ObjectiveTo observe the effect of Yangxin Tongmai Formula （养心通脉方） by midnight-noon ebb-flow administration method for rat models of premature coronary heart disease （PCHD） with blood stasis syndrome， and to explore the possible mechanism of action from the perspective of DNA methylation differential gene expression. MethodsThere were 3 SD rats in each of the blank group， model group and Yangxin Tongmai Formula group， and the rats in the model group and Yangxin Tongmai Formula group were fed with high-fat chow plus vitamin D3 by gavage plus isoproterenol hydrochloride by subcutaneous injection to construct rat models of PCHD with blood stasis syndrome. After successful modelling， rats in Yangxin Tongmai Formula group were gavaged with 18 g/（kg‧d） of Yangxin Tongmai Formula， and rats in blank group and the model group were gavaged with 4 ml/（kg‧d） of 0.9% NaCl solution， and serum samples of rats in each group were collected for DNA methylation sequencing after 3 weeks to screen for the relevant DNA methylation differentiation genes. In addition， rats with successful modelling of PCHD with blood stasis were randomly divided into model group， Yangxin Tongmai Formula with midnight-noon ebb-flow administration method group ［18 g/（kg‧d） of Yangxin Tongmai Formula was gavaged twice in the heart channel period （12：00） and pericardium channel period （20：00）］， the Yangxin Tongmai Formula control group ［18 g/（kg‧d） of Yangxin Tongmai Formula was gavaged twice at 8：00 and 18：00］ and the Atorvastatin Calcium group ［atorvastatin calcium tablets solution 1.8 mg/（kg‧d） at the same intervention time as that in Yangxin Tongmai Formula control group］， and set up a blank group of 8 rats in each group. The model group and blank group were gavaged with 0.9% NaCl solution 4 ml/（kg‧d） for the same time as the Yangxin Tongmai Formula control group. After 3 weeks of gavage， the blood lipids ［including total cholesterol （TC）， low-density lipoprotein （LDL）， high-density lipoprotein （HDL）］ levels of rats in each group were detected； the HE staining of myocardial tissues and thoracic aorta was used to observe the pathomorphological changes； the levels of serum inflammation indexes ［tumour necrosis factor alpha （TNF-alpha）， lipopolysaccharide （LPS）， and interleukin 10 （IL-10）］ were detected； immunoprecipitation-realtime fluorescence quantitative PCR was used to detect the relative expression of cardiac tissue screening differential genes. ResultsThe genes screened for differentially methylated regions were calmodulin 2 （Calm2）， calcium voltage-gated channel subunit α1s （Cacna1s）， and phospholipase Cβ1 （Plcb1）. Compared with the blank group， rats in the model group showed elevated levels of TC， LDL， TNF-α and LPS， and decreased levels of HDL and IL-10 （P＜0.05 or P＜0.01）； HE staining showed obvious swelling of myocardial fibres， accompanied by a large number of inflammatory cell infiltration， and thickening of the inner wall of the aortic vessels with internal wall damage， which was visible as a large number of lipid cholesterol crystals and obvious inflammatory cell infiltration. Compared with the model group， the TC， LDL， TNF-α and LPS contents of rats in the Yangxin Tongmai Formula with midnight-noon ebb-flow administration method group， the Yangxin Tongmai Formula control group， and the atorvastatin calcium group all reduced， and the contents of HDL and IL-10 all elevated （P＜0.05）， with the improvement of myocardial tissue damage and the reduction of inflammatory infiltration， and the improvement of the damage of the inner lining of the thoracic aorta and the reduction of lipid infiltration. Compared with Yangxin Tongmai Formula control group， LDL， TNF-α and LPS contents reduced， and IL-10 contents increased in the midnight-noon ebb-flow administration method group （P＜0.05）. Compared with the model group， the relative expression of Calm2 and Plcb1 genes decreased and the relative expression of Cacna1s gene increased in Yangxin Tongmai Formula control group and the midnight-noon ebb-flow administration method group （P＜0.05）； compared with the Yangxin Tongmai Formula control group， the relative expression of Calm2 gene decreased and the relative expression of Cacna1s gene increased in the midnight-noon ebb-flow administration method group （P＜0.05）. ConclusionThe intervention of Yangxin Tongmai Formula in the heart channel period （12：00） and pericardium channel period （20：00） was more effective in improving the blood lipid level， inhibiting inflammation， and improving myocardial tissue damage in rats of PCHD with blood stasis syndrome， and Calm2 and Cacna1s genes may be the key targets of Yangxin Tongmai Formula in intervening the blood stasis syndrome of PCHD.

Objective:To investigate the clinical and biological characteristics of relapsed childhood low-risk acute B lymphoblastic leukemia (B-ALL).Methods:The clinical and laboratory data of 34 children who admitted in Beijing Boren Hospital from July 2017 to July 2018 were retrospectively analyzed, and 127-339 mutations of hematological malignancy related genes were analyzed.Results:The median time from the diagnosis to the recurrence was 871 d (87-1 446 d). The recurrence at early stage and late stage had 26 cases (76%) and 8 cases (24%), respectively. The recurrence before maintenance treatment, during maintenance therapy and after withdrawal of chemotherapy had 3 cases (9%), 12 cases (35%) and 19 cases (56%) (13 cases relapsed within 1 year after withdrawal, 6 cases relapsed after withdrawal 1-2 years and no one relapsed after withdrawal 2 years). The sites of recurrence included bone marrow alone accounting for 26 cases (76%), both intramedullary and extramedullary disease (EMD) accounting for 6 cases (18%), EMD alone accounting for 2 cases (6%). Flow cytometry showed that 9 patients presented minimal residual disease (MRD)-positive (6 cases with one positive, 2 cases with twice positive and 1 case with 3 times positive), including 8 cases occurred at early stage and 1 case occurred at late stage; and the level of MRD was 0.02%-3.82%. Complex chromosomal karyotype appeared in 6 relapsed children with normal or hyperdiploid karyotype at first diagnosis. Hematological malignancy related gene mutation detection was made in 28 cases, and the results showed that each patient had at least one gene mutation, and 2 or more gene mutations were detected in 25 cases (89%). The high frequency of gene mutations were as follows: CREBBP (7 cases, 25%), NRAS (7 cases, 25%), KRAS(7 cases, 25%), TP53 (4 cases, 14%), and NT5C2 (4 cases, 14%).Conclusions:The recurrence of childhood low-risk B-ALL occurs mostly in the maintenance treatment or in two years of withdrawal of chemotherapy. Positive MRD after complete remission is likely to show the risk of early recurrence. The gene mutations after the poor prognosis in cancer cells may be related to the recurrence of childhood low-risk B-ALL, and the common gene mutations include CREBBP, RAS signaling pathways genes and TP53, NT5C2.

Castleman Disease ; virology ; Epstein-Barr Virus Infections ; complications ; Herpesvirus 4, Human ; Humans

Objective To investigate the association of genetic polymorphisms of ATIC and GSTP1 with plasma concentrations and adverse reactions of high-dose methotrexate( HD-MTX)in children with acute lymphoblastic leukemia (ALL). Methods A total of 70 peripheral blood samples were obtained from ALL children for extraction of genome DNA.The gene polymorphisms of ATIC T26293C and GSTP1 A313G locus were examined by using PCR and direct sequencing.Enzyme multiplied immunoassay technique(EMIT)was employed to determine the plasma concentration of MTX in 48 h.Clinical data of patients were collected during HD-MTX chemotherapy,and the adverse reactions were statistically analyzed.The associations of ATIC and GSTP1 genotypes with MTX plasma concentration and adverse reactions were investigated. Results There were genetic polymorphisms at the SNP of ATIC T26293C and GSTP1 A313G.At the SNP of ATIC T26293C,the percentages of TT, CT and CC genotypes in ALL children were 4.35%,39.13% and 56.52%,respectively,and the frequencies of T and C alleles were 23.91% and 76.09%.At the SNP of GSTP1 A313G,the percentages of AA,GA and GG genotype were 68.57%,28.57%and 2.86%,respectively,in ALL children. The frequencies of A and G alleles were 82. 86% and 17. 14%,respectively. No statistically significant difference was found in the ratio of blood MTX concentration to MTX dose at 48 h between children with different genotypes(P>0.05).In the GSTP1 A313G site,genotypes that induced the gastrointestinal reactions in the order from low to high were AA,GA,GG,and there was a significant association between gene polymorphism and gastrointestinal side effects(P<0.05).In the GSTP1 A313G site,genotypes that induced myelosuppression in the order of low to high were GG,AA, GA,and a significant association was noted between gene polymorphism and myelosuppression(P<0.05). Conclusion There are significant associations between GSTP1 A313G polymorphism and gastrointestinal side effects or myelosuppression after HD-MTX chemotherapy in ALL children.

Objective To explore the effect of serpine1, α-serpine1 protein and KLF2 in patients with lower extremity venous thrombosis by Fufang Xueshuantong capsule.Methods 44 patients were diagnosed with lower extremity venous thrombosis were collected, and randomly divided into experimental group and control group, 22 cases in each group, giving the conventional antiplatelet therapy, on the basis of this, the experimental group was given Fufang Xueshuantong capsule 1.5 g, three times a day orally, and the control group was given context Shutong granules 20 g, three times a day orally.2 weeks for a course of treatment, three courses of treatment.Before and after treatment in the detection of two groups of patients with serum serpine1, α-serpine1 KLF2 and protein contents, blood rheology, coagulation function, and carries on the analysis.ResuIts Compared with the control group, experimental group patients has better curative effect, the performance of: serum serpine1and α-serpine1 protein content were significantly decreased(P<0.05).KLF2 expression was significantly higher (P<0.05).Blood viscosity, erythrocyte aggregation index, erythrocyte sedimentation rate decreased significantly (P<0.05).Fib, D-D content significantly decreased (P<0.05).ConcIusions Fufang Xueshuantong capsule can reduce serum serpine1, α-serpine1 protein content and promote KLF2 expression, decrease the blood viscosity, erythrocyte sedimentation rate, improve patients with hypercoagulable state in patients with lower extremity venous thrombosis, so as to achieve good clinical efficacy.

Objective To explore the role of murine double minute 2 (MDM2) in apoptosis of acute lymphoblastic leukemia EU-4 cells induced by berberine.Methods EU-4 cells at logarithm growth phase were chosen to carry out the experiments.EU-4 cells were incubated with different doses of berberine for 72 hours,and the final doses of berberine were 0,1,10 and 100 μmoL/L,respectively.Then the apoptosis rate of EU-4 cells were detected by flow cytometry,and the expression levels of MDM2 protein were tested by Western blot.The levels of MDM2 gene were examined by means of real-time PCR at 0,24,48 and 72 hours after incubation of EU-4 cells with 100 μmol/L berberine,respectively.EU-4 cells in MDM2 small interfering RNA(siRNA) group were transfected with 150 nmol/L MDM2 siRNA,and the control siRNA group were transfected with 150 nmol/L control siRNA.The apoptosis rates of EU-4 cells were detected 48 hours after transfection by flow cytometry.Results The apoptosis rates of EU-4 cells were increased in a dosedependent manner,and the highest apoptosis rate was up to(60.13 ± 4.21)％,which was incubated with 100 μmol/L berberine.Significant differences were found in all groups (F =280.56,P ＜ 0.05).Berberine treatment suppressed the protein expression of MDM2 in a dose-dependent manner(F =73.82,P ＜ 0.01).The mRNA expression of MDM2 was inhibited in a time-dependent manner by 100 μmol/L berberine(F =45.37,P ＜0.01).The apoptosis rates of control siRNA group and MDM2 siRNA group were(11.09 ± 1.63) ％ and (29.84 ± 1.75) ％,respectively.Significant difference was found between the 2 groups (t =-13.57,P ＜ 0.01).Conclusions Berberine can inhibit the expression of MDM2 in a dose-and time-dependent manner,which may be one of the mechanisms of apoptosis induced by berberine in leukemia cells.

Objective To analyze the clinical features of pediatric patients with acute lymphoblastic leukemia(ALL) with bcr-abl fusion gene transcript,and discuss the treatment,prognosis factors of this kind of ALL.Methods Clinical features,treatment and prognosis were studied retrospectively in 7 bcr-abl fusion gene positive ALL patients.bcr-abl fusion gene was detected by reverse transcription polymerase chain reaction (RT-PCR).Results The average age of the 7 patients was 8 years and 1 month old.All of them were common B-immunology ALL.The rate of complete response was 50 ％ after 33 days' treatment.Conclusions The incidence rate of bcr-abl fusion gene positive ALL in pediatric is low.This type of ALL has poor remission,high relapse rate and poor prognosis.

Objective To study the molecular mechanism of berberine induced apoptosis in chemoresistant EU-4 acute lymphocytic leukemia cells.Methods EU-4 cells were treated with 0,10 and 100 μmol/L berberine for 72 h.The apoptosis induced by berberine was detected by flow cytometry.The expression of Caspase-3,PARP and X-linked inhibitor of apoptosis protein (XIAP) were determined by Western bolt assay.Caspase-3 activity was measured using microplate reader.After transfected with XIAP siRNA,the apoptosis was detected by flow cytometry.Results After treated with 0,10 and 100 μmol/L berberine for 72 h,the apoptosis rates of EU-4 cells were (9.08±1.20) ％,(22.36±2.16) ％ and (59.81±4.17) ％,respectively.Berberine induced potent apoptosis in a dose-dependent manner.The apoptosis involved activation of Caspase-3.The Caspase-3 activities were 1.70±0.25,1.92±0.10 and 2.89±0.25,respectively.Berberine inhibited XIAP expression in a dose-and time-dependent manner (P ＜ 0.05).Down-regulation of XIAP by siRNA increased apoptosis of EU-4 cells.The apoptosis rates were (9.23±1.66) ％ and (22.15±0.63) ％.Conclusion Berberine could induce apoptosis of EU-4 cells,and inhibition of XIAP leading to Caspase-3 activation is responsible for the apoptotic effect on EU-4 cells.

The expression of silence of death domains (SODD) and its clinical significance and relationship with phospho-NF-kappaB-p65 proteins in bone marrow cells of childhood acute lymphoblastic leukaemia (ALL) were explored, and the expression of SODD and phospho-NF-kappaB-p65 in Jurkat cells treated with chemotherapeutic drugs was detected in order to find a new chemotherapeutic target. The expression of SODD and phospho-NF-kappaB-p65 proteins in bone marrow cells was detected by immunohistochemistry in 25 children with ALL. The apoptosis rate was measured by Annexin-V-Fluorescence/PI double-labeling flow cytometry and the expression of SODD and phospho-NF-kappaB-p65 proteins determined by Western blotting in the Jurkat cells. It was found that the expression of SODD and active P65 in ALL was significantly higher than that in normal control group (P<0.05). The expression of the SODD and phospho-NF-kappaB-p65 proteins in the high-risk (HR) group was significantly higher than that in the standard-risk (SR) group (P<0.05). The Pearson rank correlation analysis revealed that there was a positive correlation between SODD and phospho-NF-kappaB-p65 expression (P<0.01, r=0.69). VCR could effectively induce the apoptosis of Jurkat cells, and down-regulate the expression of SODD and phospho-NF-kappaB-p65 proteins in a time-dependent manner, but DNR could not down-regulate the expression of SODD effectively. It was concluded that SODD may be closely related to the clinical classification and prognosis of ALL in children. The expression of SODD and phospho-NF-kappaB-p65 had a definite synergistic relationship with the onset and development of ALL. VCR could down-regulate the expression of SODD and inhibit the NF-kappaB activation, which could recover the sensibility of apoptosis in leukemic cells.

The expression of silience of death domains (SODD) and its clinical significance and relationship with phospho-NF-κB-p65 proteins in bone marrow cells of childhood acute lymphoblastic leukaemia (ALL) were explored, and the expression of SODD and phospho-NF-κB-p65 in Jurkat cells treated with chemotherapeutic drugs was detected in order to find a new chemotherapeutic target. The expression of SODD and phospho-NF-κB-p65 proteins in bone marrow cells was detected by immunohistochemistry in 25 children with ALL. The apoptosis rate was measured by Annexin-V-Fluorescence/PI double-labeling flow cytometry and the expression of SODD and phospho-NF-κB-p65 proteins determined by Western blotting in the Jurkat cells. It was found that the expression of SODD and active P65 in ALL was significantly higher than that in normal control group (P＜0.05). The expression of the SODD and phospho-NF-κB-p65 proteins in the high-risk (HR) group was significantly higher than that in the standard-risk (SR) group (P＜0.05). The Pearson rank correlation analysis revealed that there was a positive correlation between SODD and phospho-NF-κB-p65 expression (P＜0.01, r=0.69). VCR could effectively induce the apoptosis of Jurkat cells, and down-regulate the expression of SODD and phospho-NF-κB-p65 proteins in a time-dependent manner, but DNR could not down-regulate the expression of SODD effectively. It was concluded that SODD may be closely related to the clinical classification and prognosis of ALL in children. The expression of SODD and phospho-NF-κB-p65 had a definite synergistic relationship with the onset and development of ALL. VCR could down-regulate the expression of SODD and inhibit the NF-κB activation, which could recover the sensibility of apoptosis in leukemic cells.