| Oxymatrine (OM), a kind of alkaloid isolated from Sophia Flavecens Alt (SFA), has been proven good and prospective for the treatment of patients with chronic hepatitis B by lots of clinical and laboratoiy researches. But until now its anti-viral mechanism is uncettain. Furthermore, how to evaluate the efficiency of an and-viral drug and the reaction for patients receiving anti-viral therapy are questions to be answered. Besides liver biopsy, the quantitative detection of HBV DNA may be the most significant; direct; credible and valuable method. Objective In this study, we established a method for HBV DNA quantification: ecp-alent competitive PCR combining with DNA hybridization quantitative detection technique (PCR-ELISA). Utilizing this method, we investigated the anti-viral mechanism and therapeutic effects of OM. Methods (1) Establishing equivalent competitive PCR combining with DNA hybridization quantitative detection technique (PCR-ELISA): Mutated fragment was prepared by PCR, in which a pair of primers was synthesized to amplify the mutated fragment from wild HBV DNA template. The fragment was coloned to a plasmid vector, which worked as Internal Standard (IS). The IS was added into every standard and sample tube at known number copies and was carded through all amplification and detection steps. With enzyme-linked DNA hybridization method, the quantitation of IiBV DNA was performed by an external standard curve generated with every nm. (2) To assess the anti-viral effects of OM, the level of HBV DNA in HepG2.2. 15 cells incubated with different concentration of OM was determined. The in vitm and-viral effects were evaluated and quantified by calculating the inhibiting rate. Results (1) There were several advantages of the equivalent competitive PCR combining with DNA hybridization quantitative detection technique (PCR-ELISA) as follows: First, the preparation of IS was very simple and convenient Second, because the length and base number of both mutated and wild fragment were the same, the amplification efficiency of each could be equivalent; thus improving the precision of quantitative detection. Third, the IS contained the same primer binding sites as the HBV target and a unique probe binding region that allowed IS amplicon to be distinguished from HBV amplicon and quantified respectively. Fourth. the method possessed high sensitivity, specificity and reliability. (2) The anti-HBV effects of OM were investigated. The results indicated that the inhibiting rate of OM on the replication of HBV in HepG2.2. 15 cells increased with the concentration. The inhibiting rate reached maximum, 79.6%, at 1000 jig/mI of OM. Because large amounts of virus were released from dead cells caused by drug toxicity, the result might be inaccurate when the concentration increased. We observed that the stable concentration of the drug in medium is one of the most important factors to keep inhibiting effects. The inhibiting effects of the OM were similar in the 1st, 2nd and 3rd 24 hours at the same concentration. The anti-viral activity of the drug declined sharply if the incubation time prolonged to 48h, 72h or longer. As the result of continuous release of virus from the injured and dead cells, the quantity of HBV DNA in the supernatant of nine-day-treated cells was nearly equal to the OM-free control. Our results could not reveal the relationship between the continuous action of OM and the level of HBV DNA secreted from HepG2.2.15 cells.
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