OBJECTIVE: To determine the concentration of phenobarbital(PBB) in serum by RP-HPLC.METHODS: The analytical column was C18.The mobile phase consisted of methanol-water(44∶56) at a flow rate of 1.0 mL?min-1.The detection wavelength was set at 205 nm and the temperature of column was set at 35℃.RESULTS: A good linear relationship was obtained for PBB at a concentration range from 5.08 ?g?mL-1 to 63.50 ?g?mL-1(r=0.999 9).The mean relative recovery was 99.75% and the mean extraction recovery was 96.34%.The intra-day RSD and the inter-day RSD were all less than 4%.CONCLUSION: This method is simple,rapid,accurate,and suitable for therapeutic drug monitoring.

Objective:To establish an HPLC method for the content determination of salvianolic acid B in Ansheng Yizhi cap-sules. Methods:A Hypersil ODS2 C18 column(150 mm × 4. 6 mm,5 μm) was used and methanol-water-formic acid (40∶60∶1) was used as the mobile phase. The detection wavelength was at 286 nm. The flow rate was 1. 0 ml·min-1 and the sample size was 10 μl. Results:The calibration curve of salvianolic acid B was linear within the range 7. 75-77. 51 μg·ml-1(r=0. 999 6). The average re-covery was 98. 17%(RSD=1. 79%, n=6). Conclusion:The method is simple, accurate and repeatable, which can be used in the quality control of Ansheng Yizhi capsules.

Objective To investigate the optimal cryopreservation liquid of the stable transfected cell line named hepatic carcino-ma HepG2 transfected by lentivirus .Methods Glycerol and DMSO as cryopreservation liguid ,The HepG2 transfected by lentivirus and HepG2 cells were both cryopreserved with four various proportion of cryopreservation liquid .After thawing ,the survival rate of the two cells were observed by inverted contrast microscope and the viability and proliferation were detected with MTT assay .Re-sults Using glycerol as cryoprotectant liquid ,the survival rate of the HepG2 cells transfected by lentivirus was obviously higher than other 3 groups when the proportion of the cryopreservation liquid(DMEM ∶FBS∶Glycerol)was 0∶9∶1(P=0 .001) ,where-as there was no significant difference among the groups of HepG2 cells with various proportion of cryopreservation liquid (P=0 .293) .Using DMSO as cryoprotectant liquid ,the survival rate of the cells transfected by lentivirus was 0% ,whereas the survival rate of the HepG2 cells was higher than 60% ,and there was no significant difference between groups of various proportion of cryo-preservation liquid(P=0 .487) .The MTT assay demonstrated that the higher the serum levels was ,the better proliferation capacity those cells had(P<0 .05) .Conclusion The optimal cryopreservation liquid for the hepatic carcinoma cell line HepG2 transfected by lentivirus is used glycerol as cryoprotectant solution ,and the proportion of cryopreservation solution was 90% FBS + 10%Glycerol .

Objective To research the expression of Sp3 andβ‐Catenin in HCC and study the assessable factors of them for prognosis in patients with hepatocellular carcinoma .Methods Western blot and RT‐PCR methods were used to detect the expres‐sion of Sp3 andβ‐Catenin in HCC and the liver tissue beside tumor among 49 cases .We analyzed the difference of these two indexes expressed in HCC and the liver tissue beside tumor .Then we detected the correlation between these two indexes and the character of clinic pathology ,and researched the correlation between Sp3 and the prognosis of HCC .Results The high expression rate of Sp3 in HCC was higher than that of liver tissue beside tumor(P<0 .05) according to Western blot and RT‐PCR ,the same toβ‐Catenin (P<0 .05) .Expression of Sp3 andβ‐Catenin were both related with size of tumor and degree of differentiation .Positive correlation existed between these two indexes according to Western blot method(r=0 .681 ,P=0 .000) and RT‐PCR method(r=0 .641 ,P=0 .000) .The prognosis of cases with high expression of Sp3 was poorer than the low expression cases(P<0 .05) .Conclusion Sp3 plays a promoter role in occurrence of HCC ,which is correlated with the grade malignancy of HCC .Sp3 might participated in occur‐rence and development of HCC via the Wnt pathway .

AIM: To observe the effects of high-dose hepatitis B surface antigen (HBsAg) vaccine on cellular immune response in BALB/C mice. METHODS: The mice were immunilized separately with low-dose and high-dose HBsAg vaccine by intramuscular injection two times. The specific proliferative activities of T lymphocytes were measured by [ 3H]-TdR incorporation assay. IL-2 as well as IFN-? levels in the culture supernatant of T cells and anti-HBs IgG2a lever in sera were detected by enzyme-linked immunoabsorbent assay. RESULTS: After first vaccination with high-dose HBsAg, the proliferative activities of T cells in the experimental group were significantly stronger, both levels of IL-2 and IFN-? were markedly higher than that in the control group and the percentage of mice to produce serum anti-HBs IgG2a was significantly higher compared to that of mice immunilized by low-dose HBsAg. All data in experimental groups were further increased after second dose of vaccine. CONCLUSION: Vaccination of mice with high-dose HBsAg can induce cellular immune responses tended to Th1(T helper 1 subset) response.

AIM: To study the influence of ginkgolides on expression of inducible nitric oxide synthase (iNOS) in rats. METHODS: Middle cerebral artery occlusion(MCAO) model was induced by ligating internal carotid artery of Sprague Dawley(SD) rats.In this model,the behavioral deficits performance of SD rats was analyzed after given ginkgolides and nimo dipin, respectively, and iNOS gene expression changes in MCAO group were investigated. RESULTS:The expression of iNOS was increased in MCAO models and inhibited in ginkgolides groups. CONCLUSION:Ginkgolides have inhibitive efficacy of iNOS on cerebral ischemia and can reduce brain damage.

AIM: To investigate the effects of auricularia auricular polysaccharide (AAP) on chronic cerebral ischemia injury in rats. METHODS: The chronic cerebral ischemia mode1 was made by permanent middle cerebral artery occlusion (MCAO) on the right side. AAP at different doses (50 mg/kg and 100 mg/kg) was intragastrically administered at the onset of ischemia and in the following days after operation, once a day for 4 weeks. After 4 weeks of MCAO, Morris water maze test was introduced to examine the learning and memory functions. Nissl staining was performed to detect the survival neurons in hippocampal slices. Level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in brain tissue were measured. RESULTS: Rats treated with AAP showed a shorter escaping latency in spacial navigation test because the AAP treated rats spent less time to find the platform in spatial probe test. More survival neurons in hippocampal slices were observed from AAP treated rats. Also, the MDA level in brain tissue was reduced and SOD activity in brain tissue was increased in the AAP treated rats with MCAO. CONCLUSION: AAP protects rats from chronic brain ischemic injury, in which its anti-oxidative effect might be involved.

OBJECTIVE:To work out the quality control standard of Penqiangyan tablets.METHODS:The major herbs in the preparation were identified by TLC.The contents of total ferulic acid in Radix Angelia Sinensis and Rhizoma Chuanxiong were determined by HPLC.RESULTS:Ferulic acid had a good linearity in the range of1.244～124.4?g/ml(r=0.9984).The recovery and RSD were97.06%and1.44%(n=6)respectively.CONCLUSION:The method is simple and feasible and had a good repeatability.It can be used for the quality control of Penqiangyan tablets.

OBJECTIVE:To establish a method for the content determination of icariin in Kangguzhi zengsheng tablet,and pro-vide a basis for improving the quality standard. METHODS:HPLC was performed on the column of Hypersil ODS2 C18 with mo-bile phase of acetonitrile- water(27∶73,V/V)at a flow rate of 1.0 ml/min,detection wavelength was 270 nm,column temperature was 25℃,and injection volume was 10μl. RESULTS:The linear range of icariin was 3-60μg/ml(r=0.999 9);limit of quantita-tion was 11.2 μg/ml,limit of detection 4.3 μg/ml;RSDs of precision,stability and reproducibility tests were lower than 2.0%;re-covery was 99.00%-100.67%(RSD=0.63%,n=6). CONCLUSIONS:The method is simple,accurate and reproducible,and can be used for the content determination of icariin in Kangguzhi zengsheng tablet.

Objective To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes.Methods Total RNA was isolated from unfed female ticks,mRNA was purified and a library of oligo(dT)-primed cDNA with added directional EcoR Ⅰ/Hind Ⅲ linkers was constructed from the purified mRNA.The constructed cDNA was ligated to the EcoRⅠ/HindⅢ arms of the ?SCREEN vector.Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8.Recombinant plasmids that were subcloned to E.coli BM25.8 were isolated and transformed into E.coli JM109.Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing.Rusults The recombinant phage DNA was packaged by using phage-marker packaging extracts,resulting in a primary cDNA library with a size of 1.8?106 pfu.Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4?109 pfu/ml.Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library.Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H.longicornis tropomyosin mRNA,Rhipicephalus annulatus unknown larval protein mRNA,chromosome 2R of Drosophila melanogaster,mitochondrial DNA of H.flava,clones HqL09 unkown mRNA and Hq05 mRNA of H.qinghaiensis,and myosin alkali light chain protein mRNA.Conclusion The cDNA expression library from unfed female H.longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.