Clinical Significance and Expression of PD-1/PD-L1 and Treg Cells in JAK2 V617F Positive Myeloproliferative Neoplasms / 中山大学学报(医学科学版)

@#【Objective】To investigate the expression and clinical significance of programmed death- 1（PD- 1），programmed death-ligand 1（PD-L1）and CD4+ CD25+ Foxp3+ regulatory T cells（Treg）and CD4+/CD8+ ratios in JAK2V617F mutation positive myeloroliferative neoplasms patients（MPN）.【Methods】45 cases of JAK2 V617F positive MPN patients were selected including 17 cases of essential thrombocythemia（ET），13 cases of polycythemia vera（PV）and 15 cases of primary myelofibrosis（ET）. 30 cases of the newly diagnosed group and 15 cases of treatment group were from them. 15 cases of healthy volunteers were selected as control group. The ratio of mutant and wild type of JAK2 was detected by fluorescence quantitative polymerase chain reaction（FQ-PCR）. The expression levels of p-JAK2，PD-1 and PD- L1 in pathological tissues of bone marrow were detected by immunohistochemistry. The changes of treg cells and lymphocyte subsets in peripheral blood of MPN patients and controls were detected by flow cytometry. 【Results】 The expression levels of p-JAK2，PD-1，PD-L1，and Treg in the newly diagnosed group were significantly higher than that of treatment group and control group（P<0.05），while the expression levels of CD4 +/CD8 + ratio were significantly lower than treatment group and control group（P<0.05）. JAK2 V617F mutation burden was positive correlation with PD-1 and PD- L1，and was negative correlation with CD4 +/CD8 + ，the correlation coefficients were r=0.593，P<0.01；r=0.723，P<0.01；r=-0.771，P<0.01，respectively.【Conclusion】p-JAK2，PD-1，PD-L1，Treg，CD4+/CD8+ and JAK2 V617F were involved in the pathogenesis of myeloroliferative neoplasms.

Effect of catalpol on " glucose deprivation" cardiomyocyte injury based on estrogen receptor / 中国药理学通报

Aim: To investigate the effect of catalpol-mediated autophagy on oxidative damage of cardiomyocytes induced by glucose deprivation based on estrogen receptor(ER) and the related mechanism. Methods: The cardiomyocytes (H9c2) injury model in rat was induced by glucose deprivation for 6 h. The protective effect of catalpol on H9c2 injury and its mechanism were observed. The cells were divided into five groups: control group, model group, catalpol group (0. 28, 2. 8, 28 μmol · L-1). The effects of catalpol on reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) in cells were detected, and the effect of catalpol on autophagy was assessed by electron microscopy. ER blockers was used to detect whether the protective effect of catalpol on oxidative damage was related to ER. Lentivirus transfection was used to lower the expression of ER alpha, and the oxidative damage and autophagy of H9c2 treated with catapol or not were observed. Results: Compared with control group, the levels of ROS and MDA increased and SOD decreased in model group, and there was no significant difference in the expression of autophagy related proteins. Compared with model group, catalpol could reduce oxidative damage and increase autophagy level. After transfection of ER alpha with lentivirus, catalpol inhibited the level of oxidative damage and promoted the effect of autophagy compared with the empty virus catalpol group. Conclusions: Catalpol can activate autophagy by up-regulating the expression of ER alpha and inhibiting the oxidative damage of cardiomyocytes.

Effect of Arsenic Trioxide on K562 Cell Proliferation and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of arsenic trioxide(ATO) inhibiting K562 cell proliferation, and explore the new targets for treating chronic myeloid leukemia(CML).</p><p><b>METHODS</b>human CML cell line K562 cells were cultured in vitro, and were treated with different concentrations of ATO; MTT was used to detect the cell proliferation; flow cytometry(FCM) was used to determine cell apoptosis, cell cycle and the expression of CD44; Transcriptional levels of β-catenin and cyclin D1 were assayed by RT-PCR.</p><p><b>RESULTS</b>2 µmol/L ATO could inhibit the cell proliferation obviously in a time-and-dose-dependent manner. With drug concentration increasing and time prolonging, the expression rate of CD44 was declined gradrually. FCM with AnnexinV/PI double staining showed that K562 cells were induced to apoptosis after exposure to 2.5-10 µmol/L ATO for 48 hours and in dose-dependent manner. Treating with different concentration ATO for 48 hours, cell ratio of G/Gphase increased and cell ratio in S phase decreased gradually. RT-PCR showed that the expression of β-catenin and CyclinD1 decreased with increasing of drug concentration.</p><p><b>CONCLUSION</b>ATO in certain concentration range can inhibit K562 cell proliferation, and induce the cell apotosis, the mechanismin influencing the Wnt/β-catenin pathway may be the downregulation of CD44 expression, arresting K562 cells in G/Gphase, and affecting the gene transcription, thus inhibiting K562 cell proliferation.</p>

Effect of Anti-CD44 Monoclonal Antibody A3D8 on Expression of AP-1 in HL-60 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of anti-CD44 monoclonal antibody A3D8 on expression of transcription factor AP-1 in acute myeloid leukemia cells.</p><p><b>METHODS</b>After acute leukemia cell line HL-60 was treated by different concentrations of A3D8, the proliferation and cell cycle were detected by MTT and FCM respectively. The expressions of c-JUN and c-FOS at mRNA and protein level were detected by RT-PCR and Western Blot respectively.</p><p><b>RESULTS</b>The proliferation of HL-60 was inhibited by A3D8. The A3D8 treatment increased the percentage of G/Gcells. The expressions of c-JUN at mRNA and protein level were both decreased in HL-60 cells treated with A3D8. The expressions of c-FOS at mRNA and protein level in rapamycin treatment groups showed no statistically significant difference as compared with that in control group.</p><p><b>CONCLUSIONS</b>A3D8 can affect the activity of AP-1 through inhibiting the expressions of c-JUN at mRNA and protein level.</p>

Effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of HL-60 cells and its mechanism / 中国实验血液学杂志

This study was aimed to investigate the effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of human acute myeloid leukemia cell line HL-60 and its mechanism. HL-60 cells were cultured with different concentrations of celecoxib for 24 h. Cell proliferation was analyzed by CCK-8 assay, cell apoptosis and cell cycle distribution were detected by flow cytometry. Cyclin D1, cyclin E1 and COX-2 mRNA expressions were determined by RT-PCR. The results showed that after the HL-60 cells were treated with different concentrations of celecoxib for 24 h, the cell growth was significantly inhibited in a dose-dependent manner(r = 0.955), IC50 was 63.037 µmol/L of celecoxib. Celecoxib could effectively induce apoptosis in HL-60 cells also in dose-dependent manner(r = 0.988), blocked the HL-60 cells in the G0/G1 phase. The expression of cyclin D1, cyclin E1 and COX-2 mRNA were downregulated. It is concluded that celecoxib can inhibit the proliferation of HL-60 cells in dose-dependent manner, celecoxib causes cell G0/G1 arrest and induces cell apoptosis possibly through down-regulation of the cyclin D1 and cyclin E1 expression, and down-regulation of COX-2 expression respectively.

IL-23 alone or with IL-2 induces the killing effect of hPBMNC on K562 cells / 中国实验血液学杂志

This study was aimed to explore the killing effect of PBMNC induced by IL-23 alone or combined with IL-2 on K562 cells and its mechanism. The PBMNC were induced in vitro by IL-23 (50 ng/ml) alone or IL-23 combined with IL-2 (100 U/ml) for 72 h, and then were co-cultured with leukemia cell line K562. The CCK-8 method was used to detect the effect of PBMNC induced at different times on K562 cells, the ELISA was performed for detecting IFN-γ level in culture supernatant, and the perforin and granzymes B were detected by RQ-PCR. The results showed that the killing effect of PBMNC induced by IL-23 alone or IL-23 combined with IL-2 on K562 cells was observed, and obviously enhanced with prolonging of time, moreover, there was statistical difference among different time points (P < 0.05). The IFN-γ level in supernatant of PBMNC cultured with cytokines significantly increased, and the IFN-γ levels in group of IL-23 combined with IL-2 were higher than that in other groups (P < 0.05). The mRNA expressions level of perforin and granzymes B of the expanded PBMNC in groups cultured with cytokines were higher than that in control group (P < 0.05), and the mRNA expressions of perforin and granzymes B in group of IL-23 combined with IL-2 were significantly higher than that in others (P < 0.05). It is concluded that IL-23 can promote the killing effect of PBMNC on K562 cells. The combination of IL-2 with IL-23 displays synergic effect and a time-dependent manner. IL-23 also enhances the expression of IFN-γ, perforin and granzyme B in PBMNC. Its combination with IL-2 displays synergistic effect, suggesting that the anti-leukemic activity of IL-23 may be realized through inducing PBMNC to express IFN-γ, perforin and granzyme B.

Artesunate induced G2/M phase cell cycle arrest in multiple myeloma RPMI8226 cells / 第二军医大学学报

Objective To investigate the effect of artesunate on the cell cycle of human multiple myeloma RPMI8226 cells and explore the related molecularmechanisms. Methods RPMI8226 cellswere treated with 0, 25, and 50 pg/mL artesunate. The morphology change of cells was observed under transmission electron microscope, the cell cycle distribution was detected by flow cytometry, and the expression of cyclin B1 and p34cdc2 protein was examined by Western blotting analysis. Results After treated with 50 pg/mL artesunate for 48 h, most RPMI8226 cells showed characteristic morphology of apoptosis. With the increase of artesuate concentration, the proportion of RPMI8226 cells in G0/G1 phase was significantly decreased (P<0. 05) and that of cells in G2/M phase was significantly increased (P<0.05), suggesting that artesunate induced noticeable G2/M arrest. Cyclin B1 level was increased and the p34cdc2 level was decreased with the increase of artesuate concentration. Conclusion Artesunate can induce G2/M arrests in RPMI8226 cells, which may be related to the increasedcyclin B1 expression and decreased p34cdc2 expression.

Antioncogene PTEN-a new target for myeloma therapy / 中国实验血液学杂志

Pten gene is the first antioncogene with dual phosphatase activity discovered so far, pten gene regulates the cell cycle progress, apoptosis, metastasis and invasion of the tumor cells through negatively regulating the multiple signaling transduction pathways. Multiple myeloma (MM) is a malignant tumor occurring in terminal stage of B cell differentiation. The genetic changes are considered as the important factors in MM pathogenesis, among which the deletion of antioncogene is a critical genetic change. However, little is known about the genetic change of pten in MM. This review summarizes the research advance on pten in MM including structure of pten, mechanism of pten effect and correlation of pten with MM in order to provide some references for the investigating new gene target to treat the MM.

The role of PTEN-FAK signaling pathway in metastasis and invasive ability of leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of the wild type phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor-suppressor gene on the proliferation and apoptosis of human chronic myeloid leukemia (CML) cells line (K562) in vitro and explore the influence of PTEN-FAK signaling pathway on invasion and metastasis of leukemia cells.</p><p><b>METHODS</b>The recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562 cells and fresh leukemia cells from CML patients in blast crisis. The growth of K562 cells was assayed by MTT assay; the apoptosis rate was assessed by flow cytometry (FCM). PTEN and FAK mRNA levels were detected by real-time fluorescent relative- quantification reverse transcriptional PCR (FQ-PCR) and its protein levels by Western blot. The metastasis and invasive ability was examined by transwell chamber assay.</p><p><b>RESULTS</b>The growth of K562 cells was suppressed markedly when Ad-PTEN-GFP was transfected into K562 cells at the 200 multiplicity of infection (MOI). The maximum growth inhibition rate was 35.2%. Transwell results showed the number of cells entered the lower chamber in Ad-GFP group was 9.1 fold more than that in Ad-PTEN-GFP group;The ability of metastasis and invasion of fresh leukemia cells was also suppressed after transfection with Ad-PTEN-GFP. FAK and p-FAK proteins were down-regulated by 0.72 and 0.16 fold lower after transfected with Ad-PTEN-GFP compared with Ad-GFP group.</p><p><b>CONCLUSIONS</b>PTEN gene might inhibit the proliferation, metastasis and invasive ability of leukemia cells via down-regulating FAK expression.</p>

Efficacy of shenqi fuzheng injection combined with chemotherapy in treatment of acute leukemia and its effect on T-lymphocyte subsets, serum IFN-gamma, IL-10 and IL-2 / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the clinical efficacy of Shenqi Fuzheng Injection (SFI) combined with chemotherapy in treatment of patients with acute leukemia and its effect on the levels of T-lymphocyte subsets (CD4, CD8, CD4/CD8) and serum interferon-gamma(IFN-gamma), interleukin-10 (IL-10) and IL-2.</p><p><b>METHODS</b>Sixty-five patients with initial treating acute leukemia were randomly divided into 2 groups, the SFI group (n = 32) treated with SFI plus chemotherapy (CT), the control group (n = 33) treated with CT only. The remission rate, changes of peripheral mature neutrophilic granulocyte (PMNG) count, T-lymphocyte subsets, serum IL-10 and IL-2 before and after treatment were determined.</p><p><b>RESULTS</b>The remission rate in the two groups showed no obvious difference (P > 0.05). After CT for the 1st, 2nd and 3rd weeks, the PMNG count decreased in both groups, showing significant difference as compared with that before CT (P < 0.01 or P < 0.05). The PMNG count at the end of the 3rd and 4th week of CT remounted to higher than that at 1st and 2nd week, and the increment in the SFI group was significantly higher than that in the control group (P < 0.05). The levels of CD4, CD4 /CD8, IFN-gamma and IL-2 all increased in the two groups after treatment (P < 0.05, P < 0.01), however, that of IL-10 was significantly decreased (P < 0.01). The difference between the two groups in these criteria after treatment was also significant (P < 0.05).</p><p><b>CONCLUSION</b>SFI can improve and regulate the immune function of the patients with acute leukemia undergoing CT, it could promote bone marrow cells proliferation and enhance the efficacy.</p>

Clinical efficacy and T-lymphocyte subset, serum interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2(IL-2) levels on treatment of chronic aplastic anemia patients by shenfu injection combined with stanozol and cyclosporin A / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of Shenfu injection (SFI) and influence on T-lymphocyte subset, serum level of interferon-gamma(IFN-gamma), tumor necrosis factor-alpha(TNF-alpha), interleukin-2(IL-2) in patients with chronic aplastic anemia (CAA) based on treating with stanozol and cyclosporin A.</p><p><b>METHOD</b>60 patients with CAA were randomly divided into two groups, 30 patients in the SFI group were treated with SFI (100 mL which contains Ginsenoside 0.8 mg x mL(-1) and aconitine 1.8 microg x mL(-1) by adding it in 500 mL of 5% glucose every day) plus stanozol and cyclosporin A and 30 patients in the control group treated with slanozol and cyclosporin A alone for 2 months. The clinical efficacy was observed. The change of T-lymphocyte subset analyzed by flow cytometry and the levels of serum IFN-gamma, TNF-alpha, IL-2 measured with ELISA method were also observed before and after treatment.</p><p><b>RESULT</b>After treatment, the total effective rate of the SFI group was higher than that in the control group, but it did not showing significant difference. The CD4/CD8 levels were significantly increased (1.76+/-0.49, P< 0.01) and CD8 levels were significantly lowered (22.57+/-6.30, P < 0.01) in the SFI group after treatment. Serum levels of lFN-gamma, TNF-alpha and IL-2 were lower in both groups, and the level of TNF-alpha and IL-2 in the SFI group (0.710+/-0.213) ng x L(-1) and (0.639+/-0.247) ng x L(-1) was significantly lowered than that in the control group (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>SFI might believe the hemopoietic inhibition so as to promote the recovery of hemopoietic function through improving the T-lymphocyte subset and reducing the release of hemopoietic negative regulatory factors such as IFN-gamma, TNF-alpha and IL-2.</p>

Study on efficacy of treatment of acute leukemia by shengfu injection in combination with chemotherapy and the effect on cellular immunity, serum interleukin-6 and tumor necrosis factor-alpha levels / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the efficacy of treatment of acute leukemia by Shengfu Injection (SFI) in combination with chemotherapy and the effect of treatment on T-lymphocyte subsets (CD4, CD8, CD4/CD8), serum interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha).</p><p><b>METHODS</b>Sixty-one patients with acute leukemia of initiatory treating were randomly divided into two groups, the 31 patients in the treated group were treated with SFI plus chemotherapy and the 30 patients in the control group treated with chemotherapy alone. The remission rate, changes of absolute number of peripheral mature neutrophils, T-lymphocyte subsets, serum levels of IL-6 and TNF-alpha before and after treatment were observed.</p><p><b>RESULTS</b>The remission rate was higher in the treated group than that in the control group but the difference was insignificant (P > 0.05). The restoring of peripheral mature neutrophils in the treated group was higher than that in the control group, from the 3rd week of treatment, the difference was significant (all P < 0.05). CD4 and CD4/CD8 ratio after treatment increased in both groups, the increment was more obvious in the treated group than that in the control group significantly (P < 0.05). Serum levels of IL-6 and TNF-alpha lowered in both groups after treatment significantly(all P < 0.01), but the decrement was greater in the treated group with significant difference to the control group (P < 0.05).</p><p><b>CONCLUSION</b>SFI could improve and regulate the immune function in acute leukemia patients undergoing chemotherapy and enhance the therapeutic effect.</p>