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In this article, the authors expounded the necessary and problems of animal experiment post-ap-proval monitoring ( PAM) , which included who should do PAM, how to do PAM, and what the PAM can do. The authors also exposed the following suggestion: formulating the detailed rules, regulations and SOP, strengthening the training of PAM team member and animal experiment personnel, and monitoring the whole process of animal protocol review using the software.
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Objective To set up the rolling circle amplification (RCA) system for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to evaluate the specificity and sensitivity of this system. Methods Plasmids containing full-length of wild-type HBV genome were treated with restriction enzyme and T4 DNA ligase, and then were concentrated. The DNA fragments were recovered by the nucleic acid purification kit and severed as standard HBV cccDNA. Total DNA was extracted from hepatic tissues of seven chronic hepatitis B patients. RCA method was used to amplify genomes from tissue samples. Standard HBV cccDNA, 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of patients with chronic HBV infection were used as controls to determine the specificity of RCA. Ten-fold serial dilutions of standard HBV cccDNA were used for determining the sensitivity. Results The standard HBV cccDNA was successfully constructed and could be detected by RCA method. HBV cccDNA could be amplified from 2 mg hepatic tissue samples at least of HBV infected patients, and could be detected as low as 1 ×102 copy/μL. cccDNA was not detected in 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of chronic HBV infected patients. Conclusion RCA method can be used for rapid and simple detection of HBV cccDNA with high specificity and sensitivity.
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Objective Plant vaccine is a new kind of vaccine with good safety profiles, low cost and convenience. Our purpose was to establish a good plant system to express HBsAg and pave the way for further research on plant vaccine. Methods Transfer HBV s gene into binary vector pCAMBIA1301 which had plant promoter and hygromycin resistant gene. The recombinant vector was transferred into agrobacterium LBA4404. Then we used agrobacterium to infect tobacco. Results The GUS staining result of transformant leaves showed blue, which meant the target gene had been transferred into tobacco and expressed HBsAg seccessfully. We confirmed the insertion of S gene by PCR and its expression further by ELISA and Western blot. Conclusions HBsAg can be expressed successfully in tobacco.
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Objective To evaluate the effect of total parenteral nutrition (TPN) on hyperemesis gravidarum (HG) and pregnancy ending.Methods 27 cases of HG were treated with TPN and 30 cases with the routine infusion to adjust water electrolyte and acid base balance for the purpose of observation of vomiting stopping,time of turning ketonic negative and pregnancy ending.Results After 3 day treatment with TPN,stopping vomiting rate was 55.6%,time of turning ketonic negative was (3.1?0.84) days and neonatal weight was (3.4?0.38) kg,which was 13.3%,(5.2? 1.06) days and (2.9?0.45) kg respectively in control group.There was significant difference between the two groups (P
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Objective To explore the fluctuation of cytokine mRNA expression level in a novel T-cell-mediated immune hepatic fibrosis model induced by repeatedly injections of Concanavalin A in BALB/c mice. Methods BALB/c mice were divided into different groups. Model group mice were injected weekly up to 20 weeks with Concanavalin A (15mg/kg), via retro-orbital venous plexus under ether anesthesia. Normal control group mice were treated in the same manner weekly with normal saline. Twenty-four hours after Concanavalin A challenge at 1, 5, 12 and 20 week, 8 mice from each time were killed by cervical dislocation, repectively. The livers of different group were excised and fixed in 10% formalin for HE staining and Gomori Ag staining or frozen in optimal cutting temperature (O.C.T.) media in liquid nitrogen for immunohistochemical staining for CD4 +T or CD8 +T cell. After extracting total RNA from liver tissues, IL-2, IL-4, IL-10 and transforming factor ?1 messenger RNA were amplified by reverse transcription polymerase chain reaction. PCR products were electrophoresed on agrose containing ethidium bromide and visualized under ultraviolet light. Densitometric RT-PCR data were standardized with ?-actin signals. Results The histological change of HE staining and Gomori Ag staining indicated the fibrogenesis in model group mice. Immunohistochemical staining for CD4 + or CD8 + T cell indicated that the infiltrating lymphocytes in liver parenchyma were mainly CD4 +T lymphocytes. IL-2 mRNA expression level only increased after the first injection of Concanavalin A. The expression levels of IL-4, IL-10 and transforming growth factor ?1 mRNA significantly increased over the whole experiment period as compared with control group. Conclusions Repeated administration of Concanavalin A can induce T-cell-mediated immune hepatic fibrosis model in BALB/c mice. The expression levels of IL-4, 10 and TGF-?1 increase over the whole experiment period and may play an important role in creating mouse fibrotic model.