Effect and Involved Mechanism of RSL3-induced Ferroptosis in Acute Leukemia Cells MOLM13 and Drug-resistant Cell Lines / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and involved mechanism of RSL3 on ferroptosis action in acute leukemia cells MOLM13 and its drug-resistant cells.@*METHODS@#After MOLM13 treated with RSL3, CCK-8 assay was performed to detect cell viability, flow cytometry was used to detect the reactive oxygen species (ROS) level of the cells, RT-qPCR and Western blot were used to detect the expression of glutathione peroxidase 4 (GPX4). After MOLM13/IDA and MOLM13/Ara-C, the drug-resistant cell lines were constructed, the ferroptosis induced by RSL3 was observed. Bone marrow samples were collected from patients with acute monocytic leukemia. RT-qPCR and Western blot were performed to detect the expression of related genes and proteins involved in ferroptosis pathway.@*RESULTS@#RSL3 significantly inhibited the cell viability of MOLM13 and increased the intracellular ROS level, which were partially reversed by ferrostatin-1. The mRNA and protein expression of GPX4 decreased in MOLM13 treated with RSL3. RSL3 inhibited the viability of MOLM13/IDA and MOLM13/Ara-C cells more strongly than that of non-drug resistant cells, also increased the intracellular ROS level . The cytotoxic effects were partially reversed by ferrostatin-1. The mRNA and protein expressions of GPX4 in MOLM13/IDA and MOLM13/Ara-C cells were higher than those in non-drug resistant cells. The mRNA and protein levels of GPX4 in bone marrow of relapsed/refractory acute mononuclear leukemia patients were higher than those of ordinary acute mononuclear leukemia patients.@*CONCLUSION@#RSL3 can induce non-drug resistant cells MOLM13 ferroptosis by inhibiting GPX4 activity. MOLM13/IDA and MOLM13/Ara-C are more sensitive to RSL3 compared with non-drug resistant cells MOLM13, which may be caused by the differences in GPX4 expression. The expressions of GPX4 mRNA and protein in relapsed/refractory acute mononuclear leukemia are higher than those in ordinary acute mononuclear leukemia.

Protective effect of cornel iridoid glycoside on hepatocytes injured by D-galactosamine/tumor necrosis factor-α / 中国药理学通报

Aim To determine the effect of cornel iri-doid glycoside ( CIG ) on human hepatocyte cell line (L-02) injured by D-galactosamine (GalN) and tumor necrosis factor-α( TNF-α) .Methods Firstly, CIG was extracted , separated and purified . Cell lesion model injured by D-GalN/TNF-αwas tested by MTT method.T-AOC, SOD, MDA and calcium ion concen-tration were taken as indicators to study the effects of CIG on L-02 cell injured by D-GalN/TNF-α.The ex-pression of p-PERK, p-eIF-2α, caspase-3 protein were detected by Western blot .Results 44 mg · L-1 D-GalN and 100 μg · L-1 TNF-αwere suitable for L-02 cell lesion model.CIG high, middle, low concentra-tion group could significantly increase the L-02 cell ac-tivity by 21％, 13％, 8％, respectively and SOD activity and T-AOC ability as well compared with model group.At the same time, they markedly reduced the MDA activity except the low concentration .Three con-centrations of CIG could reduce the expression of endo-plasmic reticulum stress related protein PERK , eIF-2αand apoptosis-associated protein caspase-3. Conclu-sions CIG could protect L-02 cells injured by D-GalN/TNF-α.Increasing the cellular antioxidant abili-ty, reducing the damage of endoplasmic reticulum stress and the expression of apoptosis-associated protein may be the possible mechanism .

Effect of puerarin on inflammation-related diseases / 中国药理学通报

Puerarin is a kind of isoflavone extracted from the root of Radix Pueraria .Studies showed that puerarin had a variety of functional activities, such as anti-inflammation, antioxidation, anti-osteoporosis , lowering blood glucose , anti-tumor and so on . The effects of puerarin for inflammation-related diseases are mainly summarized in this research , including cardiovascular disease, osteoporosis, diabetes and cancer .Realizing that anti-inflammation is the common mechanism for puerarin in treatment of these diseases , reference basis is expected to be offered for puerarin to play a broad application prospect in clinical practice .

Establishment and Identification of MDS Mouse Model with Irom Overload / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a MDS mouse model with iron overload and to study the effect of iron overload on MDS.</p><p><b>METHODS</b>The exogenous mutant gene RUNX1-S291fs was inserted into the mice bone marrow mononuclear cell's genome in mice by retrovirus and transplanted into C57BL/6 mice irradiated by Co γ-ray. After 8 weeks,intraperitoneal injection of iron was performed to establish an MDS mouse model with iron overload. After 24 weeks of transplantation, the peripheral blood, bone marrow, femur, liver and spleen of mice were taken, then the morphological characteristics of peripheral blood and bone marrow cells were observed by Wright's staining; the liver, spleen and bone marrow were stained with Prussian blue to observe the iron deposition. The surface antigens of bone marrow cells were detected by flow cytometry. Bone marrow mononuclear cells and spleen tissue proteins were detected by Western blot to confirm the transfection of RUNX1-S291fs gene and expression of protein. The blood routine and transplanted cell chimeric rate of mice were monitored periodically.</p><p><b>RESULTS</b>Compared with the empty plasmid control mice, levels of leukocyte and hemoglobin as well as platelet were decreased in RUNX1-S291fs mutant mice; the peripheral blood cells and bone marrow cells showed pathological hematopoiesis; the liver and spleen enlarged significantly; the tissue structure of femur, liver and spleen was abnormal; the expression of bone marrow cell surface antigens was abnormal. Bone marrow cells and spleen tissue expressed the RUNX1-S291fs protein. Compared with the controlled mice injected with normal saline, iron deposition occurred in the bone marrow, liver and spleen stained with Prussian blue in the mice injected with iron agent.</p><p><b>CONCLUSION</b>Mice engineered to carry exogenous mutant gene RUNX1-S291fs and injected with iron showed pathologic features of MDS and iron overload, resulting in establishing MDS iron overloaded mouse model successfully, which lays a foundation for studying the effect of iron overload on MDS.</p>

Expression and Significance of HIF-1α in Erythropoiesis of Secondary Iron Overload Disease after Irradiation Damage / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the expression of hypoxia inducible factor 1α(HIF-1α) of iron-overloaded in irradiated mice and its effect on erythropoiesis.</p><p><b>METHODS</b>Twenty mice were randomly divided into 4 groups: Ctrl (control group), IR (irradiation group), IO (irradiation + iron overload group), and RAPA (rapamycin treatment group). The iron overload model was verified. The CFU-E (colony forming unit-erythroid) and BFU-E(burst colony forming unit-erythroid) were cultured; flow cytometry was used to detect the ratios of early stage (Ter119CD71) to late stage (Ter119CD71) of primitive erythroblasts; RT-PCR was used to detect the mRNA expression of HIF-1α and its related signal molecules in bone marrow cells.</p><p><b>RESULTS</b>The expression of HIF-1α in IR and IO group was significantly higher than that in Ctrl group, and that in IO group was significantly higher than IR group (P<0.05). The ratio of late stage primitive erythroblasts, the number of CFU-E and BFU-E in both IR and IO group were lower than those in Ctrl group, and those in IO group were significantly lower than those in IR group (P<0.05). Compared with Ctrl group, the expression of HIF-1α related signal pathway molecules in both IR and IO group was significantly decreased (P<0.05). Compared with IO group, the expression of HIF-1α and its related signal molecules in RAPA(mTOR inhibitor) group was decreased significantly (P<0.05), the number of BFU-E was increased significantly(P<0.05).</p><p><b>CONCLUSION</b>Irradiation induces the increase of HIF-1α and the decrease of the ability of hematopoietic colony formation and the ratio of late stage primitive erythroblasts. Iron overload can aggravate the injury. mTOR inhibitor rapamycin can partially alleviate the injury, suggesting that iron overload can lead to injury of erythropoiesis through HIF-1α.</p>

Research on the Influence of new oncogenic CSF3R mutations in Chronic Neutrophilic Leukemia / Эрүүл мэндийн лаборатори

Background:Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN), since the first description of CNL in 1920, more than 150 cases have reported in the literature. The World Health Organization (WHO) recognizes CNL, as a MPN and, for the frst time, provides recognized criteria to permit the operational classifcation of this poorly defned disease. Until recently, the molecular pathogenesis of CNL was unknown and the diagnosis was based on morphological aspects, clinical criteria and exclusion of known genetic entities like the Philadelphia translocation indicative of CML, or JAK2 mutations indicative of MPNs. Recent discovery of highfrequency granulocyte-colony stimulating factor receptor (CSF3R) mutations in CNL identifes a new major diagnostic criterion, and lend more specificity to the WHO diagnostic criteria for CNL, which are variably applied in routine clinical practice. In 2013 Maxson et al., and Pardanani and colleagues identified granulocyte-colony stimulating factor 3 receptor (CSF3R) mutations in 8 of 9 (89%), and in 13 of 13 (100%) patients with CNL, respectively. CSF3R mutations fall into 2 classes: nonsense or frameshift mutations that lead to premature truncation of the cytoplasmic tail of the receptor (truncation mutations) and point mutations in the extracellular domain of CSF3R (membrane proximal mutations). These nonsense or frameshift mutations truncate the cytoplasmic tail of CSF3R, impair its internalization,and alter its interactions with proteins such as SHP-1/2 and SOCS family members. These structural and functional alterations are thought to perturb the capacity of CSF3R to regulate granulocyte differentiation and to increase granulocytic proliferative capacity. Thetwo types of CSF3R mutations may have differential susceptibility to classes of tyrosine kinase inhibitors,with CSF3R truncation mutations showing activation of SRC family–TNK2 kinase signaling and sensitivity to dasatinib, and CSF3Rmembrane proximal mutations strongly activate the JAK/signal transducer and activator of transcription pathway and are sensitive to JAK kinase inhibitors such as ruxolitinib.The most common CSF3R mutation in CNL is themembrane proximal mutation: T618I. On September 2012 we got a case (a 67-years-old Chinese man), which had fulflled the WHO diagnostic criteria for CNL with a novel mutation site of colony stimulation factor 3 receptor (CSF3R). In our case was identifed a membrane proximal mutations CSF3RT618I and also a unreported novel mutation site of CSF3R-H54A in the CD34+ and CD15+ cell fractionsby sorting bone marrow samples (BD FACSAria™ III; BD Biosciences) using a PCR-based DNA pyrosequencing method. Thus, we sought to determine CSF3R-FL, CSF3R-T618I, CSF3R-H54A mutations have some correlation with molecular pathogenesis of CNL.Objective:To determine CSF3R-FL, CSF3R-T618I, CSF3RH54A mutations that have some signifcance on the molecular pathogenesis of CNL.Materials/Methods:1. Plasmid construction. Plasmids were constructed by PCR amplifcation of the insert, restriction digestion and ligation using standard molecular biology methods, briefly: the host vectors pLV-EF1α-EYFP-N, pLP-2, pVSV-G, pLP-1 gag pol were purifed with Omegabiotek maxi prep kit and digested by restriction enzymes (ECO RI, NOTI). The linearized vectors were purified from agarose gel using a AxyPrep TM DNA Gel Extraction Kit and the concentration of the samples was estimated on an agarose gel stained with ethidium bromide.The inserts (CSF3R-FL, CSF3R-T618I, CSF3R- H54A) were generated by PCR, the sequences of the primer-pairs used and the conditions of the PCR reactions. The amplifed DNA fragments were purifed from agarose gels using AxyPrep TM DNA Gel Extraction kits and digested by ECO RI, NOTI was used to linearize the acceptor vector. Enzymatic reactions in the case of the i) insert: 100–3000 ng purifed PCR product was digested by appropriate amount of enzyme and 4 µl of 10x reaction buffer in 40 µl of fnal volume for overnight at the appropriate temperature; ii) vector: 2000–4000 ng plasmid DNA was digested by appropriate amount of enzyme and 2 µl of 10x reaction buffer in 20 µl of fnal volume for 4 hours at the appropriate temperature. When necessary Research on the Influence of new oncogenic CSF3R mutations in Chronic Neutrophilic Leukemia Otgonbat Altangerel1, Ming Feng Zhao2, Wu Ri Mao21Department of Internal Medicine, Division of Hematology, Mongolian National University of Medical Sciences, Mongolia 2Department of Hematology, Tianjin First Central Hospital, First Central Clinical College of Tianjin Medical University,P.R. China11 the digested fragments were purifed again and the concentrations of the inserts were estimated on agarose gels, as described above. A 1:3 vector: insert molar ratio was used for the ligation reactions. Chemically competent DH5α Escherichia coli bacteria were transformed with the products of the ligation reactions and were grown on Luria Bertrani (LB) agar plates containing the required antibiotic, such as ampicillin (Sigma). A day later single colonies were picked from the plate, inoculated into and grown overnight in LB medium containing with ampicillin. Plasmids were purifed from the overnight cultures as above and tested by restriction mapping for the presence of the insert. Selected clones were sequenced by Sanger sequencing.2. Lentiviral packaging system we used 3 main components, such as the lentiviral expression vector(Plasmid DNA of CSF3R-FL, CSF3R-T618I), the lentiviral packaging plasmids (pLP-1, pLP-2 plasmids that encode for gag, pol, and rev from the HIV or FIV genome and pVSV-G), 293TNN producer cells. We seed 1X105 293TNN cells per 10 cm2 culture plate in 2-3 ml of culture medium containing DMEM medium supplemented with 4 mM L-glutamine, 4.5 g/l glucose, and fetal bovine serum (10%) without antibiotics. Grow for 18-24 hours at 37 °C with 5% CO 2 so that the cell density reaches ~60 - 80% confluency at the time of transfection. We used a GFP as a positive control, to confrm transfection experiment was successful. Then we collected the cell culture supernatant, which is containing infectious pseudoviral particles.3. Transduction of Pseudotyped Viral Particles into the primary cell of mouse. In the fnal step we have used the Mouse Colony Forming Unit Assay using MethoCultTM to assess the effects of CNL-associated oncogenes on the morphology and number of primary murine cells derived bone marrow. For this purpose cells are transduced with either control, which is without viral construct or a construct expressing the oncogene of interest (CSF3R-FL, CSF3R-T618I).Results:1. On the Plasmid construction step we successfully extracted and purifcated of recombinant plasmids ofCSF3R-FL and CSF3R-T618I cloning, but we still didexperiment to obtain the recombinant plasmid CSF3R- H54A cloning.2. After 24 hours of transfection 293TNN Cells with Packaging Plasmids and the Expression Construct, cells we visualized with green fluorescence protein under the fluorescence microscope.3. The both two of CSF3Rcloning were capable of transforming murine colony forming cells. After transforming, CFU-GM colonies were counted manually by light microscopy seven days after plating. We found that the membrane proximal mutation (T618I) transformed CFU-GM colonies number was more than the full length non-mutants (CSF3RFL), which indicates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.Conclusions:1. The establishment of the plasmid reconstruction, lentiviral packaging system and Mouse Colony Forming Unit Assay were done successfully.2. We confrmed the transformation capacity of the CSF3R mutations, especially CSF3R-T618I was higher than CSF3R-FL. This result demonstrates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.3. Further studies will be continued and are needed to prove the effects of the novel mutation site CSF3RH54A on the transduced murine bone marrow progenitor cells by using CFC assay

Effects of Iron Overload on the Apoptosis and Function of Splenic CD8+ T Cells in Mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of iron overload on apoptosis and function of splenic CD8+ T cells in mice.</p><p><b>METHODS</b>Forty C57BL/6 mice were randomly divided into control groups, Iron overload (IO), IO+NAC and IO+DFX groups. The iron overload model was established by intraperitoneal injection of iron dextran, and saline was injected as the control. The levels of intracellular reactive oxygen species (ROS) and labile iron pool (LIP) were analyzed by measuring the mean fluorescence intensity (MFI) of 2-7 dichlorofluorescein (DCF) or calcein. The ratio of CD8+ T cells and the levels of IFN-γ, TNF-α, Granzyme-B, and perforin in CD8+ T cells were detected by flow cytometry. The CD8+ T cell apoptosis was determined by flow cytometry with Annexin V/PI double staining. Real-time PCR was used to detect the expression of IFN-γ, TNF-α, Granzyme-B, perforin, BCL-2, and bax at mRNA level in CD8+ T cells.</p><p><b>RESULTS</b>Iron overload was found by spleen iron staining and flow cytometry. The level of intracellular ROS in iron overload (IO) groups was higher than that of the control groups (P<0.01). The percentage of CD8+ T cells in spleen from mice with IO was lower than that in control groups (P<0.05). The expression of IFN-γ and Granzyme-B in CD8+ T cells in IO group were lower than that in control group, the expression of IFN-γ and Granzyme-B at mRNA level in CD8+ T cells was lower than that of control group (P<0.05). CD8+ T cell apoptosis in iron overload group was significantly higher than that in control groups (P<0.01); the expression of BCL-2 at mRNA level was lower than that in control group, but the expression of BAX at mRNA level was higher than that in control group (P<0.05). These effects could be reversed after treating iron-overloaded mice with DFX or NAC.</p><p><b>CONCLUSION</b>Iron overload can inhibit the ratio of CD8+ T cells of splenic cells in mice, decrease the expression of IFN-γ, Granzyme-B, increase the apoptosis of CD3+ CD8+/CD8-. These effects may be regulated through increasing the intracellular ROS level, and can be partially reversed after treating iron-overloaded mice with DFX or NAC.</p>

Colony Stimulating Factor 3 Mutations and Myeloid Malignancies / 中国医学科学院学报

Granulocyte colony stimulating factor (G-CSF) plays a major role in the proliferation, differentiation, and activation of neutrophil cell line hematopoietic cells. G-CSF exert the function depending on its binding to colony-stimulating factor 3 receptor (CSF3R), a homo-dimer receptor located on the surface of effector cells. Some recent studies have demonstrated that CSF3R mutations play a significant role in many diseases. Some of the hematopoietic diseases, especially myeloid malignancies (e.g. chronic neutrophilic leukemia) are related to the presence of various CSF3R mutations, which leads to abnormal G-CSF signal pathways. Also, the downstream kinases can be the treatment targets for these diseases. This review summarizes CSF3R mutations, mechanisms of mutations, and their contributions to the myeloid malignancies, with an attempt to further reveal the pathogenesis of myeloid malignancies, inform the diagnosis and clinical treatment of the myeloid malignancies, and provide clues for the research and development of new molecular target drugs.

Establishing aplastic anemia animal model based on different pathogenesis / 中国实验血液学杂志

Aplastic anemia(AA) is a disease,including congenital AA and acquired AA, characterized by an extremely hypocellular marrow and peripheral blood pancytopenia due to bone marrow failure. Congenital AA is a autosomal recessive disease due to gene mutation. Persently, acquired AA is recognized as a disease caused by destruction of hematopoietic stem cells, defective marrow microenvironment and aberrant T cellular-immunity. In order to further study its pathogenesis and to choose effective therapeutic target, it has important clinical significance to establish correspondent animal model based on different pathogenesis. This article summarizes the congenital AA amimal models including Fanc A(-/-) mouse, Fanc C(-/-) mouse, Fanc G(-/-) mouse, Fanc D1(-/-)/Fanc 2(-/-) mouse, Fanc D2(-/-) mouse and other gene deficiency mouse AA models, and the acguired AA models resulting from the hematopoietic stem cell decrease, hematopoietic microenvironment injury, immune mediation and combination of hematopoietic stem cell dicrease with immune mediation.

Establishment of an mouse model of iron-overload and its impact on bone marrow hematopoiesis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a mouse model of iron overload by intraperitoneal injection of iron dextran and investigate the impact of iron overload on bone marrow hematopoiesis.</p><p><b>METHODS</b>A total of 40 C57BL/6 mice were divided into control group, low-dose iron group (12.5 mg/ml), middle-dose iron group (25 mg/ml), and high-dose iron group (50 mg/ml). The control group received normal saline (0.2 ml), and the rest were injected with intraperitoneal iron dextran every three days for six weeks. Iron overload was confirmed by observing the bone marrow, hepatic, and splenic iron deposits and the bone marrow labile iron pool. In addition, peripheral blood and bone marrow mononuclear cells were counted and the hematopoietic function was assessed.</p><p><b>RESULTS</b>Iron deposits in bone marrow, liver, and spleen were markedly increased in the mouse models. Bone marrow iron was deposited mostly within the matrix with no significant difference in expression of labile iron pool.Compared with control group, the ability of hematopoietic colony-forming in three interventional groups were decreased significantly (P<0.05). Bone marrow mononuclear cells counts showed no significant difference. The amounts of peripheral blood cells (white blood cells, red blood cells, platelets, and hemoglobin) in different iron groups showed no significant difference among these groups;although the platelets were decreased slightly in low-dose iron group [(780.7±39.60)×10(9)/L], middle dose iron group [(676.2±21.43)×10(9)/L], and high-dose iron group [(587.3±19.67)×10(9)/L] when compared with the control group [(926.0±28.23)×10(9)/L], there was no significant difference(P>0.05).</p><p><b>CONCLUSIONS</b>The iron-overloaded mouse model was successfully established by intraperitoneal administration of iron dextran. Iron overload can damage the hepatic, splenic, and bone marrow hematopoietic function, although no significant difference was observed in peripheral blood count.</p>

Effect and mechanism of iron-catalyzed oxidative stress on mesenchymal stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore effect of iron overload on the proliferation and apoptosis of mesenchymal stem cell(MSCs) and the possible mechanism.</p><p><b>METHODS</b>Iron overload model of MSCs was established by adding ferric ammonium citrae (FAC) into the culture medium at different concentrations (100, 200, 400 Μmol/L) and incubated for different lengths of time (12, 24, 48 h). The levels of labile iron pool (LIP) and reactive oxygen species (ROS) were measured to confirm oxidative stress state in the model. Changes in cell proliferation and apoptosis after iron overload were measured through population double time(DT)and annexin V-PI assay. Finally, the expressions of phosphorylated p38 mitogen activated protein kinase (P-p38MAPK), p38MAPK, protein kinase B (AKT), and p53 were determined through Western blot analysis to investigate which ROS-mediated signaling pathway was involved in this process.</p><p><b>RESULTS</b>The LIP level of MSCs was significantly increased by FAC treatment at 400 Μmol/L (mean fluorescence intensity 482.49±20.96 vs. 303.88±23.37, P<0.05). The level of intracellular ROS was positively correlated with the concentration of FAC and reached a peak level when cultured with 400 Μmol/L FAC (P<0.05).After treatment with 400 Μmol/L FAC at different time points (12 h, 24 h, and 48 h), the DT of MSCs was (1.47± 0.11) d, (1.80±0.13) d, and (2.04±0.14) d, respectively, which was signifcantly longer than that of the control, which was(1.20±0.05)d (P<0.05).The apoptosis rate was also significantly higher in iron overload group[(3.51±1.17)% vs.(0.66±0.62)%, P<0.05]with consequent increase in the expressions of P-p38MAPK, p38MAPK, and p53 proteins in iron overload group, while no significant difference was found in the expression of AKT.</p><p><b>CONCLUSION</b>Iron overload can inhibit the proliferation of MSCs and induce their apoptosis through the generation of ROS, which is probably due to the stimulation of p38MAPK- p53 signaling pathway.</p>

Effects of humanized interleukin 21 on anti-leukemic activity of cytokine induced killer cells and the mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of humanized interleukin 21 (IL-21) on anti-leukemic activity of cytokine induced killer(CIK) cells derived from peripheral blood(PB) and the mechanism.</p><p><b>METHODS</b>Mononuclear cells were separated from peripheral blood and cultured with cytokines to induce CIK cells. Proliferation of CIK cells with or without IL-21 stimulation and their cytotoxic activity against K562 cells was measured by MTT method. IL-21 receptor (IL-21R) and immunophenotypes of CIK cells were measured by flow cytometry. The expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative RT-PCR. FasL on the surface of CIK cells and intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry. The concentration of IFN-γ and TNF-α in the cultured supernatant were measured by enzyme immunoassay. JAK-STAT signalling pathway of CIK cells were measured by Western-blot.</p><p><b>RESULTS</b>After IL-21 stimulation, the proportion of CIK cells increased from (17.5 ± 4.7)% to (26.5 ± 2.1)%. Cytotoxic activity against K562 cells by CIK cells increased from (22.8 ± 2.8)% to(44.6 ± 8.3)%. The expression of IL-21R increased about 2 folds. The mRNA expression of IFN-γ increased almost 2 folds from (0.3760 ± 0.2358) to (0.7786 ± 0.2493), TNF-α increased almost 2 folds from (0.6557 ± 0.1598) to (1.3145 ± 0.2136), perforin increased almost 1.5 folds from (0.6361 ± 0.1457) to (0.9831 ± 0.1265), granzyme B increased almost 2 folds from (0.4084 ± 0.1589) to (0.7319 ± 0.1639), FasL increased almost 2 folds from (0.4015 ± 0.2842) to (0.7381 ± 0.2568), the expression of granzyme A, TNF-β and NKG2D were similar with control. Flow cytometry analysis showed that the expression of FasL of CIK cells was higher than that of control (0.19% vs 0.04%), the expression of perforin increased from 35.28% to 53.16%, and the expression of granzyme B increased from 43.16% to 78.82%. The concentration of IFN-γ in the culture supernatant increased almost 2 folds from (25.8 ± 6.1) ng/L to (56.0 ± 2.3) ng/L, and TNF-α increased almost 3 folds from (5.64 ± 0.61) µg/L to (15.14 ± 0.93) µg/L. Western blot showed that the expression of STAT1 and STAT5a had no significant differences, but the expression of STAT3 and STAT5b were higher than that of control.</p><p><b>CONCLUSION</b>Humanized IL-21 could enhance the anti-leukemic activity of CIK cells via increasing IL-21R, perforin, granzyme B, FasL, IFN-γ and TNF-α, as well as activating JAK-STAT signaling pathway. These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemic immunotherapy.</p>

Reactive oxygen species and bone marrow hematopoietic stem cell senescence / 中国实验血液学杂志

Reactive oxygen species (ROS) are bioactive oxygen molecules produced after exposure to exogenous oxidants or endogenously through cellular aerobic metabolism. Hematopoietic stem cells (HSC) are multipotent, self-renewing stem cells residing in hematopoietic tissues. Recent studies show that an abnormal increase in ROS production is associated closely with HSC senescence. Many signaling molecules such as FoxOs, ATM, mTOR, TSC1, Bmi1 and AKT play a significant role in ROS-induced HSC senescence. The roles of p53-p21 and p16-Rb pathways can induce hematopoietic dysfunction and lead to ROS-induced HSC senescence. This review summarizes the recent progress of studies on ROS-induced HSC senescence, and further elaborates the potential signaling molecules and pathways, aiming to provide a new target and thread for clinical treatment.

Effect of oxidative stress on bone marrow mesenchymal stem cells / 中国医学科学院学报

Bone marrow mesenchymal stem cells (MSCs) are somatic stem cells that can differentiate into progenies of multiple lineages. They play an important role in hematopoiesis and stem cell therapy due to their multi-lineage potentials and immunomodulatory properties. Oxidative stress is a disturbed redox state caused by accumulation of reactive oxygen species. It can induce the senescence and apoptosis of MSCs via phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and p53 pathways, and inhibit the proliferation and differentiation of MSCs through apurinic/apyrimidinic endonuclease/redox factor 1 (APE/REF-1) and extracellular signal-regulated kinase (ERK) pathways. Furthermore, using anti-stress medication and hypoxic preconditioning, the functions of MSCs can be further enhanced. Accordingly, further studies on the effect of oxidative stress on MSCs and its signaling pathways may be meaningful for the treatment of hematologic diseases and for improving stem cell therapy.

Reactive oxygen species and fibrosis in tissues and organs - review / 中国实验血液学杂志

Reactive oxygen species (ROS) is a kind of molecules derived by oxygen in the metabolic process of aerobic cells, which mainly includes superoxide, hydroxyl radicals, alkoxyl, hydrogen peroxide, hypochlorous acid, ozone, etc. They can destroy the structure and function of cells through the damage of biological macromolecules such as DNA, proteins and the lipid peroxidation. ROS also can regulate the proliferation, differentiation and apoptosis of cells through several signaling pathways and participate in fibrogenesis of many organs including hepatic and pulmonary fibrosis. Recent study shows that ROS might have an important effect on the forming of myelofibrosis. Consequently, ROS plays a significant role in the fibrogenesis of tissues and organs. In this review, the relevance between ROS and common tissues and organs fibrosis is summarized.

In vitro effect of iron overload on bone marrow cell function by inducing the reactive oxygen species / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the in vitro effect of iron overload on the generation of reactive oxygen species (ROS) and of bone marrow (BM) cell function.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Establishment of iron overloaded bone marrow model in vitro and its impact on hematopoiesis / 中国实验血液学杂志

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.

Clinical significance of leukemia stem/progenitor cell related gene expression in acute leukemia / 中国实验血液学杂志

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.

Effects of interleukin 21 on anti-leukemia activity of cytotoxic T lymphocytes induced by dendritic cells / 中国实验血液学杂志

This study was aimed to explore the effects of interleukin 21 (IL-21) on the anti-leukemia activity of cytotoxic T lymphocytes (CTL) induced by dendritic cells (DCs) in vitro. The peripheral mononuclear cells from leukemia patients in complete remission were cultured with the specific cytokines to induce the production of DCs. The DCs loaded with RNA from autologous leukemic cells as antigen, and co-cultured with autologous T lymphocytes to get leukemia specific CTL. The cytotoxic activity of CTL against autologous leukemic cells was measured by LDH release method. The concentration of IFN-gamma and TNF-alpha in the culture supernatant was measured by enzyme immunoassay. The effects of IL-21 on the mature DCs were also studied by the measurement of the phenotype of DC and the allogenic mixed lymphocytic reactions induced by DCs. Experiments were divided into 2 groups: test group in which IL-21 (200 ng/ml) was added in coculture of DC/CTL and control group in which no IL-21 (200 ng/ml) was added. The results showed that when cultured with IL-21, the quantity of CTL increased from (56.73 +/- 10.21)% (control group) to (73.43 +/- 18.01)% (p < 0.01); The concentration of IFN-gamma and TNF-alpha in the culture supernatant increased from (154.91 +/- 67.20) ng/L (control group) to (310.62 +/- 141.15) ng/L (p < 0.01) and from (8.77 +/- 5.09) microg/L (control group) to (15.25 +/- 6.56) microg/L (p < 0.01) respectively. At the effector: target ratio of 20:1, the cytotoxic activity against autologous leukemic cells by CTL increased from (50.22 +/- 5.07)% (control group) to (75.38 +/- 9.47)% (p < 0.01). IL-21 had neither effect on the phenotype (CD1a, CD83, CD86, CD80 and HLA-DR) of mature DCs nor the allogeneic mixed lymphocytic reactions induced by DCs. It is concluded that IL-21 can strengthen the proliferation of CTL, and improve the production of IFN-gamma and TNF-alpha, thus enhance the anti-leukemia activity of CTL. Nevertheless, there is no effect of IL-21 on the function of mature DCs. These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemia immunotherapy.

Comparison between CMV quantitative PCR and CMV-pp65 antigen test for detection of CMV infection in allogeneic hematopoietic stem cell transplantation / 中国实验血液学杂志

The study was aimed to compare the efficiency of cytomegalovirus (CMV) quantitative PCR and CMV-pp65 antigen test for detection of CMV infection and their clinical significance in patients received allogeneic hematopoietic stem cell transplantation (HSCT). 84 patients received allogeneic HSCT were enrolled in study. Anticoagulant blood samples were obtained from the recipients before and after transplantation and in the convalescence. CMV quantitative PCR and CMV-pp65 antigen test were performed weekly. The results showed that out of 84 patients, 26 cases were positive (30.95%) by CMV quantitative PCR method. Of the 26 cases, 9 cases were CMV antigenemia and 13 cases were CMV disease, the median positive time was 37.1 (7 - 105) days after HSCT. 22 cases were positive (26.19%) by CMV-pp65 antigen test method, the median positive time was 46.6 (10 - 128) days after HSCT. All the 22 positive cases detected by CMV-pp65 antigen test were also positive by CMV quantitative PCR method. Nevertheless, 4 positive cases detected by CMV quantitative PCR but negative detected by CMV-pp65 antigen test method did not develop CMV disease. The CMV disease was found in the cases either with moderate to high copies of CMV quantitative PCR or moderate to high level CMV antigenemia by CMV-pp65 antigen test method. The clearance median time was 17.5 (11 - 28) days by CMV quantitative PCR method after receiving antiviral therapy and was 10.0 (7 - 21) days by CMV-pp65 antigen detection method. It is concluded that both CMV quantitative PCR and CMV-pp65 antigen test can detect the infection of CMV early and effectively in patients received HSCT. CMV quantitative PCR is more sensitive, and CMV-pp65 is more specific. It can be more effective to guide the antiviral treatment and evaluate its efficacy when combining the two methods.