Blocking Two Signals of Immuno-Activated T Cells from Antigen Presenting Cells Can Prevent Chronic GVHD of Murine / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of blocking TCR-CD3 and B7-CD28 signals on immune function of mice with chronic GVHD by using TJU103 and CTLA4-Ig.</p><p><b>METHODS</b>On the basis of foregoing murine model of chronic GVHD, according to interference modes after infusion 6×10 spleen cells of donor mice, the recipients were divided into 5 groups: blank control, cGVHD, TJU103 interference, CTLA4-Ig interference and TJU103+CTLA4-Ig interference groups. The score of clinical manifestation and tissue histopathology were used to evaluate the effects of all the interferences on chronic GVHD.</p><p><b>RESULTS</b>TJU103 and CTLA4-Ig could not influence the formation of the mouse chimera. The analysis of Kaplan survival curve of mice with chronic GVHD showed that the CTLA4-Ig and TJU103+CTLA4-Ig reduced the incidence of chronic GVHD, the TJU103 could delay the occurrence of chronic GVHD, but all the interference factors could not change the severity of chronic GVHD.</p><p><b>CONCLUSION</b>TJU103 can delay the onset time of chronic GVHD, and the CTLA4-Ig can reduce the incidences of cGVHD, the combining use of TJU103 and CTLA4-Ig can significantly reduce the incidence of chronic GVHD, but can not change the severity of chronic GVHD.</p>

Synergistic Killing Effects of Arsenic Trioxide Combined with Itraconazole on KG1a Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of arsenic trioxide combined with itraconazole on proliferation and apoptosis of KG1a cells and its potential mechanism.</p><p><b>METHODS</b>The cell morphology was observed with Wrighe-Giemsa staining; cell survival rate was examined by CCK-8; and colony formation capacity was measured by methylcellulose colony formation test; the flow cytometry was used to analyse the cell apoptosis rate and cell cycle; the protein expressions of BCL-2,caspase-3,BAX,SMO,Gli1 and Gli2 were detected by Western-blot.</p><p><b>RESULTS</b>The arsenic trioxide and itraconazole alone both could inhibit the KG1a cell proliferation in dose-and time-dependent manner. In comparison between single and combined drug-treatment group, both the cell survival rate and the colony number of the single drug-treatment group were significantly lower(P<0.05), and the apoptosis rate was higher in the combined drug-treatment group. In the combined-treatment group, the protein expression of Caspase-3 and BAX was upregulated, while the protein expression of BCL-2,SMO,Gli1 and Gli2 was downregulated.</p><p><b>CONCLUSION</b>Arsenic trioxide combined with itraconazole can inhibit the KG1a cell proliferation and induce apoptosis, which may be related with the inhibition of Hh signaling pathway and upregulation of both Caspase-3 and BAX protein expression, and provided experimental data of arsenic trioxide combined with itraconazole for the treatment of refractory AML.</p>

Honokiol combined with Gemcitabine synergistically inhibits the proliferation of human Burkitt lymphoma cells and induces their apoptosis / 中国实验血液学杂志

This study was aimed to investigate the effect of Honokiol (HNK) combined with Gemcitabine (GEM) on the proliferation and apoptosis of human Burkitt lymphoma Raji cells. Cell proliferation was detected by CCK-8 method to study the role of Honokiol and Gemcitabine in Raji cells. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The level of apoptosis-related protein BCL-2 was measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (P < 0.01) and the inhibition rate was related to the concentration and action time of HNK; and apoptosis rate markedly increased (P < 0.01), while most Raji cells were arrested at G0/G1 phase and decreased in S phase after treatment with combination of two drugs; the expression of BCL-2 protein decreased (P < 0.01). It is concluded that Honokiol combined Gemcitabine can synergistically inhibit the proliferation, induce cell apoptosis, and down-regulate the expression of BCL-2 in Raji cells. The possible mechanism of synergistic effect may be related with arrest of cell cycle at G0/G1 phase and downregulation of the expression of BCL-2.

Synergistic killing effect of arsenic trioxide combined with curcumin on KG1a cells / 中国实验血液学杂志

This study was aimed to explore the effect of arsenic trioxide combined with curcumin on proliferation and apoptosis of KG1a cells and its potential mechanism. The cell survival rate was mesured by MTT; colony formation capacity was examined by methylcellulose colony formation test; flow cytometry was used to analyse the cell surface molecules, cell apoptosis rate and cell cycle; the cell morphology was observed with Wright-Giemsa staining and the protein expression of BCL-2, BAX, PARP was detected by Western blot. The results showed that the phenotype of KG1a cells was CD34(+)CD38(-), while the phenotype of HL-60 cell was CD34(+)CD38(+). The former possessed a stronger colony ability than the latter. Effect of curcumin and arsenic trioxide alone on cell proliferation and inhibition was in dose-dependent manner. Compared with single drug-treatment group, the cell survival rate and colony number were lower, and the apoptosis rate was higher in combined drug-treatment group. Protein expression of BCL-2 and PARP was upregulated, while the protein expression of PARP was downregulated in the combined treatment group. It is concluded that compared with HL-60 cells, KG1a cells are the earlier leukemia stem/progenitor cells. Arsenic trioxide combined with curcumin can effectively inhibit the KG1a cell proliferation and induce apoptosis, which may be associated with the downregulation of BCL-2 and PARP protein expression and the upregulation of BAX protein expression.

Effect of honokiol on proliferation and apoptosis in HL-60 cells and its potential mechanism / 中国实验血液学杂志

This study was aimed to investigate the effect of Honokiol (HNK) on proliferation and apoptosis of acute myeloid leukemia HL-60 cells and its potential mechanism. Inhibitory effect of HNK on the HL-60 cell proliferation was detected by MTT assay. Flow cytometry was used to detect the change of cell cycle and AnnexinV/PI staining was used to detect apoptosis. Western blot was applied to analyze the cell cycle protein (cyclins), cyclin-dependent kinase (CDK), P53, P21, P27, BCL-2, BCL-XL, Bax, caspase-3/9 and proteins for MAPK signal pathway. The results showed that HNK could inhibit the proliferation of HL-60 cells in time- and dose dependent ways. HNK arrested HL-60 cells in G0/G1 phase, and S phase cells decreased significantly (P < 0.05). The expression of cyclin D1, cyclin A, cyclin E and CDK2/4/6 were significantly down-regulated (P < 0.05), the expression of P53 and P21 was significantly upregulated after treating for 24 h with HNK (P < 0.05). After 24 h treatment with HNK, HL-60 cell apoptosis increased significantly with the upregulation of activated caspase-3, -9, BAX expression and the downregulation of BCL-2, BCL-XL expression. The MAPK subfamily, P38 and JNK were not significantly changed, but the expression of MEK1/2-ERK1/2 was significantly downregulated (P < 0.05). It is concluded that HNK arrestes the cells at G0/G1 phase and induces HL-60 cell apoptosis through the intervention of MEK1/2-ERK1/2 signaling pathway.

NVP-BEZ235 inhibits proliferation and colony-forming capability of CD34(+)CD38(-) human acute myeloid leukemia stem cells / 中国实验血液学杂志

This study was aimed to explore the effect of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle and colony forming capability of CD34(+)CD38(-) human acute myeloid leukemia (AML) KG1a cells. Flow cytometry was used to detect expression of CD34 and CD38 on the surface of human AML KG1a cells; Trypan blue assay was used to analyze the effect of NVP-BEZ235 at various concentrations on proliferation of KG1a cells; flow cytometry was performed to examine the cell cycle of KG1a cells after NVP-BEZ235 treatment; Soft agar colony-forming experiment was used to detect the colony forming ability of KG1a cells treated with NVP-BEZ235 at various concentrations. The results indicated that the percentage of CD34(+)CD38(-) AML KG1a cells was (98.02 ± 0.72)%. NVP-BEZ235 (0.125 - 1 µmol/L) inhibited the proliferation of KG1a cells in a time-and dose-dependent manner (P < 0.05) and the 50% inhibition concentrations (IC50) at 24 h and 48 h were 0.597 µmol/L and 0.102 µmol/L, respectively. KG1a cells were arrested at G0/G1 phase after treating with 0.5 µmol/L NVP-BEZ235 for 24 h, it was significantly higher than that of control group (83.2 ± 3.80)% vs (43.47 ± 9.60)% (P < 0.05). KG1a cells treated with NVP-BEZ235 (0 - 1 µmol/L) for 14 d and 21 d, the number of colony decreased respectively from (375.67 ± 21.46) per 2500 KG1a cells and (706.33 ± 87.31) per 2500 KG1a cells to 0, with statistical significance (P < 0.05). It is concluded that NVP-BEZ235 can inhibit proliferation and colony-forming capability of CD34(+)CD38(-) human AML KG1a cells.

Cytotoxicity of Naja Naja Actra Venom Component combined with activated immune cells on leukemia cell line KG1a / 中国实验血液学杂志

This study was aimed to investigate the cytotoxic effect of the Naja Naja Actra Venom Component (NNAVC) combined with activated immune cells on human acute myeloblastic leukemia line KG1a cells. The cytotoxic effects of NNAVC at different concentrations on KG1a cells were measured by CCK-8 method. LDH releasing assay was used to detect the cytotoxic effects of activated immune cells, NNAVC combined with activated immune cells on KG1a cells and the sensitivity of KG1a treated with NNAVC to activated immune cells. The results showed that the inhibitory rate of NNAVC on KG1a cells increased with the concentration enhancement, the cytotoxicity of activated immune cells at the different effector to target (E:T) ratios(6.25:1, 12.5:1, 25:1) on KG1a cells were 12.30%, 24.85% and 52.26%. The cytotoxicity of NNAVC combined with activated immune cells at the different E:T cell ratios (10:1, 20: 1) on KG1a cells were 56.21% and 85.59%, which were higher than that of NNAVC or activated immune cells alone. The cytotoxicity of activated immune cells at the E: T cell ratio of 10:1 on KG1a cells treated with NNAVC at different concentrations were 25.65%, 31.33%, 28.63% and 16.78%, respectively, and that at the E:T cell ratio of 20: 1 were 40.62%, 44.70%, 44.62% and 40.72%. It is concluded that:both of NNAVC and activated immune cells have lethal effect on KG1a cells, and the combination of NNAVC and activated immune cells can strengthen their effect on KG1a.

A case report of myelodysplastic/myeloproliferative disease unclassifiable with karyotype aberration of trisomy 8 and JAK2 mutation / 中国实验血液学杂志

This study aimed to investigate the relationship between clinical features of myelodysplastic/myeloproliferative disease, unclassifiable (MDS/MPD-U), karyotype of chromosome and JAK2 mutation in 1 case. The clinical features, karyotype and JAK2 mutation of the patient with MDS/MPD-U were studied by means of bone marrow biopsy, karyotype analysis and ARMS-PCR technique. The results indicated that the typical micromegakaryocytes and thrombocytosis, karyotype aberration of trisomy 8 as well as JAK2 V617F mutation were found in this patient. It is concluded that the patient was diagnosed as MDS/MPD-U with trisomy 8 and JAK2 V617F mutation. The data of this patient will provide evidence for studying correlation of chromosome karyotype aberration with JAK2 V617F mutation and for evaluating prognosis of MDS/MPD-U.

Effect of bone-marrow mesenchymal stem cells on the immune function of aging rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of transplantation of bone-marrow mesenchymal stem cells (MSCs) on the immune functions of aging rats.</p><p><b>METHODS</b>Healthy SD rats were randomized into normal control, aging model group and MSCs group. The aging model was established by daily subcutaneous injection of D-galactose for 4 consecutive months. MSCs were isolated from the bone marrow of adult SD rats and injected (3×10(6) MSCs) in rats in the MSCs group via the tail vein once a week for 4 weeks. The spleen index, activity of T lymphocytes and the levels of IL-2 and IL-10 in spleen were measured, and the pathological changes of the spleen were observed after the treatments.</p><p><b>RESULTS</b>MSCs transplantation enhanced the cellular immune function of aging rats manifested by obviously increased spleen index, activity of T lymphocyte and the level of IL-2, and lowered level of IL-10 in the spleen. The rats in the aging model group showed serious spleen injury, which was obviously lessened by MSCs injection.</p><p><b>CONCLUSION</b>MSCs transplantation can improve the cellular immune function of aging rats and ameliorate spleen injury induced by D-galactose.</p>

Establishment of an erythroleukemia model in CB6F1 mice by transplant with haploidentical mouse leukemic cell line FBL-3 / 中国实验血液学杂志

The purpose of study was to investigate the feasibility for establishing erythroleukemia model in CB6F1 mice by transplant with haploidentical mouse leukemic cell line FBL-3 and to explore the biological characteristics of FBL-3 cells in CB6F1 mice, CB6F1 and C57BL/6 mice were inoculated intravenously at doses of 1×10(3)-1×10(7) FBL-3 cells respectively. The survival time, the count of peripheral white blood cells, the percentage of erythroblasts and chromosome of these mice were observed. The liver, spleen, lung and kidney were obtained from the dying CB6F1 mice for pathological examination. The ultrastructure of erythroblasts in bone marrow and spleen was observed by transmission electron microscopy as soon as these mice died. Expression of MHC molecules and karyotype of spleen and bone marrow cell were measured. The results showed that 100% and 92.5% incidences of erythroleukemia were observed when 1×10(3)-1×10(7) FBL-3 cells had been administrated intravenously to CB6F1 and C57BL/6 mouse, respectively. There was a linear relationship between the survival time and the number of inoculated leukemic cells. The survival time of CB6F1 was longer than C57BL/6 mice inoculated the same number cell. The main targets for FBL-3 cell infiltration were liver, spleen, marrow, lung and kidney. The reaction of FBL-3 cells to glycogen staining was positive, while the to reaction peroxidase, alkaline phosphatase and butyric acid staining were negative, reaction to chloroacetic acid staining partially was positive. Virus-like particles were found in the spleen and bone marrow cells under electron microscope. Chromosomes of spleen and bone marrow cells in the majority were non-diploid, and the expression of H-2b increased, H-2d expression decreased. It is concluded that the erythroleukemia mouse model can be established in CB6F1 mice transplanted with leukemic FBL-3 cells, that provides a convenience experimental erythroleukemia model for study.

An enzyme-linked immunosorbent assay for determining serum anti-themocyte globulin concentration / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an enzyme-linked immunosorbent assay (ELISA) for determining anti-themocyte globulin (ATG) levels in serum samples.</p><p><b>METHODS</b>The microplate was coated with mouse anti-rabbit IgG monoclonal antibody, and sheep anti-rabbit polyclonal antibody conjugated with HRP was used as the second antibody for detecting the serum ATG levels in patients undergoing allogeneic hematopoietic stem cell transplantation.</p><p><b>RESULTS</b>The optimal concentration of the coating antibody and dilution ratios of the serum samples and IgG-HRP conjugate were 0.2 microg/ml, 1:40 and 1:2500, respectively. The lower sensitivity limit of the assay was 31.25 ng/ml for ATG detection. A linear relationship was established within the concentration range from 40 to 1000 ng/ml, with the coefficients of variation of 7.91 within assay and 5.22 between assays, respectively. Seven patients undergoing stem cell transplantation with ATG pretreatment showed gradually decreased concentration of ATG, and after 90 days ATG could still be detected.</p><p><b>CONCLUSION</b>The sandwich ELISA we established provides a specific and sensitive method for quantitative measurement of ATG in the clinical setting. In patients undergoing stem cell transplantation with ATG pretreatment, the ATG concentration gradually decreases but remains detectable 90 days after the administration.</p>

The action of donor-derived NK cell in leukemic mice MHC haplotype-mismatched bone marrow transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the role of donor-derived NK cells in haploidentical bone marrow transplantation (BMT) in leukemic mice.</p><p><b>METHODS</b>CB6F1(H-2b/d) mice model of EL9611 (H-2d) erythroleukemia was established by injection of EL9611 (H-2d) cells via tail vein. CB6F1(H-2b/d) mice were used as recipient, and C57BL/6(H-2b) mice as donor. Five days later, 70 CB6F1(H-2b/d) mice were randomly divided into 7 groups (10 mice per group) as follows: group 1: without treatment; group 2: simple-irradiated group; group 3: treated with cytarabine (Ara-C) at 50 mg/kg x6 d; group 4: simple BMT; group 5: haploidentical BMT with graft-versus-host disease (GVHD) that injected with bone marrow cells and spleen cells of C57BL/6(H-2b) mice 4 hours after irradiation; group 6: after irradiated with 9 Gy, mice were injected with C57BL/6(H-2b) NK cells (1 x 10(6)) and 4 hours later with BM cells, group 7: after irradiation of 9 Gy, mice were injected with C57BL/6(H-2b) NK cells (1 x 10(6)) and 4 hours later with BM cells and spleen cells. The blood routine test, survival time, body weight, and histopathology in the recipients were observed and compared among these group.</p><p><b>RESULTS</b>The survival time was (10.1 +/- 0.9), (9.8 +/- 0.9), (22.7 +/- 3.2) and (20.1 +/- 1.7) days in groups 1, 2, 3, and 5 respectively; was (30.1 +/- 16.0) days in group 4, out of which 2 mice survived for more than 30 days. The survival time was (39.1 +/- 18.1) and (49.3 +/- 17.2) days in groups 6 and 7 respectively, out of which 4 mice in group 6 and 7 mice in group 7 survived for more than 30 days. The survival time in group 6 was much longer than that in group 1, 2, 3 and 5 (P < 0.01). The survival time in group 7 was much longer than that in other groups (P < 0.05). The liver and spleen enlargement, organ destruction and infiltration with leukemic cells were observed in mice died from leukemia. The chimerism of Y chromosome appeared (80%-90%) in long-term survival mice in groups 6 and 7.</p><p><b>CONCLUSION</b>Donor-derived NK cells have the antileukemia ability and reduce GVHD in haploidentical BMT in erythroleukemia mice (EL9611, H-2d).</p>

Effectiveness of allogeneic NK cells in haploidentical bone marrow transplantation for leukemic mice / 中国实验血液学杂志

The aim of study was to investigate the effectiveness of allogeneic natural killer (NK) cells in haploidentical bone marrow transplantation (BMT) for leukemia mice. CB6F(1)(H-2b/d) murine model of EL9611 (H-2d) erythroleukemia was established by intravenous injection of EL9611 (H-2(d)) cells. CB6F(1)(H-2b/d) mice were transplanted with bone marrow (BM) cells from C57BL/6(H-2b) mice. Seventy CB6F(1)(H-2b/d) mice were randomly divided into 7 groups with 10 mice per group. 5 control groups were: group 1, in which no treatment was performed; group 2, in which mice were lethally irradiated (9 Gy); group 3, in which mice were treated with cytarabine with dose of 50 mg/kg for 6 days followed by the infusion of EL9611 (H-2(d)); group 4, in which mice were transplanted with BMT and group 5-the GVHD-control group, in which mice were transplanted with BM and spleen cells from C57BL/6(H-2b) mice 4 hours after irradiation. Experimental groups were divided into 2 groups: group A, in which mice were injected with C57BL/6(H-2b) NK cells (1 x 10(6)) after irradiation and were transplanted with BM from C57BL/6(H-2b) 4 hours later, and group B, in which mice were transplanted with BM cells and spleen cells from C57BL/6(H-2b) 4 hours after irradiation. The effect was assessed and compared by blood picture, survival time, body weight, and histopathology in the recipients. The results showed that the survival times in control group 1, 2, 3 and 5 were (10.10 +/- 0.88), (9.80 +/- 0.92), (22.70 +/- 3.23) and (20.10 +/- 1.73) days respectively. The survival time of control group 4 was (30.10 +/- 15.95) days and was over 30 days in 2 mice. The survival times in experimental group A and B were (39.10 +/- 18.11) and (49.30 +/- 17.24) days respectively. 4 mice in experimental group 1 and 7 mice in group 2 survived over 30 days. The survival time of experimental group 1 was significantly longer than that of control group 1, 2, 3 and 5 (p < 0.01). The survival time of experimental group 2 was significantly longer than that of other groups (p < 0.05). Histopathologic examination showed that splenohepatomegalia and disorganization of liver and spleen with infiltration of a large amount of leukemia cells in mice dead of leukemia. Chimerism of Y chromosome was shown in mice of experimental groups with long survival time. It is concluded that the injection with donor-derived NK cells can both eliminate leukemia cells and decrease the severity of GVHD after haploidential BMT.

Matrine enhances the cytotoxic sensitivity of ABCG2high nasopharyngeal carcinoma cells to natural killer cells by inducing high expression of NKG2D receptor ligands / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism underlying the effects of matrine in enhancing the cytotoxic sensitivity of CNE2/DDP cells highly expressing ATP-binding cassette superfamily G member 2 (ABCG(2)(High)) to allogenic natural killer (Allo-NK) cells.</p><p><b>METHODS</b>ABCG(2)(High) CNE2/DDP cells and Allo-NK cells were isolated by magnetic activated cell sorting (MACS). Flow cytometry was used to evaluate the purity of the isolated cells and the expression of NKG2D ligands on the target cells before and after incubation with matrine. The cytotoxic sensitivity of the treated and non-treated ABCG(2)(High) CNE2/DDP cells to Allo-NK cells was measured by LDH releasing assay.</p><p><b>RESULTS</b>The expression rate of ABCG2 was (91.40-/+2.32)% in ABCG(2)(High) CNE2/DDP cells. More than 90% of the isolated NK cells were identified to be CD3(-)CD16(+)CD56(+) cells. The expression rates of MICA, MICB, ULBP1, ULBP2, and ULBP3 on the target cells incubated with matrine increased from (2.92-/+0.33)%, (4.27-/+0.33)%, (5.80-/+0.62)%, (11.10-/+3.15)%, and (7.75-/+1.14)% to (11.30-/+0.89)%, (14.29-/+2.61)%, (12.56-/+1.06)%, (43.24-/+4.43)%, and (12.77-/+1.06)%, respectively. At the E: T ratio of 10:1 and 20:1, the cytotoxic sensitivity of ABCG(2)(High) cells to Allo-NK cells increased from (15.32-/+1.34)% and (27.26-/+6.81)% in un-treated cells to (28.53-/+1.37)% and (42.72-/+2.80)% in matrine-treated cells, respectively, showing significant differences in the cytotoxic sensitivity of the target cells in both groups produced by matrine treatment (F=29.05, P=0.000).</p><p><b>CONCLUSION</b>Matrine can up-regulate the expressions of NKG2D ligands (MICA/B and ULBP1-3) in ABCG(2)(High) nasopharyngeal carcinoma cells, which results in increased cytotoxic sensitivity of the cells to Allo-NK cells.</p>

In vitro tracing of rat bone marrow mesenchymal stem cells using the fluorescent dye CFSE / 南方医科大学学报

<p><b>OBJECTIVE</b>To determine the optimal condition for labeling rat mesenchymal stem cells (MSCs) using the fluorescent dye CFSE and the maximum time length allowed by CFSE staining for MSC tracing in vitro.</p><p><b>METHODS</b>Rat MSCs were labeled with CFSE at different concentrations (2.5, 5.0, 10.0, 20.0 and 40.0 micromol/L) for 1, 5 or 10 min. The transfection efficiency and fluorescence intensity in the cells were measured by flow cytometry and fluorescence microscope to determine the optimal condition for MSC labeling. Under the optimal condition, the effect of CFSE on the growth of MSCs was evaluated by MTT assay, and flow cytometry and fluorescence microscope were used to determine the maximum time length following CFSE labeling to allow MSC tracing.</p><p><b>RESULT AND CONCLUSION</b>Staining with CFSE at 20.0 micromol/L for 5 min was optimal for labeling rat MSCs in vitro, which allowed detection of the MSCs as long as 21 days after the labeling without obviously affecting the cell growth (P>0.05).</p>

Role of paternal veto cells in preventing graft-versus-host disease after HLA-haploidentical stem cell transplantation in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of paternal veto cells in preventing graft-versus-host disease (GVHD) after related HLA- haploidentical stem cell transplantation in mice.</p><p><b>METHODS</b>MHC-haploidentical recipient B6CF1(H-2 b/d) mice pretreated with total body irradiation at 9.0 Gy for 4 h before transplantation. The recipient mice were divided into 4 groups, and in the irradiation group, only injection of 0.3 ml D-Hank's liquid was given through the tail vein; in the control group, the mice received injection through the tail vein of 4.5x10(6) bone marrow cells mixed with 3.0x10(7) spleen cells from C57BL/6 mice without the preventive measures of GVHD; the mice in the two experiment groups received cell transplantation in the same manner, and on day 4 after transplantation, 5.0x10(6) and 1.0x10(7)spleen cells from BALB/c mice were injected through tail vein, respectively. The hematopoietic recovery, engraftment and GVHD of the recipient mice were observed.</p><p><b>RESULTS</b>Without any treatment, all mice in the control group developed GVHD and died after transplantation. In the 10 mice with injection of 5.0x10(6) spleen cells, GVHD occurred in 5 mice with a 30-day survival rate of 50%; the median survival time of the mice with GVHD was 20 days, significantly longer than that of the control mice (14 days, P<0.05). In the 10 mice injected with 1.0x10(7) spleen cells, 2 developed GVHD and the 30-day survival rate was 80% (8/10) with a median survival time of 30 days, significantly longer than that of mice with injection of 5.0x10(6) spleen cells and the control mice (P<0.05).</p><p><b>CONCLUSION</b>Paternal veto cell transplantation can decrease the occurrence of GVHD after related HLA haploidentical stem cell transplantation in mice.</p>

Cytotoxicity of allogenetic natural killer cells against CD34+ acute myelogenous leukemia cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the cytotoxic effect of allogenetic natural killer (NK) cells in vitro on human CD34+ acute myelogenous leukemia cells.</p><p><b>METHODS</b>CD34 expression on acute myelogenous leukemia KG1a cells was detected by flow cytometry. KG1a cells were co-cultured at different effector-to-target (E:T) ratios with NK cells isolated from 5 healthy individuals using magnetic cell sorting. Lactate dehydrogenase (LDH) release assay was employed to examine the cytolysis of KG1a cells in the co-culture, and the inhibition rate of the KG1a cell colony formation in methylcellulose was determined with K562 cells sensitive to NK cells as the control.</p><p><b>RESULTS</b>A expression rate as much as (98.0-/+1.1)% was detected for CD34 antigen on KG1a cells, and the isolated NK cells (CD3(-)CD16+CD56+ cells) had a purity of (93.2-/+3.7)% after magnetic cell sorting. Allogenetic NK cells exhibited obvious cytotoxicity and colony inhibition in vitro against KG1a cells at different E:T ratios, and the effects were significantly enhanced as the E:T ratios increased (P<0.05). At the same E:T ratio, the cytotoxicity and colony inhibition rate of allogenetic NK cells against KG1a cells was lower than those against K562 cells (P<0.05).</p><p><b>CONCLUSION</b>Allogenetic NK cells exhibit obvious cytotoxicity and colony formation against CD34+ acute myelogenous leukemia cells.</p>

Effect of small interfering RNA targeting multidrug resistance-related protein and bcl-2 on drug resistance and apoptosis of K562 and K562/ADM cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of small interfering RNA (siRNA) targeting multidrug resistance-related protein (MRP) and bcl-2 genes in modulating drug resistance and apoptosis of K562 and K562/ADM cells.</p><p><b>METHODS</b>Two siRNA constructs targeting respectively bcl-2 and MRP genes, were synthesized and transfected either alone or in combination into K562 and K562/ADM cells via lipofectamine2000. MTT assay was used to evaluate the viability of the transfected cells at 24, 48 and 72 h Post-fransfection, and RT-PCR was performed to determine the mRNA levels of bcl-2 and MRP. The effects of MRP siRNA and bcl2 siRNA on the apoptosis and the protein expression of Bcl-2 and MRP were evaluated with flow cytometry.</p><p><b>RESULTS</b>In K562/ADM cells, the IC (50) decreased from 12.81 microg/ml (ADM group) to 3.74 microg/ml (ADM+MRP siRNA group), 6.82 microg/ml (ADM+bcl2 siRNA group) and 2.51 microg/ml (ADM+MRP siRNA+bcl2 siRNA). Similarly, in K562 cells, the IC50 decreased significantly from 6.75 microg/ml (ADM) to 3.22 microg/ml (ADM+MRP siRNA), 3.56 microg/ml (ADM+bcl2 siRNA) and 1.84 microg/ml (ADM+MRP siRNA+bcl2 siRNA) (P<0.05). Flow cytometry demonstrated significantly increased apoptosis of the cells following MRP siRNA and bcl2 siRNA transfection, which also resulted in significantly decreased expressions of MRP and bcl-2 proteins (P<0.05).</p><p><b>CONCLUSION</b>Treatment with both MRP and bcl-2 siRNAs inhibits the target gene expression, and increases the drug sensitivity and apoptosis of K562 and K562/ADM cells.</p>

Immunoediting of natural killer cells by human nasopharyngeal carcinoma cell line: altered expression of KIRs and NKG2D receptors leads to reduction of natural killer cell-mediated cytolysis / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the changes of inhibitory killer cell immunoglobulin-like receptors (KIRs), NKG2D receptor and the cytotoxicity of natural killer (NK) cells induced by persistent exposure to CNE2 cells.</p><p><b>METHODS</b>The HLA-class I genotypes of CNE2 cells and KIR genotypes were determined by PCR with sequence-specific primers (PCR-SSP). The expressions of KIR2DL1, KIR2DL3, KIR3DL1, and NKG2D by the NK cells (freshly isolated NK cells, NK cells cocultured with 100 U/ml IL2 or with 100 U/ml IL2 and CNE2 cells as the control, IL2 and CNE2 groups, respectively) were analyzed by flow cytometry. Cytotoxicity of NK cells against CNE2 cells were detected by LDH releasing assay.</p><p><b>RESULTS</b>The HLA genotypes of CNE2 cells were A2, 24, B18, 35, Cw4, 7. NK cells isolated from 3 healthy donors expressed KIR2DL1, KIR2DL3, and KIR3DL1. After 4, 24 and 48 h of culture, NK cells in CNE2 group displayed higher KIR2DL1, KIR2DL3 but lower NKG2D expression than those in the control and IL2 groups (P<0.01), whereas the latter two groups showed no significant difference in KIR2DL1, KIR2DL3, and NKG2D expressions (P>0.05), and no difference in KIR3DL1 expression was found between the 3 groups (P>0.05). After 24 h of culture, the cytotoxicity against CNE2 cells mediated by the NK cells in IL2 and CNE2 groups were (26.96-/+1.47) % and (2.74-/+1.64) % at E:T ratios of 10:1, and (35.74-/+3.59)% and (4.57-/+2.41) % at E:T ratio of 20:1, respectively. NK cells in CNE2 group displayed lower cytotoxicity than those in IL2 group (P<0.01).</p><p><b>CONCLUSIONS</b>Persistent exposure to tumor cells expressing NKG2D ligands can lead to downregulated expression of NKG2D receptor, increased expression of KIRs and reduction of NK-mediated cytolysis. These results elucidate the molecular mechanism of reduced cytotoxicity mediated by the edited NK cells and indicate that blocking HLA-class I-bound KIRs or enhancing the expression of NKG2D may promote NK cell-mediated cytolysis.</p>

Expression of HLA class I molecules and MHC class I chain-related molecules A/B in K562 and K562/AO2 cell lines and their effects on cytotoxicity of NK cells / 中国实验血液学杂志

The study was aimed to investigate the expression of HLA class I molecules and MHC class I chain-related molecules A/B (MICA/MICB) in K562 and adriamycin (ADM)-resistant K562 cell lines (K562/AO2) and their effect on cytotoxicity of NK cells. Expression of HLA class I molecules and MICA/MICB on the surface of K562 and K562/AO2 cell lines were analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against K562 and K562/AO2 cells were detected by LDH releasing assay at different effect-to-target cell ratios (E:T). In blocking experiments, anti-MHC class I monoclonal antibody (McAb) (W6/32, a pan anti-HLA class I antibody) and anti-MHC class I chain-related molecules McAb (BAMO-1, specifically against MICA and MICB) were added to the target cells at E:T of 10:1. The results showed that the expression of MHC class I chain-related molecules on K562 was higher than that on K562/AO2 (P=0.000), and HLA class I molecules were not detectable on both cells. Cytotoxicities of NK cells against K562 and K562/AO2 cells were (29.32 +/- 0.12)%, (45.33 +/- 0.78)%, (58.37 +/- 0.87)%, (72.37 +/- 0.96)% and (12.47 +/- 0.91)%, (24.36 +/- 1.11)%, (33.29 +/- 1.03)%, (53.87 +/- 1.27)% at E:T ratios of 5:1, 10:1, 20:1 and 30:1 respectively (P=0.000), the cytotoxicity of NK cells on K562 cells was significantly higher than that on K562/A02 cells at different E:T ratios. Blocking experiments confirmed that at E:T of 10:1 killing of NK cells against K562 and K562/AO2 cells was efficiently inhibited by BAMO-1, whereas W6/32 had no effect on K562 and K562/AO2 cells. It is concluded that the expression of MHC class I chain-related molecules on K562 and K562/AO2 cells is correlated with NK cell-mediated lysis. NK cells display higher cytotoxicity against parental K562 cells than multi-drug resistant K562/AO2 cells. Down-regulation of MICA/B in multi-drug resistant tumor cell lines leads to reduction of susceptibility to NK lysis.