Adult ; Budd-Chiari Syndrome ; complications ; Humans ; Hypereosinophilic Syndrome ; complications ; Male

The objective of the present study was to establish a method based on principal component analysis (PCA) for the study of transdermal delivery of Chinese medicinal formulae, and to choose the best penetration enhancers for Qingfei Xiaocuo gel depend on this method. Using improved Franz type diffusion cell and excised rat skin in vitro as transdermal barrier, the receptive solution fingerprint was established by HPLC, harvesting the areas of the common peaks in the fingerprint, then the total factor scores of the concentrations at different times were calculated using PCA and were employed instead of the concentrations to compute the cumulative amounts (Q12) and enhancement ratio (ER), the latter of which were considered as the indexes for optimizing penetration enhancers. Compare to the control group, the ER of the other groups increased significantly and furthermore, 2.5% azone with 2.5% menthol manifested the best effect. PCA represent most information in the receptive solution, the method above could choose the best penetration enhancers, it could be a reference for the study of transdermal delivery of Chinese medicinal formulae.
Administration, Cutaneous ; Animals ; Drugs, Chinese Herbal ; analysis ; pharmacokinetics ; Gels ; In Vitro Techniques ; Male ; Medicine, Chinese Traditional ; Mice ; Principal Component Analysis ; Skin ; metabolism

Autoantibodies ; blood ; Autoimmune Diseases ; complications ; Hepatitis C, Chronic ; complications ; immunology ; Humans

Objective To analyze the result of treatment for acute-on-chronic liver failure patients with fast high efficiency Nucleoside and to explore the relations among inhibition to virus replication , liver failure development and immune rejection .Methods Sixty-two cases of acute-on-chronic liver failure pa-tients with HBV DNA(+) were divided into study group (treated with a kind of fast and nucleoside , n =30) and control group( n =32).HBV DNA,CD4 +T,CD8 +T, C3,C4 TBIL,PTA were observed at treat-ment 0w,2w and 4w.Results All of the study and control group patients , serum HBV DNA were positive before treated.And the levels of CD4+,CD8 +,C3,C 4,TBIL,PTA of study group were not significantly compared with control group .At treatment 2w , the rate of HBV DNA diverted negative in study group 90.0%(27/30), was significantly more then control group (9.4%, 3/32)(χ2=37.14 , P <0.01).But the CD4 +,CD8 +,C3,C4,TBIL,PTA levels were not significantly however between study and control group . At treatment 4w ,the rate of HBV DNA diverted negative in study group (96.7%, 29/30), was significant-ly more then control group(12.5%,4/32) (χ2 =40.74, P <0.01).CD4 +, CD8 +,C3,C4,TBIL,PTA levels of the study group were significantly more compared with the control group .The CD4 +level of study group (495.33 ±89.91)cells/ml, was higher significantly then control group (270.34 ±97.74)cells/ml( t=9.42, P <0.01),the CD8 +level (571.03 ±120.15 ) cells/ml, was higher significantly then control group(224.88 ±79.68)cells/ml( t =13.45, P <0.01).The C3 level of the study group (0.28 ±0.11) g/L, was lower significantly then control group ( 0.68 ±0.13 ) g/L ( t =13.13 , P <0.01 ) , the C4 level (0.12 ±0.06)g/L, was lower significantly then control group (0.23 ±0.10)g/L( t =4.92, P <0.01). The TBIL level of study group ( 653.93 ±131.02 )μmol/L, was higher significantly then control group (285.63 ±154.63)μmol/L( t =10.09, P <0.01),the PTA level (17.13 ±7.07)%, was lower signifi-cantly then control group(50.94 ±13.68)%( t =12.10, P <0.01).The death rate of the study group( 57.9%) was higher significantly compared with the control group (28.1%)(χ2 =6.39, P <0.05).Con-clusion Treatment of chronic severe hepatitis with fast and high efficiency nucleoside may arise the T cell subset level and make the immune rejection strength , as a result the liver failure maybe far away from cure .

OBJECTIVETo explore the mutated KLF6 gene in hepatocellular carcinoma (HCC) and to characterize its behavior in human hepatocellular carcinoma cell line HepG2.

METHODSWe analyzed the DNA isolated from 23 hepatocellular carcinoma tissues and their adjacent nontumor tissues by polymerase chain reaction (PCR). Direct sequencing was used to establish the incidence of mutation in exon2 of the KLF6 gene. Loss of growth suppressive function of the HCC-derived KLF6 mutants was characterized by in vitro analyzing alteration of cell cycle and MTT assay. Expression of p21WAF1, a possible downstream gene of KLF6, was detected in human hepatocellular carcinoma cell line HepG2 transiently transfected with KLF6 genes.

RESULTSMutations of KLF6 were found in 2 of the 23 (8.7%) hepatocellular carcinomas. The two mutations were located in the transactivation domain and one of them resulted in single amino acid substitution of TGG (W) by GGG (G) at codon 162. Unlike the wild-type KLF6, cancer-derived KLF6 mutants neither suppressed growth nor induced p21WAF1 following transfection into culture cells.

CONCLUSIONSMutations of the KLF6 gene may play a role in the pathogenesis of HCC, but are not the dominating mechanism resulting in inactivation of KLF6 functions. KLF6 suppresses hepatocellular carcinoma cell proliferation partly through upregulating expression of the p21WAF1 gene.

Base Sequence ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; physiology ; Liver Neoplasms ; genetics ; pathology ; Molecular Sequence Data ; Point Mutation ; Proto-Oncogene Proteins ; genetics ; physiology ; Sequence Analysis, DNA

OBJECTIVETo investigate the effect of telbivudine on peripheral blood regulatory T cells and its significance in patients with chronic hepatitis B (CHB).

METHODSThirty-six HbeAg positive chronic hepatitis B patients were recruited and received telbivudine treatment for 9 months. Before and during the 3, 6, 9 months of treatment, flow cytometry was used to detect the proportion of peripheral blood Tregs; real-time PCR was used to detect the levels of HBV DNA in the serum. Markers of hepatitis B virus infection were detected by ELISA assay and levels of alanine aminotransferase in the serum were measured.

RESULTSThe proportion of peripheral blood Tregs in patients with CHB was significantly higher than that in healthy controls and decreased over 6 or 9 months of telbivudine treatment to a level comparable to that of the healthy controls. After 3 months of telbivudine treatment, the rate of undetectable HBV DNA in patients whose proportion of peripheral blood Tregs was decreased was higher than those whose Tregs had been reduced, but the difference was not statistically significant (P more than 0.05). Three, 6 or 9 months of telbivudine treatment resulted in HbeAg negativity in 4 (11.1%) patients, 7 (19.4%) patients or 9 (25.0%) patients respectively. In 7 (19.4%) patients who had seroconversion from HBeAg to anti-HBeAg, after 3 or 6 months of telbivudine treatment, their proportion of peripheral blood Tregs had decreased to a level comparable to that of the healthy controls.

CONCLUSIONTelbivudine treatment reduces HBV replication and the proportion of peripheral blood Tregs. In addition, patients who have their proportion of peripheral blood Tregs decreased quickly at the early phase of telbivudine treatment are prone to have HBeAg to anti-HBeAg seroconversion.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Case-Control Studies ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Interleukin-2 Receptor alpha Subunit ; Middle Aged ; Nucleosides ; therapeutic use ; Pyrimidinones ; therapeutic use ; T-Lymphocytes, Regulatory ; drug effects ; Thymidine ; analogs & derivatives ; Young Adult

OBJECTIVETo prepare the genetic engineering regulatory T cells (Treg) via the self-inactivating (SIN) lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression.

METHODSThe bicistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected using liposome by lentiviral packing system, which included the packaging plasmid Delta NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4(+)CD25(-) T cells, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry (FCM). The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit-8 (CCK-8) and the cytokine production was performed by ELISA.

RESULTSThe lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10(6) IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocultured, the CD4(+)CD25(-) T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3 epsilon and APCs, the proliferative capacity of Foxp3-transduced T cells and the production of IL-2, IL-4, IL-10, IFN-gamma were significantly lower than those in control group (P < 0.01); Foxp3-transduced T cells also significantly inhibited the proliferation of CD4(+)CD25(-) T cells.

CONCLUSIONSThe genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4(+)CD25(+) Treg.

Animals ; Cell Proliferation ; Cells, Cultured ; Forkhead Transcription Factors ; genetics ; metabolism ; Genetic Engineering ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; Phenotype ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism ; Transfection

OBJECTIVETo construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.

METHODSBDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.

RESULTSThe infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.

CONCLUSIONThe recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.

Cell Line ; Factor VIII ; biosynthesis ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Transfection

OBJECTIVETo rapidly and sensitively detect the four virulence-associated factors of Streptococcus suis, a multiplex PCR was developed.

METHODSIn the process of this reaction, four distinct DNA targets were amplified. One target was based on the serotype 2 (and 1/2) specific cps gene and the others were based on Streptococcus suis mrp, epf (epf*) and sly gene, encoding the MRP, EF(EF*) and Sly proteins of Streptococcus suis. 72 isolates, which including 48 strains of Streptococcus suis and 24 strains of negative control, and 49 clinical specimens were detected by the multiplex PCR assay.

RESULTSAll PCR products were detected by electrophoresis on 1.2% agarose gels. With the 48 Streptococcus suis strains, the positive detection rates of cps2+, mrp+, epf+, epf*+ and sly+ were 16/48, 14/48, 12/48, 3/48 and 26/48,respectively. The results were confirmed by bacteriological examination. There were no specific amplification products including 49 clinical specimens and 24 negative control strains.

CONCLUSIONThe results demonstrated that multiplex PCR was a highly specific and sensitive diagnostic tool for the detection of virulence-associated factors of streptococcus suis.

Bacterial Proteins ; genetics ; Polymerase Chain Reaction ; methods ; Streptococcus suis ; genetics ; pathogenicity ; Virulence Factors ; genetics

OBJECTIVETo screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).

METHODSBased on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.

RESULTCo-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.

CONCLUSIONThe expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.

Base Sequence ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Embryo, Mammalian ; Green Fluorescent Proteins ; genetics ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hepatitis B e Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; Humans ; Kidney ; cytology ; Liver Neoplasms ; pathology ; Molecular Sequence Data ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; RNA, Small Interfering ; RNA, Transfer, Val ; genetics ; RNA, Viral ; genetics ; Transfection