ABSTRACT
Objective·To compare the differences in the composition of female vaginal flora by real-time quantitative PCR and 16S rDNA sequencing.Methods·Forty-nine healthy reproductive women less than 45 years old were selected.Specimens were collected from posterior fornix.DNA were extracted and the microbiome were ananlyzed by both 16S rDNA V1V2 region sequencing and qPCR of 22 selected genes.The results detected by two methods were compared.Results·According to the classification standard of vaginal community state type (CSTs),qPCR analysis showed that 35 out of 49 samples were dominated by Lactobacillus species,among them,type Ⅰ (Lactobacillus crispatus,9),type Ⅲ (Lactobacillus iners,24),type Ⅳ (no Lactobacillus as the dominant bacteria,12),type Ⅴ (Lactobacillusjensenii,2).16S rDNA V1V2 region sequence analysis showed that of the 49 samples,13 belonged to type Ⅰ,type Ⅱ (1),type Ⅲ (23),type Ⅳ (8),type Ⅴ (2).Two methods of vaginal flora classification were consistent for 38 cases,consistent rate was 77.6％.Conclusion·Two methods analysis of vaginal flora showed the different results.If qPCR was used to classify the vaginal microbiome,it was necessary to consider the influence of relevant technical factors on the results.
ABSTRACT
MTT assay was used in this study to investigate the inhibitory effect of danshensu on the activity of 2.2.15 cells among human hepatoma cell line (HepG2); indirect fluorescence labeling method was used to measure the changes of reactive oxygen levels in the cells; ELISA method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in cellular supernatants; HBV DNA level was measured with fluorogenic quantitative PCR method. The inhibitory effect of danshensu on HBV RT(hepatitis B virus reverse transcriptase) was studied by using enzyme inhibition dynamics, and the effect of danshensu on secondary structure of HBV reverse transcriptase was monitored by using circular dichroism. The results showed that danshensu had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC₅₀) of (15.35±2.43) μmol•L⁻¹; danshensu could significantly inhibit HBsAg and HBeAg expressions, and showed an inhibitory effect on HBV DNA replication. In addition, danshensu was an effective inhibitor for HBV reverse transcriptase [IC₅₀ (21.32±2.43) μmol•L⁻¹]. The fluorescence labeling results showed that the reactive oxygen levels in the cells were increased with the increase of danshensu concentration. Circular dichroism analysis showed that danshensu could induce partial change of conformation of HBV reverse transcriptase and gradually increased α-helical content. These results indicated that danshensu could make the structure of the enzyme become closer by binding to HBV reverse transcriptase, which was not conducive to the formation of the active center, so it could finally decrease the activity of HBV reverse transcriptase. Such decrease in enzyme activity would directly affect the HBV DNA replication, and combined with the decrease of the antigen levels, the effect of danshensu on HBV was increased.