Effect of proliferation of triptolide on MV4-11 cells and its mechanism research associated with RAS/ERK/MAPK / 中国医师杂志

Objective To explore the effect of proliferation and apoptosis,and research its mechanism associated with RAS/extracellular signal regulated kinase/mitogen-activated protein kinase (RAS/ ERK/MAPK) in Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation acute lymphocytic leukemia cell line MV4-11 cells treated by triptolide (TP).Methods MV4-11 cells were respectively treated by triptolide with diverse concentrations and different times.The proliferation inhibition rate was measured by methyl thiazolyl tetrazolium,the apoptosis rate was detected by flow cytometry,the mRNA expressions of FLT3,RAS,ERK,forkhead box protein M1 (FOXM1),and c-Myc were analyzed by realtime fluorescent quantitative polymerase chain reaction (PCR).Results The proliferation inhibition rate markedly increased in a dose-time dependent manner after MV4-11 cells were treated by TP.After cells were treated with 10,and 20 nmol/L TP,respectively,the apoptosis rates at 48 h were (17.30 ± 0.56) ％,(35.77 ±0.55)％,and those at 72 h were (49.83 ±0.45)％,(68.90 ±0.75)％ correspondingly.PCR data showed that the mRNA level of FLT3 mRNA decreased obviously,and that of RAS,ERK,FOXM1,and c-Myc also decreased.Conclusions Triptolide could significantly inhibit the MV4-11 cells proliferation and induce cell apoptosis,and its mechanism might be through inhibiting the expression of related genes on RAS/ERK/MAPK signaling pathway.

Effects of triptolide on proliferation and apoptosis of acute myeloid leukemia cell line MV411 with FMS-like tyrosine kinase 3 internal tandem duplication and their mechanisms / 白血病·淋巴瘤

Objective To investigate the effects of triptolide (TP) on proliferation and apoptosis of acute myeloid leukemia cell line MV411 with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), and its effect on PI3K-Akt-mTOR pathway. Methods MTT assay was used to detect the proliferation inhibition of MV411 cell proliferation treated by different concentrations of TP in 24, 48 and 72 h. Flow cytometry was used to measure the cell apoptosis rate at 48 and 72 h. Real-time fluorescent quantitative PCR was used to detect the expression of FLT3, PTEN, PI3K, Akt, mTOR mRNA on PI3K-Akt-mTOR pathway. Results After treated by 0, 5, 10, 20, 40, and 80 nmol/L TP in 24, 48 and 72 h, the viability of MV411 cells was inhibited in a time-dose dependence manner. MV411 cells was treated by 0, 10, and 20 nmol/L TP after 48 and 72 h, the cell apoptosis rates were rising with the increasing of drug concentration, there were statistical significances [48 h:(3.30±0.20) %, (17.10±0.36) %, (35.67±0.61) %, 72 h: (7.37±0.32) %, (49.33± 0.40)%, (68.92±0.11)%, all P=0.000]. The expressions of FLT3, PI3K, Akt, and mTOR mRNA were down-regulated and PTEN mRNA expression was up-regulated by TP. Conclusion TP may inhibit the proliferation and induce apoptosis of MV411 cells by affecting the expression of PI3K-Akt-mTOR pathway related genes.

Clinical analysis of sodium valproate combined with decitabine for treatment of myelodysplastic syndrome / 白血病·淋巴瘤

Objective To observe the clinical effects and safety of sodium valproate combined with decitabine for treatment of myelodysplastic syndrome (MDS). Methods Forty-two patients with MDS were enrolled in department of hematology in Shanxi Dayi Hospital from February 2012 to February 2017. According to random number table, the patients were divided into the control group (21 cases) and the experimental group (21 cases). The patients in the control group received decitabine at the dose of 20 mg·m-2·d-1, and intravenous infusion was completed in 2 hours, continuous therapy up to 5 days, 4 weeks as a course; the patients in the experimental group received combined medication, orally given sodium valproate 0.2 g once, 3 times per day. One week later, the dosage was added to 0.4 g once, 3 times per day. Both groups received at least 4 courses of treatment. The treatment was stopped when serious adverse reactions or obvious disease progression occurred. The bone marrow smear was rechecked every 4 weeks after treatment to evaluate the efficacy. The expressions of ASXL1, DNMT3A and TET2 in bone marrow cells were detected by fluorescence quantitative PCR before and after treatment. Results The total treatment response rate of the experimental group and the control group were 76.2 % (16/21) and 57.1 % (12/21) respectively, and there was statistically significant difference (P< 0.05); the total remission rate of the two groups was 47.6 % (10/21) and 38.1 %(8/21) respectively, and there was no significant difference (P> 0.05). All patients had slight adverse reactions, and the adverse reaction rate was 42.9 % (9/21) and 38.1 % (8/21), and there was no significant difference (P>0.05). The content of TET2 mRNA and DNMT3A mRNA after treatment in both groups were decreased compared with the expressions before treatment, and there were significant differences (P<0.05). However, there was no significant difference between the two groups after treatment (P> 0.05); the content of ASXL1 mRNA had no obvious change in the control group and a dramatic decrease in the experimental group compared with that before treatment (P<0.05). Conclusion Sodium valproate combined with decitabine has favorable effects and mild adverse reactions for treatment of MDS, besides, it can influence the expressions of TET2, DNMT3A and ASXL1.

Mechanisms of MDM2-p53 pathway in apoptosis of in K562/G01 cells induced by triptolide / 白血病·淋巴瘤

Objective To explore the apoptosis of K562/G01 cells induced by triptolide through MDM2/p53 signaling pathway. Methods K562/G01 cell line was treated with different concentrations of triptolide. MTT was used to detect the cell proliferation inhibition rate. FCM was used to determine the apoptosis rate changes in 12 h and 24 h. The mRNA expression levels of bcr-abl, XIAP, MDM2, p53 were detected by real-time quantitative PCR. Results After treatment by 10, 20, 40, 80, 100 nmol/L TP in 12, 24, 48 h, the viability of K562/G01 cells was inhibited in time-dose dependence manner. K562/G01 cells was treated by 20 nmol/L, 40 nmol/L TP after 12 h, 24 h, the cell apoptosis rate was rising with drug concentration and time. The bcr-abl, XIAP, MDM2 mRNA expression was down-regulated and p53 mRNA expression was up-regulated by TP. Conclusion TP can inhibit the growth of K562/G01 cell line and induce apoptosis through XIAP-MDM2-p53 signaling pathway.

Analysis of the Correlation between the Infrared Radiation Temperature of Specific Points and the Symptoms in Electracupuncture-treated Patients with Overactive Bladder / 上海针灸杂志

Objective To observe the correlation between infrared radiation temperature of specific points of the bladder (Zhongji, Pangguangshu, Weizhong and Shenshu) and OAB symptom severity in patients with overactive bladder (OAB) before and after electroacupuncture.Method Eighty-six patients were treated with electroacupuncture. The infrared radiation temperatures of the points were measured using an infrared thermograph in the patients before and after electroacupuncture. The patients’ symptoms were scored using the OAB Symptom Score (OABSS). The correlation between the infrared radiation temperature and the symptom score was analyzed according to the changes in the two.Result In the patients, the OAB symptom score (OABSS) was 8.00 (7.00, 9.00) before treatment and 2.00 (4.00, 6.00) after. There was a statistically significant difference between the two (P<0.05). There were statistically significant pre-/post-treatment difference in the infrared radiation temperatures of the points (Zhongji, Pangguangshu, Weizhong and Shenshu) (P<0.05). The OAB symptom score (OABSS) and Zhongji infrared radiation temperature had a rank and positive correlation.Conclusion Zhongji infrared radiation temperature has important reference value for the assessment of OAB symptom severity.

Tetrandrine affects the expression of DNMT in HL-60 cells / 中国医师杂志

Objective To investigate the expression of DNA methyltransferase (DNMT) in HL-60 cells induced by tetrandrine (Tet).Methods HL-60 cells were treated with different concentrations of Tet and decitabine (DAC) alone and in combination with both.Methyl thiazolyl tetrazolium (MTT) assay was used to assess cytotoxic effect.Flow cytometry (FCM) was used to determine apoptosis rate.Real-time quantitative polymerase chain reaction (PCR) assay was used to quantify mRNA levels of DNMT.Western blot was used to quantify the expression of DNMT protein.Results Tet inhibited the growth and proliferation of HL-60 cells in a time-and dose-dependent manners (both P ＜0.01).Tet treated HL-60 cells after 48 h at the concentration of 2 μmol/L,and 4 μmol/L,the levels of DNMT gene and protein in the drug administration group decreased compared to the control group.After incubation for 48 h with Tet 2 μmol/L combined with DAC 4 μmol/L,the combination group was significantly depressed.Conclusions Tet could potently inhibit the growth and proliferation of HL-60 cells,reduce the expression levels of DNMT mRNA and protein,and have a more obvious effect in the combination group.

Bromodomain4 inhibitor JQ1 inhibits the proliferation of Ph positive acute lymphocytic leukemia cells and its mechanism / 白血病·淋巴瘤

Objective To observe the effect of bromodomain4 (brd4) inhibitor JQ1 on proliferation inhibition and apoptosis of Ph positive acute lymphocytic leukemia (Ph+ ALL) cells, and to explore the influence on the expression of brd4 and its downstream genes (myc and p53), and the reverse effect on bcr-abl.Methods Different concentrations of JQ1 were used on SUP-B15 cells.The proliferation inhibition rate was detected by MTT, the apoptosis rate was determined by flow cytometry (FCM), and the expressions of bcr-abl mRNA, brd4 mRNA, myc mRNA and p53 mRNA were detected by real-time fluorescent quantitative PCR (RT-PCR).Results Different concentrations of JQ1 inhibited SUP-B15 cells proliferation and induced cell apoptosis.The apoptosis rate was significantly increased compared with that in control group with a time-dose dependent manner.Median inhibitory concentration at 72 h was 1.0 pmol/L.At the same time, JQ1 decreased the transcription levels of bcr-abl mRNA, brd4 mRNA and myc mRNA, and increased the transcription level of p53 mRNA.Conclusions As a brd4 inhibitor, JQ1 can decrease the expression of brd4 to affect the expression of its downstream genes myc and p53, meanwhile, it can change the over expression of bcr-abl to suppress the proliferation of Ph+ ALL cells and induce apoptosis.

Triptolide affect the methylation status of HL-60cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of triptolide (TP) on the methylation status of human promyelocytic leukemia cells (HL-60) and explore a preliminary demethylation mechanism.</p><p><b>METHODS</b>Normal HL-60 cells as control group, the cell proliferation level of HL-60 cells was detected by MTT assay, being treated by different concentration TP (3.125, 6.25, 12.5, 25 nmol/L) for 24 h or 48 h respectively; Choosing the 3.125 nmol/L and the 6.25 nmol/L TP affected HL-60 cells for 48 h, the cell apoptosis rate and cell cycle were determined by flow cytometry, the expressions of death-associated protein kinase 1 (DAPK-1) and methyltransferase DNMT1, DNMT3B mRNA were measured by real time-PCR (RT-PCR), LINE-1, DAPK-1 genes'methylation variations were analyzed by methylation specific PCR (MSP).</p><p><b>RESULTS</b>Compared with control group, the different concentration TP could significantly inhibit the proliferation of HL-60 in a time-dose dependent manner (P<0.05, P<0.01). After being treated by TP for 48 h, the cell early apoptosis rate of control group and 6.250 nmol/L TP group were (2.07 ± 1.91)%, (9.77 ± 3.52)%, respectively (P<0.05); When the TP concentration increased, DAPK-1mRNA expression increased (P<0.01), DNMT1, DNMT3B mRNA expression significantly dampened (P<0.01); the promoter of LINE-1, DAPK-1 genes were hypermethylation state in the control group, after being treated by TP for 48 h, the brightness of LINE-1, DAPK-1 genes'methylation strips weakened, and the non-methylation strips enhanced all in a dose-dependent manner.</p><p><b>CONCLUSION</b>TP could down-regulate the transcriptional expression of methyltransferase DNMT1/3B genes, indirect action to reduce the degree of DAPK-1, LINE-1 genes mathylation, thus promote DAPK-1 gene expression level and inhibit the HL 60 cell growth.</p>

Study on mechanism of mitochondrial in K562/G01 cells apoptosis induced by triptolide / 白血病·淋巴瘤

Objective To investigate the possible mechanism of mitochondrial in chronic myeloid leukemia cells K562/G01 cells apoptosis induced by triptolide.Methods K562/G01 cells were treated with different concentrations of triptolide.MTT assay was used to assess cytotoxic effect.FCM was used to determine apoptosis rate,mitochondrial membrane potential and the activity of Caspase-9 of each experimental group.Real-time quantitative PCR assay was used to quantify mRNA levels of Caspase-9 and cytochrome C and Western blot assay was used to determine protein levels of cytochrome C.Results Triptolide inhibited the growth and proliferation of K562/G01 cells in a time-and dose-dependent manner (both P ＜ 0.001).Meantime,triptolide could make the mitochondria membrane potential fade away and enhance the activity of Caspase-9 (F =566.431,2 555.485,P ＜ 0.001).In addition,triptolide could dose-dependently up-regulated the transcription of Caspase-9 and cytochrome C (F =61 007.702,452 121.760,P ＜ 0.001),and the protein expression of cytochrome C,whose gray value in each experimental group was 21.54±0.59,39.63±0.58,53.29± 1.47 and 75.68±1.87 (F =5 677.928,P ＜ 0.001) respectively.Conclusion Triptolide could potently inhibit the growth and proliferation of K562/G01 cells,and the mitochondria apoptosis pathway might be one of the important apoptosis mechanisms in chronic myeloid leukemia cells induced by triptolide.

Effects of hydroxychloroquine on the apoptosis of peripheral blood mononuclear cells and Th17 in systemic lupus erythematosus / 中华风湿病学杂志

Objective To observe the effect of Hydroxychloroquine (HCQ) on the apoptosis of peripheral blood mononuclear cells (PBMCs) of Systemic lupus erythematosus (SLE) patients and on the level of Th17 cells and IL-17.Methods The peripheral blood of 20 incipient SLE patients in active stage were taken,and PBMC were separated for cell culture.Using HCQ and Chlorambucil (CLB) as an intervention,and after cultured for 24 h and 48 h,the apoptosis of PBMC and the level of Th17 were tested using Flow cytometry (FCM),the supernatants were collected to test for the level of cytokine IL-17 by ELISA.One-way ANOVA was used and SNK-q was used in the comparison between every two groups.Results There was significant difference in the apoptosis rate of mononuclear cells between the HCQ and CLB group at 24 h [HCQ:(10.3±0.7)％,CLB:(8.5±1.1)％] and48 h [HCQ:(13.9±0.6)％,CLB:(11.8±0.8)％] (P＜0.05).There was significant difference between HCQ [24 h:(0.81±0.13)％,48 h:(0.73±0.45)％] and CLB group [24 h:(0.78±0.26)％,48 h:(0.68±0.20)％] in Th17 percentage (P＜0.05).The levels of IL-17 in the supematants of the HCQ group [24 h:(26.3±0.97)％,48 h:(24.2±0).91)％] and CLB group [24 h:(24.6±0.7)％,48 h:(22.6±1.1)％] were significandy different between the two groups(P＜0.05).Conclusion HCQ has apoptosis-induction effect on PBMC,and it can decrease the number of Th17 and IL-17 level in the PBMCs.

Morphological and phenotypic analysis of monocyte-derived dendritic cells with rhCD40L in acute myeloid leukemia with complete remission and the healthy persons in vitro / 白血病·淋巴瘤

ObjectiveTo induce monocyte-derived dendritic cells(MoDC)from acute myeloid leukemia (AML) and healthy persons by rhCD40L in normal human AB serum system in vitro and to identify the morphology and phenotype of MoDC. MethodsPeripheral blood mononuclear cells(PBMNC)of AML and healthy persons were cultured in RPMI 1640 media including human AB serum, GM-CSF, rhIL-4 and rhCD40L, respectively. MoDC were identified by morphological features, surface antigen expression and the ability to stimulate T cells. ResultsAfter cultured for 7 days, MoDC displayed typical morphology with elongated dendritic process,and upregulation of the costimulatory molecules CD40,CD80,CD86 and CD83.The morphology and expression of costimulatory molecules were not significantly different between AML and healthy persons (P＞0.05),but were significantly different between rhCD40L group and without rhCD40L group (P＜0.05). MoDC had the ability to activate T cells, and there were no statistical differences between AML and healthy persons(P ＞0.05), but were significant differences between rhCD40L group and without rhCD40L group (P＜0.05). MoDC started to secrete IL-12 on day 5, and there was no statistical differences between AML and healthy persons(P＞0.05),and had differences between rhCD40L group and without rhCD40L group (P＜0.05).ConclusionMoDC can be cultured from the peripheral blood of AML and healthy persons.There were no significant differences in morphology and phenotype.Monocyted-derived DC can be used as an alternative to generate leukemia-specific cytotoxic T cells,especially in the presence of rhCD40L.

The clinical significance of immune-related marker detection in idiopathic thrombocytopenic purpura / 中华内科杂志

Objective To assess the clinical significance of detecting the immune markers in idiopathic thrombocytopenic purpura (ITP). Methods The frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombopoietin in 64 ITP patients and 31 healthy controls were measured with enzyme-linked immunospot assay (ELISPOT),modified monoclonal antibody immunobilization of platelet antigens assay (MAIPA), flow cytometry and sandwich enzyme-linked innnunosorbent assay respectively. Results Compared with the controls[1.3 ± 0. 5/105 peripheral blood mononuclear cell (PBMC), (0.33±0.06,0.41±0.03), (22.08±4.54)% and (8.19±2.46)%], the frequencies of circulating B cells secreting platelet-specific antibody (7.6±4.6/105 PBMC in acute ITP group, 5.3±3.0/105 PBMC in chronic ITP group), platelet-specific antibody (including the anti-GP Ⅱ b/Ⅲa antibody, anti-GP Ⅰ b/X antibody) (0.51 ±0.11, 0.48±0.06 in acute ITP group; 0.49±0.10,0.46±0.09 in chronic ITP group), the percentage of CD8+ T Lymphocyte (27.09±9.86 ) %, the percentage of reticulated platelet in ITP patients[the megakaryocyte cytosis group (24. 85 ± 19. 18)%, the normal megakaryocyte group (23.89±18.90)%]were significantly increased ( all P＜0.05).The frequencies of circulating B cells secreting platelet-specific antibody in acute ITP patients were notably increased (P＜0.05) compared to the chronic ITP patients. In T lymphocyte subsets, the percentage of CD3+T lymphocyte and CD4+ T lymphocyte and the ratio of CD4+/CD8+ in the patients with ITP[(60.88±14.59)%, (28.41±10.55)%, 1.18±0.59]were notably decreased than those in the healthy controls [(69.89±6.43)%, (35.38±5.05) %, 1.64±0.29, P＜0.05]. There was no apparent difference of the level of thrombopoietin between ITP patients with megakaryocyte cytosis (72. 09 ± 41.64 ) and health controls (75.37± 26. 32, P ＞ 0. 05 ), however, the level of thrombopoietin of ITP patients with normal megakaryocyte apparently increased (118.60±70.72, P＜0.05). Conclusion Detecting the frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombepoietin in the patients with ITP may improve the diagnosis and guide clinical therapy.

Experimental and clinical study on overexpression of multidrug resistance proteins in the elderly acute myeloid leukemia / 白血病·淋巴瘤

Objective To investigate the expression of P-glycoprotein(P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP) and predicts treatment outcome in the elderly acute myeloid leukemia. Methods Multi-parameter flow eytometric assay was used to quantify expression of P-go, muhidrug-associated resistance protein (MRP) and lung resistance-related protein (LRP) in bone marrow viable blasts from 12 eldely acute myeloid leukemia patients at diagnosis (M2a:4, one is come from MDS, M3a:2, M5a:5, M6a:1). Correlation of the MDR protein expression with treatment outcome were analysed. Results The frequency of expression of P-gp, MRP and LRP was 58.33 %, 8.33 %, 50 %, respectively and P-gp(+)/MRP(+) 0, P-gp(+)/LRP(+) 33.33 %, MRP(+)/LRP(+) 0, P-gp(+)/MRP(+)/LRP(+)8.33 %, P-go(-)/MRP (-)/LRP(-) 33.33 %;respectively. The frequency of overexpression of P-gp or LRP alone and both of them were relatively higher. The rate of complete remission (CR) and overall survival (OS) at one year of P-gp(+) group were significantly lower than those of P-go(-) group, and LRP(+) group also were lower than LRP(-) group. Conclusion The Frequency of expression of P-gp or LRP and coexpression of both of them were higher in elderly patients with AML. The overexpression of P-gp and/or LRP was a poor prognotic factor for eldely myeloid leukemia in elderly patients.

Treatment of malignant serosal effusion through injecting donor lymphocytes into serosal cavity / 肿瘤研究与临床

Objective To investigate the clinical efficacy and side effects of malignant serosal effusion through injecting donor lymphocytes into serosal cavity once for all.Methods First,the serosal effusion was drained thoroughly,then donor lymphocytes were injected into pleural cavity once for all.Results Nine cases of malignant serosal effusion were treated;significant effectiveness included five cases,partial effectiveness four cases.The effect rate was 100 %.The main side effects were fever and chest pain.Conclusion Treatment of malignant serosal effusion through injecting donor lymphocytes into serosal cavity once for all has definite effectiveness,with acceptable side effects.