Gut microbiota at different stages of HBV infection / 实用医学杂志

Objective To investigate the correlation between gut microbiota and hepatitis B virus(HBV) infection at different stage. Methods 84 patients aged 25 ~ 40 were enrolled in the study,the ALT,AST and HBV antigens and antibodies qualitatively detected. Then they were divided into four groups based on HBV related antigens and antibodies qualitative detection according to guideline for chronic HBV infection:48 in the protective antibody positive group where vaccine injection was performed for producing protective antibodies ,14 in the HBV recovery group 9 in chronic HBV infection group And 13 in antibody negative group where vaccine injection was done. Their fecal samples were collected and total DNA were extracted and subjected to enterobacterial repetitive intergenic consensus sequence polymerase chain reaction(ERIC-PCR). After ERIC-PCR,gel electrophoresis and genetool software were used to analyse the differences in the amount of bands and the position of main bands between groups. Results The amounts of bands in the antibody negative and chronic HBV infected groups were significantly smaller than the protective antibody group and HBV recovery group. The position of main band in the protective antibody group and HBV recovery group were wildly spread within groups ,and most main bands in the HBV recovery group were above 282 bp. The main bands in the antibody negative and chronic HBV infected groups were centralized in 60 ~ 70 bp. Conclusions Compared with the protective antibody group and HBV recovery group ,the richness of gut microbiota in the antibody negative and chronic HBV infected groups are significantly decreased and lack of microbiota above 200 bp in ERIC-PCR.

Rapid selection of recombinant orf virus expression vectors using green fluorescent protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein (GFP) as the reporter gene.</p><p><b>METHODS</b>The flanking regions of the ORFV132 of orf virus DNA were amplified by PCR to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The shuttle plasmid was transfected into OFTu cells and GFP was incorporated into orf virus IA82Delta 121 by homologous recombination. The recombinant IA82Delta121-V was selected by green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro.</p><p><b>RESULTS</b>The recombinant orf virus IA82Delta121-V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the replication of the virus in vitro but reduced its virulence.</p><p><b>CONCLUSION</b>Green fluorescent protein is a selectable marker for rapid, convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82Delta121-V can serve as a new expression vector for exogenous genes.</p>

A comparative study of tumor-associated macrophages isolation in colorectal carcinoma by Ficoll-Hy-paque density gradient centrifugation and Percoll density gradient centrifugation / 实用医学杂志

Objective Percoll density gradient centrifugation and Ficoll-Hypaque density gradient cen-trifugation, which are frequently-used methods for separation of tumor-associated macrophages (TAMs) from solid carcinoma were compared, in order to find an effective way to separate TAMs from colorectal carcinoma (CRC). Furthermore, we studied the best adherence time of separating macrophage among mononuclear cells. Methods specimens were collected from CRC patients , after digesting into single cells , TAMs were separated from the same specimen by 100% Ficoll, 35% percoll and 25% combined with 65% percoll respectively. After these pre-liminary separation, the collected cells were purified a second time by adherence separation. The purity of TAMs were detected by immunofluorescence. Results TAMs purity from Ficoll-Hypaque density gradient centrifugation was 80.18%, statistically higher than that from Percoll density gradient centrifugations (54.33% and 10.93% re-spectively). Conclusion Compared to Percoll density gradient centrifugation, Ficoll-Hypaque density gradient centrifugation is a more effective and simple way to isolate TAMs from colorectal carcinoma , suggesting it can be wildly used in clinical and basic medical research. 2-4 hours is the best adherence time for isolating macrophage.