Objective To study the relationship of accumulated dose of anthracycline(ANTH) with the cTnT level in acute leukemia patients. Methods The cTnT levels and the accumulated dose of anthracycline (ANTH) of 88 acute leukemia patients who were treated with anthracycline in our hospital from 2004-2009 who were treated with anthracycline. All the patients were divided into two groups according to a certain cTnT level,and the each incidence of elevated cTnT was obtained. Results 8 of 37 patients who received ≥200 mg/m2 of ANTH versus 1 of 51 patients who received <200 mg/m2 of ANTH had a higher incidence of elevated cTnT (P <0.05). Conclusion Incidence of elevated cTnT increases when the ANTH reaches the certain dose.

Objective To construct the miRNA‐21 eukaryotic overexpression vector pmR‐21 and to explore its regulation effect on the expression of c‐myc gene in HepG2 .2 .15 cells .Methods The miRNA‐21 precursor gene fragment pre‐miRNA‐21 was amplified by PCR ,then connected to the pmR‐mCherry plasmid vector after double enzyme digestion ,the accuracy of the recombi‐nant vector was verified by double enzyme digestion and sequencing ;then the recombinant vector was transfected into HepG2 .2 .15 cells ,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was used to detect the cell proliferation in each group .Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;miRNA‐21 expression level of the pmR‐21 recombinant vector group was significantly increased;c‐myc gene expression was increased in the pmR‐21 recombinant vector group at 72 h after transfection ,the cell proliferation in the pmR‐21 recombinant group was faster than that in the control group(P<0 .05) .Conclusion The pmR‐21 eukaryotic overexpression vector is successfully con‐structed ,this recombinant vector can express miRNA‐21 stably ;miRNA‐21 can up‐regulate c‐myc gene expression ,c‐myc gene is one of miR‐21′s targets for playing a cancer‐promoting action .

Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.

Objective To explore prokaryotic expression,purification of hemolysin coregulatory protein(Hcp)of Vibrio cholerae,and preparation of its polyclonal antibodies.Methods PCR was used to amplify Vibrio cholerae Hcp gene and clone it into pET28a vector to construct recombinant expression vector.The recombinant vector pET28a-hcp was transformed into E.coil BL21(DE3)for expression condition optimization and expression form identification.The soluble Hcp protein was purified by Ni-NTA column.The purified Hcp protein was used to immunize BALB/c mice to prepare polyclonal antibodies.The antibody titer was detected by indirect enzyme-linked immunosorbent assay(ELISA)to evaluate its immunogenicity.Western blot was used to analyze the specific recognition of antibodies to Hcp protein in Vibrio cholerae.Results The enzyme fragment digested by recombinant vector pET28a-hcp was consistent with the expected,the sequencing results were consistent with the Hcp gene sequence in the GenBank database,and the pET28a-hcp recombinant plasmid was successfully constructed.The recombinant plasmid was induced by isopropyl-β-D-thiogalactopyranoside(IPTG)to express the target protein with a relative molecular weight of 28 kD.The pure Hcp protein was obtained after purification by Ni-NTA column,and then Hcp polyclonal antibody(anti-Hcp)with a titer of 1∶512 000 could be obtained from immunized mice.Western blot results showed that anti-Hcp had specificity in recognizing Hcp protein in Vibrio cholerae.Conclusion The soluble expression of Hcp protein is successfully obtained,and high-titer polyclonal antibodies against Hcp are obtained after immunization of mice,which may lay a foundation for subsequent studies on the role of Hcp protein in the pathogenesis of T6SS in non-O1/non-O139 V.cholerae.

Objective To observe the clinical efficacy and safety of GINA regimen and GINA regimen combined with oral Huaiqihuang granule in the treatment of children with bronchial asthma.Methods A ran-domized,single blind,multicenter,parallel controlled clinical trial wascarried out.A total of 1 128 patients with bronchial asthma in children were randomized into two groups.The observation group were treated with GINA regimen combined with oral Huaiqihuang granule.The GINA regimen treatment group was treated by GINA reg-imen.Clinical assessment and C-ACT scores was observed in first month,third month,sixth month after treat-ment.Clinical assessment included the times of upper respiratory tract infection occurrence,bronchitis and pneu-monia,asthmatic attacks,application of emergency medicine,hospitalizations due to asthmatic.Drug adverse effect in the two groups was compared.Results The times of upper respiratory tract infection,bronchitis and pneumonia,asthmatic was significantly decreased(P <0.05 ),and C-ACTscores were significantly higher(P <0.05)in the first month,the third month,and the sixth month after treatment.There were 16 cases of drug relat-ed adverse reactions.Seven cases were in observation group(n ﹦7)(1.15%)and 9 cases in the GINA regimen treatment group(n ﹦9)(1.73%).There was no significant difference of adverse effect between the two groups (P >0.05).Conclusion The treatment of bronchial asthma in children with GINA regimen combined with oral Huaiqihuang granule can significantly reduce the incidence of respiratory infections and the number of asthmatic attacks dramatically and safely improve clinical curative effect,asthma control.

Objective: To construct the mmu-miR-155 eukaryotic overexpression vector pmR-155 and to investigate its effect on HBV replication and expression of PTEN in vivo. Methods: The mmu-mir-146a precursor gene fragment pre-mmu-mir-146a was amplified by PCR, then connected to the pmR-mCherry plasmid vector after double enzyme digestion, the accuracy of recombinant vector was verified by colony PCR、double enzyme digestion and sequencing; then the recombinant vector was transfected HBV transgene mice(Experimental Group)with hydrodynamics-based injection via vena caudalis, and pmR-mCherry plasmid、PBS were respectively transfected into the mice as Empty plasmid Group、Blank Group. The concentration of IFN-γ in the serum was detected by ELISA. The expression of SOCS1、PTEN mRNA in the liver was detected by qPCR at 30d post-transfectioned. The Western blot was performed to detect the changes in SOCS1、PTEN、HBX in the liver tissue at 30 d post-transfectioned. The results were analyzed with Student’s t-test, or one-way analysis of variance and the least significant difference test. Results: the colony PCR、double enzyme digestion and sequencing verified that the gene was inserted into the pmR-mCherry vector. Compared with Blank Group, the expression of miR-155 in the Experimental Group was significantly increased(t = 8.90, P < 0.01); the concentration of IFN-γ in the Experimental Group was significantly increased(F = 26.58, P < 0.01); the mRNA(F_{SOCS1 mRNA} = 19.72, P < 0.01; F_{PTEN mRNA} = 7.38, P < 0.05) and protein(F_SOCS1 = 50.30, P < 0.01; F_PTEN = 129.00, P < 0.01) expression of COCS1、PTEN was significantly decreased in the Experimental group and the protein of HBX was also significantly(F_HBX = 77.97, P < 0.01). Conclusion The pmR-155 eukaryotic overexpression vector is successfully constructed, this recombinant vector can express miR-155 stably; miR-155 can down-regulate cocs1、PTEN gene expression and up-regulate the expression of IFN-γ, it can inhibit the replication of HBV and a potential targets to treating hepatocellular carcinoma.