AIM To establish a quantitative analysis of multi-components by single marker(QAMS) method for the content determination of six catechins in Xinnaojian Capsules (Tablets) (tea extract).METHODS The analysis of 50％ methanol extract of this drug was performed on a 35 ℃ thermostatic Shimadzu Wonda Cract ODS-2 column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of 0.5％ acetic acid (A)-acetonitrile (B) flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 280 nm.With epigallocatechin gallate as an internal standard,the relative correction factors of epigallocatechin,catechin,epicatechin,gallocatechin gallate and epicatechin gallate were calculated,from which the content determination was made.RESULTS Six constituents showed good linear relationships within their own ranges (r ≥ 0.999 8),whose average recoveries were 96.00％-98.47％ with the RSDs of 2.09％-2.91％.The results obtained by QAMS method approximated those obtained by external standard method.CONCLUSION This simple and reliable method can be used for the quality control of Xinnaojian Capsules (Tablets).

Objective To study the effects of wedelolactone on the expression of P-glycoprotein (P-gp) and to explore the multi-drug resistance reversing mechanism of wedelolactone in K562/A02 cells in vitro.Methods The half maximal inhibitory concentration of ADM in K562/A02 was determined by MTT method.The protein expression level of P-gp was determined by Western blot after wedelolactone pretreatment.Results Wedelolactone remarkably enhanced chemo-sensitivity to ADM of K562/A02 cells.After 0.2, 2, 20 μmol/L different concentration of wedelolactone treatment in 24 h, the relative reversal efficiency of K562/A02 to ADM was 23.5％, 47.1％ and 67.7％, respectively.according to the results of Western blot, wedelolactone was shown to efficiently inhibit the expression of P-gp (P＜0.05).The relative efficiency of K562/A02 to ADM was 25.4％,46％ and 55.6％, respectively.Conclusion Wedelolactone could modulate P-gp expression, and P-gp expression down regulation may be one of the MDR reversal mechanisms in K562/A02 cells by wedelolactone.

BACKGROUND:Many studies reported the relationship of the mechanical properties and water content about bone tissue, which is one of organizations containing the lowest water content on human body. Researches on effect of water on biological tribology behavior of bone tissue have been rarely reported and are the experimental study generally. OBJECTIVE:To explore the influence and the damage mechanism of water on biological tribology behavior of bone tissue, by comparing multiscale numerical model established with the experiment. METHODS:Dehydration of the bone tissue was studied by nanoindentation test and both reciprocating sliding and impact wear tests. A multi-scale finite element model was constructed under a flat-on-ball configuration. RESULTS AND CONCLUSION:The viscoelasticity and the tribological properties of bone tissue significantly decreased as well as the different wear mechanisms under applied loading after drying. The analytical results indicated that there were high stress condition, which incurred the micro-crack initiation and the appearance of peeling and wear, around the Haversian canal, circumferential lamellas and the interstitial tissues. Meso-scale:dehydration weakened the function of absorption and interruption of stress, which facilitated crack extension in pore. Micro-scale:the high stress gradient of structure of canaliculi and lacunae is an important cause of tissue damage.

Objective To study the influence of HBV replication regulator,enhancer I and Pre- S2,on the immune response of HBV DNA vaccine.Methods DNA fragments of HBsAg,PreS2 HBsAg,HBsAg-enhancer I and PreS2-HBsAg-enhancer I region of HBV were amplified by PCR using the complete genome DNA of HBV adr subtype,and inserted into VR1012 vectors,respective- ly.The recombinant plasmids were transfected into HepG2 cells,and injected into Balb/C mice.The expression of HepG2 cells and the cellular and humoral immune response of mice were tested by cell immuno-chemistry,ELISA and ELISPOT.Results The target protein were expressed by transfected HepG2 cells,enhancer I and Pre-S2 can promote the expression of HBsAg in transfected cells.The HBsAb and the HBsAg specific CTL in inoculated mice were found in the second week after injection, PreS2 but not enhancer I can promote the immune response in inoculated mice.Conclusions When inserted into HBV DNA vaccine,enhancer I and PreS2 can promote the expression of HBsAg in transfected HepG2 cells,PreS2 can promote the immune response in inoculated Balb/C mice.

AIM:To observe whether modified epitopes from hepatocellular carcinoma antigen MAGEC 2 have HLA-A2-restricted antitumor ability.METHODS:HLA-A2 epitopes from MAGEC2 protein were predicted by NetCTL 1. 2,SYFPEITHI and IEDB.The change of binding anchor motifs by replacing anchor residues created the modified peptides from MAGEC2.The binding affinity of the peptides to HLA-A*0201 molecule was evaluated by T2 cells binding assay. ELISPOT assay and intracellular cytokine staining were used to investigate the ability of the peptides inducing specific re -stricted cytotoxic T-lymphocytes(CTLs)to release interferon-γ(IFN-γ).The ability of the peptides to induce T-cell re-sponse was investigated by cytotoxicity assay in vitro.RESULTS:The candidate peptides P248, P248-1Y,P356,P356-1Y,P356-2L and P356-1Y2L showed moderate affinity toward HLA-A2 molecule.T2 binding assay showed that P248-1Y and P356-1Y2L showed significantly higher affinity for HLA-A2 than the native peptides.ELISPOT assay and intracellular cytokine staining showed P248, P248-1Y, P356 and P356-1Y2L were able to induce specific CTLs to release IFN-γ. ELISPOT assay showed that significantly higher levels of IFN-γrelease were induced by P248-1Y and P356-1Y2L than the native peptides.The CTLs induced by P248,P248-1Y,P356 and P356-1Y2L lysed HepG2 cells,and P248-1Y and P356-1Y2L peptide-specific CTLs showed higher cytotoxicity against HepG 2 cells than the native peptide-specific CTLs(P<0.05).CONCLUSION: Compared with the native peptides, modified epitopes P248-1Y and P356-1Y2L have higher binding affinity with HLA-A2 and retain immunogenecity.In addition, the antitumor immunity effects of modified epitope P248-1Y and P356-1Y2L are stronger than the native peptides.The peptides P248-1Y and P356-1Y2L are excellent HLA-A2-restricted CTL epitopes from tumor antigen MAGEC 2, which could serve as new candidates towards antitumor peptide vaccines.

Objective To analyze the differentially expressed genes between the NCAM + c-Kit +RBE and NCAM-c-Kit-RBE of intrahepatic cholangiocarcinoma (ICC) cell lines,and to screen out the differentially expressed genes that are related to the stem cell signaling pathways.Methods Magnetic activated cell sorting was used to isolate the NCAM + c-Kit +/NCAM-c-Kit-subset cells,and then Agilent Whole Human Genome Microarray Kit was used to test the difference in gene expressions between the NCAM + cKit + and NCAM-c-Kit-subset cells.The difference in gene expressions related to the stem cell signaling pathways was analyzed by the SAS system.The result of the microarray was further confirmed by RT-PCR.Results The total differentially expressed genes which could be found through gene microarray were 7270 [foldchange(fc) ≥2 or fc ≤0.5].Compared with the NCAM-c-Kit-RBE,3572 genes were upregulated while 3698 genes were downregulated.The differences in gene expressions related to the stem cell signaling pathways were 421 (fc ≥2 or fc ≤ 0.5),among which 231 genes were upregulated while 190 genes were downregulated.Conclusions High-flux microarray could be used to screen out lots of differentially expressed genes between the NCAM + c-Kit + and NCAM-c-Kit-RBE cells.The differences in gene expression in the stem cell signaling pathways could also be further analyzed using the SAS system.

Objective To compare the effects of different methods of general anesthesia on postoperative cognitive function in patients undergoing non-cardiac surgery.Methods One thousand ASA Ⅰ or Ⅱ patients,aged 18-60 years and undergoing non-cardiac surgery,were randomly divided into five groups (n=200 each):isoflurane + propofol + fentanyl group (group IPF),isoflurane + remifentanil group (group IR),sevoflurane + propofol + fentanyl group (group SPF),sevoflurane + remifentanil group (group SR),and propofol + remifentanil group (group PR).Two hundred patients receiving non-operative treatment served as control group (group C).In groups IPF and SPF,anesthesia was maintained with inhalation of 1.68％ isoflurane or 1.71％ sevoflurane,target controlled infusion (TCI) of propofol with the target plasma concentration of 2-5 μg/ml,and intermittent intravenous boluses of fentanyl.In groups IR,SR and PR,anesthesia was maintained with inhalation of 1.68％ isoflurane or 1.71 ％ sevoflurane,or TCI of propofol with the target plasma concentration of 2-5 μg/ml,and TCI of remifentanil with the target plasma concentration of 2-6 ng/ml.The patients' cognitive function was assessed with minimental state examination (MMSE) 1 day before operation,when leaving the post-anesthetic care unit (PACU),and 1 and 3 days after operation,respectively.Z score was used to identify the cognitive dysfunction as recommended by Moiler when leaving the PACU,and 1 and 3 days after operation.Results Compared with group C,the MMSE score was significantly decreased when leaving the PACU,and the incidence of cognitive dysfunction increased when leaving the PACU and 1 day after operation in the other groups (P ＜ 0.05).Compared with groups IPF,IR,SPF and PR,the incidence of cognitive dysfunction was significantly increased in group SR (P＜0.05).Conclusion General anesthesia with sevoflurane combined remifentanil exerts fewer effects on the postoperative cognitive function in patients undergoing non-cardiac surgery.

The fine structure of enoxaparin sodium samples with different degree of 1,6-anhydro derivatives were analyzed with polyacrylamide gel electrophoresis, high performance liquid chromatography, ultraviolet spectroscopy, infrared spectroscopy and nuclear magnetic resonance spectroscopy. A further study of anticoagulation activity of enoxaparins was performed, including those on their inhibition activities of coagulation factor Xa (FXa) and thrombin (FIIa). The results showed that the anti-FXa and -FIIa activities of enoxaparins with different degree of 1,6-anhydro derivatives (20.0%-39.7%) with similar structure characteristics, had decreasing tendency when the degree of 1,6-anhydro derivatives increased. Especially, the anti-FXa activity was sensitive to the change of the degree of 1,6-anhydro derivatives.
Anticoagulants ; chemistry ; Enoxaparin ; chemistry ; Factor Xa Inhibitors ; chemistry ; Thrombin ; antagonists & inhibitors

Objective To investigate mutation spectrums of α- and β-haemoglobin genes in thalassemia patients and carriers in Yunnan province,and to establish procedures on prenatal gene diagnosis.MethodsTotally 10 033 counseling couples and pregnant women,and 22 cases of children with moderate or severe thalassemia were recruited from 5 parts of Yunnan Province,middle,western,eastern,southern and northern areas, during July 2009 to July 2011.Medical records, including results of haemoglobin electrophoresis,blood routine examination,and gene diagnosis of subjects were collected and saved in an database in Excel software by the Key Laboratory for Birth Defects and Genetic Diseases.Using multiple gap-PCR and PCR-reversed dot blotting kits, DNA samples collected from 1077 cases of haematological positive thalassemia patients and carriers were tested to determine common mutations of the α-or β-haemoglobin genes.The codon regions of haemoglobin genes were sequenced by the Sanger sequencing in cases that the mutation tests were negative.Mutation spectrums of α- and β-haemoglobin genes were concluded.Prenatal gene diagnosis was offered to fetuses who had risk of thalassemia major.Results( 1 ) In 1077 cases of haemological screen positive subjects,deletions and mutations of α-haemoglobin gene were tested in 119 subjects among 347 cases suspected as α-thalassemia patients and carriers.Five kinds of deletions and mutations on α-haemoglobin gene were found.In 104 subjects,four kinds of common deletions and mutations onα-haemoglobin gene were determined:--SEA, -α3.7, αCS α,-α4.2.Other 14 subjects were double heterozygotes with haemoglobin H disease and severe α-thalassemia phenotypes.A rare mutation of insertion and deletion in α2 haemoglobin gene intron,α301-24-301-23 indel,was found in one carrier subject.(2)In 1077 cases of haemological screen positive subjects,deletions and mutations of β-haemoglobin gene were tested in 297 subjects among 730 cases suspected as β-thalassemia patients and carriers.Sixteen kinds of β-haemoglobin gene mutations were found,including 7 cases of rare abnormal haemoglobinopathy patients with β-haemoglobin gene mutations.In one case with β + phenotype patient,the Codon 5 (-CT)mutation at β-haemoglobin gene was found (firstly reported in China ). (3) Three fetuses with high riskS of α-thalassemia were accepted for prenatal diagnosis.One case of Hb Bart's hydrops syndrome fetus with the genotype --SEA/--SEA,and one case of mild α-thalassemia fetus with the genotype αCS α/αα were found.Another one fetus was found with normal α-haemoglobin.In 6 fetuses accepted for prenatal diagnosis due to high risks of β-thalassemia,one case of β-thalassemia major with the genotype CD17( A→T)/-28 (A→G) was found,3 fetuses were heterozygote carriers,and 2 fetuses had normal genotypes without mutations found in their parents.Medical terminations for 2 fetuses with severe thalassemia were made according to the choice of pregnant women.Other 7 pregnancies continued to term.Anemia or growth retardation was not found in the 7 infants when following up after given-birth 6 to 12 months.Conclusions The mutation spectrums of α- and β-haemoglobin genes of thalassemia patients and αarriers.in Yunnan province are special,in which β-haemoglobin gene exits more polymorphism in the mutation spectrum.Carrier screening in pregnant women,and offering prenatal gene diagnosis to the high risk pregnancies should be an efficient strategy to prevent thalassemia major.

To develop a core-shell structure pDNA-CaPi-PLGA nanoparticles (CS-pDNA-CaPi-PLGA-NPs), calcium phosphate-pDNA nano complexes (CaPi-pDNA) were encapsulated inside of PLGA shells. The characteristics of the nanoparticles, including morphology, average particle size, zeta potential, entrapment efficiency, loading efficiency, stability in medium, pDNA protection ability from nuclease degradation, in vitro release, cytotoxicity and cell transfection were investigated and compared with the embedded structured CaPi modified PLGA nanoparticles (embedded-pDNA-CaPi-PLGA-NPs). The results showed that the obtained CS-pDNA-CaPi-PLGA-NPs were spherical in shape with an average particle size of (155 +/- 4.5) nm, zeta potentials of (-0.38 +/- 0.1) mV, entrapment efficiency of (80.56 +/- 2.5)% and loading efficiency of (1.16 +/- 0.04)%. The CS-pDNA-CaPi-PLGA-NPs were stable in the release media and could protect pDNA against nuclease degradation. And they also exhibited sustained release of pDNA in vitro. The highest gene transfection efficiency of the CS-pDNA-CaPi-PLGA-NPs in vitro reached (24.66 +/- 0.46)% (after 72 h transfection), which was significantly higher than that of free pDNA [(0.33 +/- 0.04)%, P < 0.01] and the pDNA-PLGA-NPs [(1.5 +/- 0.07)%, P < 0.01]. Besides, the transfection lasted for longer time than that of embedded-pDNA-CaPi-PLGA-NPs and the cytotoxicity of it was significantly lower than that of PEI (P < 0.01). These results indicate that CS-pDNA-CaPi-PLGA-NPs are a promising non-viral gene vector. Key words: gene delivery system; polylactic-co-glycolic acid; calcium phosphate; nanoparticle