) T lymphocyte, CD40 B lymphocyte and Bcl-2 protein expression in model group were obviously reduced compared with those of sham operation group, but the number of apoptotic cells and Bax protein expression were elevated, reaching their nadir or peak 24 hours after the surgery [the optical densities (A value): CD4(+) T lymphocyte: 18.28±4.57 vs. 98.60±18.18; CD40 B lymphocyte: 26.96±6.26 vs. 104.87±30.97; Bcl-2 protein expression: 20.23±11.75 vs. 149.67±5.24; apoptotic cells: 241.75±44.79 vs. 14.67±5.24; Bax protein expression: 128.75±44.79 vs. 5.34±4.26, all P<0.01], then they gradually increase or decrease. CD8(+) T lymphocyte count did not significantly changed . The results showed that BYHWD could markedly elevate the level of CD4(+) T lymphocyte and CD40 B lymphocyte, and lower the protein expression of apoptotic cells and Bax. There were no significant changes in the CD8(+) T lymphocyte and Bcl-2 protein expression. Furthermore, the results in the BYHWD prevention group were better than those in BYHWD treatment group (A value: CD4(+) T lymphocyte: 94.12±15.45 vs. 72.37±8.00; B lymphocyte: 90.46±13.34 vs. 55.66±4.23; apoptotic cells: 27.63±9.91 vs. 40.83±16.09; Bax protein expression: 11.63±5.91 vs. 30.83±16.09, P<0.05 or P<0.01). Ninety-six hours after CLP, the above indexes gradually approached to the level of the sham operation group. Correlation analysis showed that cell apoptosis and Bax were positively correlated (r=0.522, P=0.000), but cell apoptosis and Bcl-2 showed negative correlation (r=-0.659, P=0.000). CONCLUSION: BYHWD improves the immunological suppression in rats with sepsis by lowering apoptosis of CD4(+) T lymphocyte and B lymphocyte in spleen, and its underlying mechanism may be that BYHWD produce a decrease of apoptosis through Bax.

[Establishment of iron overloaded bone marrow model in vitro and its impact on hematopoiesis].

This study was to establish an iron overload bone marrow (BM) model by co-culturing the mononuclear cells from BM with iron, and investigate its hematopoiesis changes. The iron overload model was set up by adding different concentration of ferric citrate (FAC) into the mononuclear cells from BM and culturing for different time, and the model was confirmed by detecting labile iron pool (LIP). Then the apoptosis of hematopoietic cells, ability of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM and CFU-mix) and percentage of the CD34(+) cells of the BM cells all were determined. The changes of these indexes were tested after the iron-overloaded BM was treated with deferasirox (DFO). The results showed that after BM cells were cultured with FAC at different concentrations for different time, the LIP increased in time-and concentration-dependent manners. The intracellular LIP reached maximum level when cultured at 400 µmol/L of FAC for 24 hours. The detection of BM cell hematopoietic function found that the apoptotic rate of the FAC-treated cells (24.8 ± 2.99%) increased significantly, as compared with normal control (8.9 ± 0.96%)(p < 0.01). The ability of hematopoietic colony forming in FAC-treated cells decreased markedly, as compared with normal control (p < 0.05). The percentage of CD34(+) cells of FAC-treated cells (0.39 ± 0.07%) also decreased significantly, as compared with normal control (0.91 ± 0.12%)(p < 0.01). And these changes could be alleviated by adding DFO. It is concluded that the iron-overloaded model has been set by adding iron into the mononuclear cells from BM in vitro, and the hematopoietic function of iron-overloaded BM is deficient. These changes can be alleviated by removing the excess iron from the BM cells through treating with DFO. These findings would be helpful to further study the mechanism of iron-overload on the hematopoiesis of BM and also useful to find the way to treat iron-overload patients with hematopoietic disorders.

[Effects of oxidative stress on hematopoiesis of hematopoietic stem and progenitor cells with iron overload].

OBJECTIVE: To establish a model of hematopoietic stem and progenitor cells with iron overload derived from umbilical cord blood (UCB) cells and explore the effects of reactive oxygen species (ROS) on the hematopoiesis of hematopoietic stem and progenitor cells with iron overload. METHODS: The model was established by adding different concentrations (50, 100, 200, 400 µmol/L)of ferric citrate (FAC) into mononuclear cells from UCB and culturing for different times (6, 12, 24 h). The UCB cells were divided into 4 groups: control group, group FAC, group FAC+N-acetyl-L-cysteine (NAC) and group FAC+ L-Glutathione (GSH). Then the changes of ROS, labile iron pool (LIP), apoptosis, the capacity of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM, CFU-mix) and the percentage and the numbers of CD34(+), CD33(+), GlyA(+) cells were detected. And the changes of these indices were tested after the treatment of iron overload UCB with antioxidants (NAC and GSH). RESULTS: UCB cells were cultured with the addition of FAC at different concentrations for different times. The level of total ROS increased in time and concentration-dependent manners. The intracellular level of ROS peaked when cultured at 200 µmol/L of FAC for 24 hours. Cells were treated with antioxidants NAC or GSH after cultured with 200 µmol/L FAC for 24 hours. Then the ROS levels of total cells, myeloid cells and erythroid cells decreased markedly versus normal controls. The LIP of total cells, myeloid cells and erythroid cells increased markedly when cells were cultured at 200 µmol/L of FAC for 24 hours versus normal controls (P < 0.05). NAC and GSH had no effect on the level of LIP. The apoptotic rates of FAC-treated cells [(20.90 ± 3.45)%] increased significantly versus normal controls [(9.20 ± 1.29)%] (P < 0.05). The capacity of hematopoietic colony forming in FAC treated cells decreased markedly versus normal controls. The percentage and numbers of CD34(+), CD33(+), GlyA(+) cells of FAC-treated cells also decreased significantly versus normal controls (P < 0.05). And these changes could be recovered by the addition of NAC or GSH. CONCLUSION: Oxidative stress plays an important role in the injuries of hematopoiesis of hematopoietic stem and progenitor cells with iron overload by inducing the generation of ROS. These findings may help us find a specific target and improve the therapeutic efficacy of ineffective hematopoiesis in patients with iron overload.

[In vitro effect of iron overload on bone marrow cell function by inducing the reactive oxygen species].

OBJECTIVE: To investigate the in vitro effect of iron overload on the generation of reactive oxygen species (ROS) and of bone marrow (BM) cell function. METHODS: BM mononuclear cells (BMMNCs) were cultured with ferric citrate (FAC) at different concentrations and for different time to create iron overload and confirmed by the detection of cellular labile iron pool (LIP). The changes of ROS, apoptosis, hematopoietic colony formation (CFU-E, BFU-E, CFU-GM and CFU-mix) and the percentage of the CD34 + cells percentage were analyzed. The differences of these index were tested after the iron overload treated with deferasirox (DFO) or antioxidants (N-acetyl-L-cysteine, NAC). RESULTS: 1) When BMMNCs were cultured with FAC, the LIP was found to increase in a time and concentration dependent manner. The intracellular LIP reached maximum at 400 micromol/L of FAC for 24 hours. 2) The ROS of total cells, leukocytes and erythrocytes increased to 1.77, 1.75 and 2.12 fold respectively compared with that of normal control when cells were cultured at 400 micromol/L of FAC for 24 hours . DFO and NAC could reduce the ROS efficiently (P<0.05). 3) The apoptotic rates of the FAC treated cells [(24.80 +/- 2.99)%] increased significantly compared with that of normal control [(8.90 +/- 0.96)%]. The capacity of hematopoietic colony formation in FAC treated cells decreased markedly compared with that of normal control (P<0.05). The percentage of CD34+ cells of FAC treated cells [(0.39 +/- 0.07)%] also decreased significantly compared with that of normal control [(0.91 +/- 0.12)%]. And these changes could be recovered by addition of NAC or DFO. CONCLUSION: Iron overload can affect the hematopoiesis by inducing the generation of ROS and this damage could be corrected by removing the excess iron and ROS of the BM cells. These findings might improve the treatment of dyshematopoiesis in patients with iron overload.

[Anti-glioma effect of combination of bFGF-siRNA and Vpr in nude mice].

OBJECTIVE: To study the anti-glioma effect of recombinant adenovirus mediated combined gene therapy of bFGF-siRNA and HIV1-Vpr in vivo. METHODS: Mouse glioma model was established by injecting 5 × 10(6) LN229 cells into BALB/c-nu nude mice. 30 nude mice were randomly divided into 5 groups: the negative control group, mock group, bFGF-siRNA group, Vpr group and combined therapy group, which at regular intervals were injected with PBS, rAd5-null, rAd5-bFGF-siRNA, rAd5-Vpr, rAd5-bFGF-siRNA plus rAd5-Vpr, respectively. The tumor volume was recorded every third day to draw a growth curve. After four weeks treatment, the mice were killed and specimens were taken. HE, immunohistochemical and TUNEL staining were performed to observe the cell morphology, detect the changes of relevant target proteins and cell apoptosis, respectively. Also the ultrastructural changes were observed by electron microscopy. RESULTS: The tumor growth inhibition rates were 36.9%, 37.2% and 58.6% in the bFGF-siRNA group, Vpr group and combined therapy group, respectively, and the combined therapy group showed the most significant effect (P < 0.05). Also the results of HE, immunohistochemical and TUNEL staining revealed that the combined therapy group had the best effects on proliferation inhibition and apoptosis induced in glioma cells (P < 0.05). The most significant ultrastructural changes were observed in the combined therapy group. CONCLUSION: The combined gene therapy of bFGF-siRNA with Vpr shows a prominent and synergistic anti-glioma effect compared with that of mono-gene therapy in nude mice.

[Study on apoptosis of splenic lymphocytes in rats with sepsis].

OBJECTIVE: To observe the change in number of spleen T lymphocytes and B lymphocytes and apoptosis of splenocytes in rats with sepsis, and explore the mechanism of immune imbalance in sepsis. METHODS: Eighty healthy male Wistar rats were randomly divided into two groups: sham operation (n=30) and sepsis model (n=50). Sepsis model was reproduced by cecal ligation and puncture (CLP). The rats were sacrificed and their spleens were obtained at 6, 12, 24, 48, 96 hours after the model was reproduced. The pathological changes in spleen were observed by optical microscopy. Immunohistochemistry method was used to examine the positive expression of CD4(+)/CD8(+)T lymphocyte and B lymphocyte, Bax and Bcl-2. Apoptotic cells of spleen were detected by means of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). RESULTS: By optical microscopy, white pulps were atrophied and splenic nodules were destroyed after CLP. At 6, 12, 24, 48, 96 hours after CLP, the number of CD(+)T lymphocyte, B lymphocyte and Bcl-2 positive expressed cells in sepsis model group were obviously reduced as compared with sham operation group (all P<0.01). CD8(+) positive lymphocytes were not significant changed (all P>0.05). Apoptotic splenocytes and Bax positive cells were obviously increased compared with those of sham operation group (all P<0.01). Correlation analysis showed that Bcl-2 expression and cell apoptosis was negatively correlated (r=-0.659, P<0.01), but Bax positive cells and apoptosis showed positive correlation (r=0.522, P<0.01). CONCLUSION: Disturbance of immunological function exists in the rats with sepsis at 24 hours after CLP. The number of CD4(+) T lymphocyte, B lymphocyte was obviously reduced, apoptotic splenocytes were obviously increased. Bax and Bcl-2 play an important role in apoptosis of splenocytes in sepsis.

[Effects of small interfering RNA targeting basic fibroblast growth factor on proliferation and apoptosis of glioma cell line U251].

BACKGROUND & OBJECTIVE: Basic fibroblast growth factor (bFGF), a member of the fibroblast growth factor (FGF) family, is a multifunctional cytokine participating in the process of wound healing, tissue repairing, proliferation and differentiation of immature neural cells. We previously found that bFGF is overexpressed in glioma cells, and could stimulate the vascularization and cell proliferation of glioma. This study was to explore the effects of bFGF small interfering RNA (siRNA) on the proliferation and apoptosis of glioma cell line U251. METHODS: Four bFGF siRNAs (siRNA1-4) and one random siRNA were synthesized using chemical method and transfected into U251 cells. The U251 cells cultured with Opti-MEM I (no serum media) were used as normal control. The expression of bFGF in transfected U251 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and SP immunohistochemistry. Cell proliferation was detected by MTT assay; cell apoptosis was detected by flow cytometry. RESULTS: At 48 h after transfection, the mRNA levels of bFGF in U251 cells were 0.95+/-0.02 in normal control group, 0.95+/-0.02 in negative control group, 0.85+/-0.02 in siRNA-1 group, 0.76+/-0.04 in siRNA-2 group, 0.65+/-0.04 in siRNA-3 group, and 0.56+/-0.03 in siRNA-4 group; at 72 h after transfection, the mRNA levels of bFGF were 0.95+/-0.04, 0.84+/-0.05, 0.81+/-0.05, 0.80+/-0.05, 0.77+/-0.04, and 0.53+/-0.05, respectively. Among the 4 siRNAs, siRNA-4 showed the strongest effect. The proliferation inhibition rate of U251 cells was significantly higher in siRNA-4 group than in negative control group [(66.39+/-1.23)% vs. (56.3+/-1.45)% at 48 h after transfection, (40+/-2.6)% vs. (14.74+/-0.62)% at 72 h, P <0.05]. CONCLUSION: The optimal bFGF siRNA can inhibit bFGF expression and proliferation of glioma U251 cells.

[Anti-endotoxin core glycolipid antibody: the preparation of immune serum of E. Coli J5].

OBJECTIVE: To prepare high titer anti-endotoxin core glycolipid (J5) antibody (CGL) for the treatment of Gram-negative bacteremia and septic shock. METHODS: Nontoxic bacterial vaccine (50 x 10(12)U/L) against E.Coli O111:B4 mutant strain J5 was prepared. J5 bacterial vaccine was injected into rabbits through ear marginal vein (saline as control preparation), one time pre three days, totally five times. Injected doses were as following: 0.1 ml, 0.2 ml, 0.4 ml, 0.6 ml, and 0.8 ml. One week after fifth injection, blood samples from heart were collected and immune serum was isolated. Indirect clotting test was used to determine the titer of antibody and cross reaction. RESULTS: Among 12 immunized rabbits, titers of antibody against E. Coli J5 were exceeding 1:1 024 in 6 rabbits, and they had cross reaction with various kinds of Gram-negative bacterial endotoxins. CONCLUSION: The titer of anti-endotoxin core glycolipid (E.Coli J5) antibody prepared by us appears to be high, and it can combine with various kinds of Gram-negative bacterial endotoxins.