Liquid-phase microextraction is a novel pretreatment technique for biological samples developed on the basis of liquid-phase extraction technology, which is simple, rapid, economical, and environmentally friendly, and has been widely used in the analysis of biological matrix samples such as blood, urine, and saliva. In this paper, we review the basic principles of the main modes of liquid-phase microextraction techniques, i.e., single-drop microextraction, dispersive liquid-liquid microextraction, and hollow-fiber liquid-phase microextraction, and the progress of their applications in biological sample pretreatment by reviewing the literature in the past five years, with a view to providing technical support and reference for sample pretreatment in the fields of in vivo drug analysis, pharmacokinetic studies and new drug development.

A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of twelve major compounds in Longdan Xiegan Pill.The chemical profile of the twelve compounds,including geniposidic acid (1),geniposide(2),gentiopicroside(3),liquiritin(4),crocin(5),baicalin(6),wogonoside(7),baicalein(8),glycyrrhizic acid (9),wogonin (10),oroxylin A ( 11 ) and aristolochic acid A (12),was acquired using high-performance liquid chromatography-diode array detector coupled with an electrospray tandem mass spectrometer (HPLC-DAD-ESI-MS).The analysis was performed on a Dikma Platisil ODS C18 column (250 mm × 4.6 mm,5 μm ) with a gradient solvent system of acetonitrile-0.1％ aqueous formic acid.The validation was carried out and the linearities ( r ＞ 0.9996),repeatability (RSD＜1.8％),intra- and inter-day precision (RSD＜1.3％),and recoveries (ranging from 96.6％ to 103.4％ ) were acceptable.The limits of detection (LOD) of these compounds ranged from 0.29 to 4.17 ng.Aristolochic acid A,which is the toxic ingredient,was not detected in all the batches of Longdan Xiegan Pill.Furthermore,hierarchical cluster analysis was used to evaluate the variation of the herbal prescription.The proposed method is simple,effective and suitable for the quality control of this traditional Chinese medicine (TCM).

OBJECTIVETo explore whether hepatitis B virus (HBV) S gene-modified dendritic cells (DCs) might induce a specific cytotoxic T lymphocyte (CTL) response.

METHODSThe recombinant adenoviruses carrying HBsAg genes were prepared and used to transfect DCs generated from cord blood. The efficacy of transfection was observed through the expression of enhanced green fluorescent protein (EGFP) in DCs and the expression of HBsAg was detected by ELISA. HBV S gene-modified DCs were co-cultured with T cells from cord blood and T cells stimulating activities were detected using mixed lymphocyte reaction (MLR). The CTL assay was carried out to assess the ability of CTL lines to lyse target cells of HepG(2)22.1.5 by measuring lactate dehydrogenase (LDH) release.

RESULTSThe results showed that HBV S genes were expressed in DCs with high efficacy by recombinant adenoviral vector. DCs had a normal shape after transfection. The result of MLR showed that HBV S gene-modified DCs could effectively stimulate naive T cells to proliferate. The induced specific CTL lines could lyse target cells of HepG(2)22.1.5.

CONCLUSIONSHBV S gene-modified DCs enhanced the function to induce a specific CTL effect, showing its promise for developing anti-viral vaccine in future.

Cell Line ; Cells, Cultured ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; virology ; Hepatitis B ; immunology ; virology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Cytotoxic ; immunology

Bone transplantation is widely used in orthopaedics, and allograft bone transplantation is being more and more emphasized. In this article, the allograft bone combined with growth factors transplantation for repairing bone defects were reviewed. Moreover, the way to compound many growth factors and other material is the tendency of allogenic bone grafting, which enhance the opportunity of success in bone transplanting.
Bone Morphogenetic Proteins ; therapeutic use ; Bone Transplantation ; Fibroblast Growth Factor 2 ; therapeutic use ; Humans ; Intercellular Signaling Peptides and Proteins ; therapeutic use ; Platelet-Derived Growth Factor ; therapeutic use ; Transforming Growth Factor beta ; therapeutic use ; Transplantation, Homologous ; Vascular Endothelial Growth Factor A ; therapeutic use

ObjectiveTo analyze the clinicopathological features and prognosis of gastric cancer patients with metastatic nodules of perigastric soft tissue. MethodsIn this study,1025 cases of gastric cancer received radical resection.According to the metastasis of perigastric soft tissue,patients were divided into metastatic group ( group MP,n =334 ),non-metastatic group ( group NMP,n =691 ).The clinicopathological features and prognosis were compared between the two groups. ResultsIn group MP,the ratio of upper,middle,lower,total gastric cancer was 25.8％,22.0％,51.4％,0.9％ and the ratio in group NMP was 33.2％,21.3％,41.3％,4.2％ respectively,showing significant higher ratio of upper and total gastric cancer in MP group(P =0.000). In group MP 47.3％ cases with tumor size ≥5 cm,significantly higher than that in NMP group(27％ ) (P =0.000).Lymph node metastatic ratio between 21％ -40％ and 41％ -100％ was found in 24.4％ and 37.3％ in MP group respectively,significantly higher than that of 12.9％,10.8％ in NMP group(P =0.000).20.1％ cases had distal metastasis in group MP,significantly higher than that of 4.1％ in group NMP(P=0.000).In group MP and NMP group,the ratio of Borrmann infiltration typing was 82.1％ vs.64.6％,the ratio of positive CEA was 21.2％ vs.11.4％,the ratio of lower or undifferentiation typing was 78.7％ vs.64.2％,all with significant difference (P =0.000 ). COX regression analysis showed the infiltration depth,organic invasion,lymph node metastatic ratio,M staging,Borrmann typing,metastatic nodules was the independent prognostic factors.Prognosis was significantly poorer in the cases with perigastric soft tissues than without ( P =0.000 ).Stratified analysis showed that irrespective of tumor size,infiltration depth,lymph node metastatic ratio,CEA value,Borrmann typing,differentiation degree,the mean survival time was significantly shorter in MP group than that in group NMP(P ＜ 0.005).In cases without distal metastasis,the prognosis was significant poorer in group MP than that in group NMP ( P =0.000 ),however,there was no significant difference between two groups in cases without distal metastasis ( P =0.076).ConclusionsPerigastric soft tissue metastasis was common in gastric cancer,more frequently seen in tumor ≥5 cm,or with organic invasion,lymph nodemetastaticration ≥ 21％, distalmetastasis, Borrmanninfiltrationtyping, loweror undifferentiation typing,positive CEA. Perigastric soft tissues metastasis was the independent prognotic factor for gastric cancer.

OBJECTIVETo study HBV preS2/S gene expression effects in mammalian cells transferred with recombinant adenoviral vector.

METHODSThe replication-deficient recombinant adenoviral vector (Ad-HBs) carrying HBV preS2/S gene were constructed by homologous recombination in bacteria. The 293 cells, Vero cells, HepG2 cells and mesenchymal stem cells (MSCs) were infected with adenoviruses. The expressions of enhanced green fluorescent protein (EGFP) were observed with fluorescence microscope and the expressions of HBsAg were detected by RT-PCR and ELISA in vitro.

RESULTSMore than 90% of 293 cells, Vero cells, HepG2 cells or MSCs expressed EGFP after transfection at the MOI of 20 and the titers of HBsAg were more than 3.229 (A value) in culture supernatant.

CONCLUSIONThe HBV preS2/S gene was not only expressed efficiently in immortalized cells, but also expressed efficiently in stem cells with the recombinant adenoviruses vector.

Adenoviridae ; genetics ; Animals ; Cell Line ; Cell Line, Tumor ; Cercopithecus aethiops ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Humans ; Microscopy, Fluorescence ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vero Cells

Objective To observe the effects of serum with drug Penthorum chinense Pursh extractum on transforming growth factor (TGF)-β1 and collagen I secretions of activated hepatic stellate cells. Methods Twenty male SD rats were divided into 2 groups, and were administered with 0.9% sodium chloride solution and Penthorum chinense Pursh extraetum via gastrogavage for 3 days respectively and then sacrificed. Serum samples of these rats were collected. HSC-T6 cells were divided into the normal group and the treatment group. The cells of the normal group were incubated in Dulbecco's modified eagle medium (DMEM)with sera of normal rats, while those of the treatment group were incubated in DMEM with sera from Penthorum chinense Pursh extractum treated rats. The HSC-T6 viability was observed by AlamarBlue assay, while the toxicity of Penthorum ehinense Pursh extractum was measured by 3-(4, 5-Dimethyhhiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The expression of collagen I and TGF-β1 mRNA were determined by real timepolymerase chain reaction (real time PCR). The protein expressions of collagen I and TGF-β1 were analyzed by Western blotting. The data were analyzed by single factor analysis of variance and pairwise comparison was done by q test. Results Different concentrations of sera from Penthorum chinense Pursh extractum treated rats could all inhibit HSC-T6 proliferation, especially when the sera concentration were 10% and the HSC-T6 cells were incubated for 24 h (P＜0.01 ). MTT assay indicated that sera from Penthorum chinense Pursh extractum treated rats showed no obvious toxicity to HSC-T6 compared with those from normal rats (P ＞0.05). After 24 h incubation, 10% sera from Penthorum chinense Pursh extractum treated rats could significantly down-regulate mRNA expression of TGF-β1 and collagen I compared with normal group (TGF-β1 2.790±0.174 vs 9. 827 ± 1.429, P＜0.01 ; collagen I 1.213 ± 0.099 vs 4.053 ± 1.005, P＜0.01 ). Mcanwhile, the protein expressions of TGF-β1 and collagen I were also obviously inhibited in drug treated group compared with normal group (P＜0.01). Conclusions Serum from Penthorum chinense Pursh extractum treated rats can significantly decrease TGF-β1 and collagen I secretions of activated hepatic stellate cells, which provides the experimental evidence for liver fibrosis treatment.

Objective To investigate the characteristics of renal hemodynamic and the esoteric prostacyclin(PGI2),thromboxane A2(TXA2)level in children with early Henoch-Schonlein purpura(HSP),and study the function of TXB_2/6-Keto-prostaglandin F(6-Keto-PGF_(1?))(T/K)numerus in early changes of kidney injury.Methods Children involved in the experiment were dicided into 3 groups.Thirty-one patients with HSP,divided into 2 groups according to routine urianlysis:children with HSP without renal damage group(n=16)and Henoch-Schonlein purpura nephritis(HSPN)group(n=15).Control group with 16 healthy children,their age and sex match with the other 2 groups.The urine of all children,including the children in control group,was sampled in 24 hours.The urinary production of the samples were kept in the freezer at-20 ℃.The radioimmunoassay was applied to determine the 6-Keto-PGF_(1?),TXB_2 quantitatively,and calculate the number of T/K.In the early morning the children accept the Doppler arteria renalis sonography with an empty stomach to determine the Vmax of the period of contraction of the arteria renalis the Vmin of diastolic phase and the resistent index(RI).SPSS 13.0 software was used to analyze the data.Results 1.The renal hemodynamic indicated a change of high velocity and resistance,the masculine rate(83.9%)was ob-viously higher than that in routine urinalysis(48.4%)(?2=5.79 P0.05).The RI in the former group(0.798?0.165)was much higher than that in the other one(0.637?0.116)(t=4.02 P

Objective To construct a recombinant adenoviral vector carrying HBcAg-HSP70 chimeric gene by homologous recombination in bacteria and to detect its expression in vitro. Methods Heat shock protein 70 gene from Mycobacterium tuberculosis were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV-HBsAg. Then the resultant pAdTrack-CMV-HBsAg-HSP70 was eotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBsAg-HSP70 gene (pAd-HBsAg-HSP70) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells. Several kinds of mammal cells (293 cells and Vero cells) were infected with adenoviruses and the expression of HBsAg-HSP70 was detected by RT-PCR and ELISA in vitro. Results The adenoviral plasmids pAd-HBsAg-HSP70 were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease Pac Ⅰ analyses. The recombinant adenoviruses Ad-HBsAg-HSP70 were packaged successfully in 293 cells. The titer of Ad-HBsAg-HSP70 was up to 2 × 1012 pfu/L after the second passage of proliferation in 293 cells. HBsAg and HSP70 were expressed efficiently in mammal cells after infection. Conclusion The recombinant adenoviruses expressing HBsAg and HSP70 were constructed successfully which can be used further in study of gene therapy for HBV.

OBJECTIVETo study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China.

METHOD30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites.

RESULTSWe successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24).

CONCLUSIONThe last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.

Adult ; China ; epidemiology ; Computational Biology ; DNA, Viral ; genetics ; Epitopes, T-Lymphocyte ; immunology ; Female ; Genotype ; HLA-A Antigens ; immunology ; Hepatitis B Core Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; immunology ; Hepatitis B, Chronic ; epidemiology ; immunology ; virology ; Humans ; Male ; Mutation ; Sequence Analysis, DNA ; T-Lymphocytes, Cytotoxic ; immunology