The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-?-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the ?-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.

Objective To study lobular involution of tissues around breast tumor,and to assess the consistency of lobular involution of tissues from different parts of the breast.Methods 22 patients receiving breast conservation surgery in Breast Surgery Department of Peking Union Medical College Hospital from Dec.1 st,2010to Dec.1 st,2011 were collected.88 pieces of HE staining were measured in terms of lobular area and number of acini per lobular,and lobular involution was evaluated.Bivatiate correlation analysis was applied to explore correlation between lobular area and acini per lobular.ANOVA,crosstabs and reliability analysis were applied to explore involution consistency of different parts of breast (P ＜ 0.05).Results The average area of lobuli was (90 248.5 +56 909.4) μm2 and the number of acini was 25.68 ± 18.86 per lobular.The lobular area and number of acini were correlated with each other significantly(Pearson r =0.78,P ＜ 0.01).Involution status of different parts of breast had good consistency(for lobular area 19 cases showed no difference in ANOVA analysis and for lobular area/number of acini 18 cases showed no difference; Kappa coefficient =0.65; ICC =0.73).Conelusions Lobuli around breast tumor have comparatively poor involution,with big lobuli and large number of acini,which are correlated with each other.Involution status of different parts of a breast has good consistency.Biopsy from one site to evaluate involution extent of the whole breast is practicable.

Objective: To prepare and in vitro evaluate the self-microemulsifying drug delivery system (SMEDDS) of adefovir dipivoxil (ADV).Methods: The optimized formula was screened by solubility, compatibility, ternary phase diagram and orthogonal design with the self-emulsifying time and particle size of microemulsion as the indices.The property of self-emulsification and the dissolution in vitro of ADV-SMEDDS were also determined.Results: The optimized SMEDDS was composed of Cremophor EL35 (37.5%), Transcutol HP (37.5%) and PECEOL (25%), and the drug loading was 3%.The ADV-SMEDDS formed stable microemulsion after the dilution by 50-fold amount of water in 24 s, the average particle size was (26.30±0.46) nm, the zeta potential was (-8.96±0.57) mV, and the dissolution was more than 85% in 5 min.Conclusion: The optimized formula of ADV-SMEDDS has significantly enhanced solubility and dissolution of adefovir dipivoxil in vitro.

Objective To explore the immunogenicity of recombinant chimeric 6Aβ15-T including the Aβ1-15 epitope and a T-helper epitope formulated with different adjuvants and to evaluate its feasibility as a candidate vaccine for Alzheimer disease (AD).Methods The recombinant chimeric antigen 6Aβ15-T formulated with Al adjuvant, Freund′s adjuvant or MF59 adjuvant was administered to two strains of mice .The 6Aβ15-T-immunized group without adjuvants ( Mock) and non-immunized group (Control) were included in this study as control groups .The specific antibody and cellular immune response of the chimeric antigen were evaluated .Results In BALB/c strain mice, three types of adjuvants could substan-tially boost the immunogenicity of chimeric antigen 6Aβ15-T and produce a high level of specific-Aβ(β-amyloid) antibod-ies.In C57BL/6 strain mice, the existence of adjuvants enhanced the immune response of 6Aβ15-T antigen, but the mice in Mock group also produced a strong antibody response .In two strains of mice, prevalence of anti-AβIgG1, which was an indicator of Th2 polarization, was observed in the 6Aβ15-T-immunized mice.Additionally, the Al adjuvant induced a high-er level of IgG1 antibody titers, and the ratio of IgG1/IgG2a was the largest.As expected, the 6Aβ15-T antigen formulated with or without adjuvants induced PADRE-specific, but not Aβ42-specific T cellular immune response .Conclusion The 6Aβ15-T antigens formulated with different types of adjuvants could induce strong Th 2-polarized Aβ42-specific antibody re-sponses without activating self-reactive Aβ42-specific T cells in two strains of mice .The results suggested that the recombi-nant chimeric antigen 6Aβ15-T is a good candidate vaccine for AD .

Objective To evaluate the value of real-time three-dimensional echocardiography(RT-3DE)in diagnosing congenital tricuspid valve anomaly.Methods Eighteen patients with congenital tricuspid valve anomaly were studied by RT-3DE,the spatial framework and neighboring structures of the tricuspid valve were analyzed and compared with result of the operation.Results The anomaly of tricuspid,chorda tendineae,the papillary muscle and their connection with neighboring structures could be displayed clearly from different directions.The diagnose accordance rate of RT-3DE was 83%.Conclusions RT-3DE may provide more information on congenital tricuspid valve anomaly than 2DE.

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.

The policy implementation model of G. C. Edwards was used to analyze the public policy health impact assessment in Zhejiang province, and summarize its practice and existing problems in four aspects of policy implementation standards, policy resources, policy executors′ intention and management organization structure, so as to provide reference for promoting the national health impact assessment pilot work. The analysis results showed that Zhejiang province has initially established the public policy health impact assessment mechanism and achieved phased results, but there were still some problems, including the imperfection of policy content and implementation strategy, the inadequacy of leadership decision-making and top-level design, the difference in attitude, understanding and implementation preference of policy implementation subjects, and the ambiguity of the authority and responsibility system of each department in cooperation. In order to further promote the smooth development of public policy health impact assessment, Zhejiang province should actively promote the top-level design to strengthen policy support, integrate and optimize policy resources, gradually establish and improve the health governance mechanism of multiple and overall coordination, and promote the high-quality development of public policy health impact assessment by taking cross departmental cooperation as the path of health co-construction.