Objective To study the prokaryotic expression of fusion gene A?-HBcAg and analyze the immunoreactivity and immunogenicity of expression protein. Methods Recombinant plasmid pBV220/A?-HBcAg was transformed into E.coli DH5?, and expressed by temperature inducing. The bacteria were split by ultrasonic wave. The expression of the fusion protein was studied by SDS-PAGE and Coomassie brilliant blue staining. The immunoreactivity of the fusion protein was determined using ELISA. After immunized intraperitoneally with the fusion protein, 5 Balb/c mice's sera titers of anti- A? and anti-HBc were evaluated by ELISA. Results Fusion protein was in sediment of the split bacteria as inclusion bodies and its expression level was 5% of the total sediment protein. The fusion protein had both immunoreactivity of A? and HBcAg. The titers of anti-A? and anti-HBc were very low after 3 times of immunization. After immunization for 5 times, the titers reached 1∶800 and 1∶3 200 for anti-A? and anti-HBc, respectively. Conclusion Recombinant gene A?-HBcAg can be expressed in E.coli DH5? and the expression protein has certain immunoreactivity and immunogenicity. It indicates that further work should be done to enhance the expression level of fusion gene A?-HBcAg and improve the immunogenicity of the fusion protein.

Objective To prepare a hybridoma secreting stab le monoclonal antibodies against ?-amyloid peptide (A? 1-42) with high titer. Methods By genetic engineering technology, A ? gene was recombined with the MIR of HBcAg to get the A? and HBcAg fusi on protein. Spleen cells from BALB/c mice immunized with A? and HBcAg f usion protein were fused with mouse myeloma cells SP2/0. Results Two strains of hybridomas (1H 7 and 1F 3) secreting stable monoclonal antibodies raised against A? 1-42 were ob tained. The subtypes of A? 1-42 antibodies were IgG 3. C onclusion The A? 1-42 monoclonal antibodies obtained have high titers and specificity.

The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Abeta(25-35) and serued as the experimental Abeta-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against A-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAv-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template, double stranded cDNA of HNG with FLAG in its C-terminal was obtained, which was cloned into the plasmid pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time, the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG, 25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h, then cell morphology, MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h, cell viability decreased to (65.8±5.3)%, and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG, Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast, pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.

The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Aβ25-35 and serued as the experimental Aβ-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against Aβ-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAV-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Objective: To evaluate the anthracyclines-induced cardiotoxicity in patients with early-stage breast cancer. Methods: This retrospective study analyzed data of 64 patients (aged from 36 to 59 years old) with early-stage breast cancer after surgery. Patients were divided into ACT group (n=21), FAC group (n=19) and EC group (n=24). The NCI CTC 4.0 scores was used to evaluate the side effects at the time of 2 weeks, 4 weeks and 6 weeks after chemotherapy. Meanwhile, the level of cTnT, the incidence of abnormal electrocardiogram (ECG) and left ventricular ejection fraction (LVEF) were used to evaluate the anthracyclines-induced cardiotoxicity, the follow-up observation points were as follows: at the acute cardiotoxicity (time A), subacute cardiotoxicity (time B), 24 months after chemotherapy (time C), 36 months after chemotherapy (time D), 48 months after chemotherapy (time E), 60 months after chemotherapy (time F). The 3-years and 5-years overall survival and progress free disease survival among three groups were compared. Results: The ages, clinical stage, the size of tumor, axillary lymph node positivity and Eastern Cooperative Oncology Group Scores were similar among three groups (P>0.05); the incidence of side effects level 4 was 0. The levels of cTnT in the three groups were significantly lower than those at the baseline and time points C, D, E and F (all P<0.05), and the levels of cTnT were significantly higher in EC group than in FAC and ACT group at the time points B, C, D, E and F (P<0.05); however, the incidence of abnormal ECG and LVEF was similar among the 3 groups (P>0.05). The 5-year overall survival was 95.2% (20/21) ,100% (19/19) and 95.8% (23/24) in ACT group, FAC group and EC group, respectively; 5-year progress free disease survival was 95.2% (20/21) ,94.7% (18/19) and 91.7% (22/24) in ACT group, FAC group and EC group, respectively (P>0.05) . Conclusions Patients with early-stage breast cancer after surgery could tolerate the anthracyclines-induced cardiotoxicity. Three chemotherapy schemes of ACT, FAC and EC, especially the EC protocol, could affect the myocardial damage. However, outcome is comparable among patients treated with above chemotherapy schemes in this patient cohort.

To explore the association between sarcopenia (SAR) and long-term prognosis of patients with chronic heart failure (CHF) aged 70 years and over,182 CHF patients from January l,2012 to December 31,2014 were included in the present study.The patients were divided into the SAR group and the non-SAR group.The median follow-up period was 36 (3,57) months.The endpoint was any heart failure-related event (HFRE).There were significant differences in age,body mass index,hemoglobin,B-type natriuretic peptide,hypersensitive troponin T (hs-TnT),left ventricular ejection fraction (LVEF) and cardiac function class between the two groups (all P＜0.05).The Kaplan-Meier analysis showed that the survival time of the non-SAR patients was much longer than that of the SAR patients (P＜0.05).The multivariate Cox regression analysis indicated that SAR was an independent risk factor for HFRE suggesting a role of sarcopenia on long-term prognosis of patients with chronic heart failure.