OBJECTIVETo establish human colorectal tissue model in HIV-1 mucosal infection and by using pseudotyped virus to simulate the biological process of HIV-1 mucosal infection from HIV-1 entering into mucosa to local infection establishment.

METHODSTumor adjacent normal colorectal tissues were obtained with informed consent. After excised the muscularis externa, the mucosa and submucosa were dissected into the same blocks and cultured in 12-well cell culture plates. The cultured tissue structure and morphology were observed from day 0 to day 13 by staining with the hematoxylin eosin (HE), and the tissue activity was detected by 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The established tissues explants were infected by a single cycle replicated pseudotyped virus and propagated for 6 - 7 days, then subjected to the detection of p24 production within supernatant to verify the applicability of the model for the studying of HIV-1 mucosal infection. The applicability of the established explants for safety and reactivity evaluation of mucosa topical drugs was conducted by the using of first generation antiseptic Nonoxynol-9 (N-9) as an example.

RESULTSHE staining showed the structure of colorectal tissue was remained well until 5(th) day and still evident until 13(th) day. The tissue activity of cultured mucosa was above 80% at day 4, and still remained over 50% at day 7 as detected by MTT assay. After infected by pseudo virus, the increased level of p24 was detected from supernatant collected on 1(st), 4(th), 8(th) day, which indicated a local infection was created. In addition, the dose changing of N-9 was reflected sensitively by the activity of this model.

CONCLUSIONEx vivo human colorectal tissue model mimic HIV-1 mucosal infection was established that can be used to replicate the bioprocess of human HIV-1 mucosal infection.

Colon ; pathology ; virology ; HIV Infections ; pathology ; virology ; HIV-1 ; Humans ; Intestinal Mucosa ; pathology ; virology ; Models, Biological ; Rectum ; pathology ; virology ; Tissue Culture Techniques ; methods ; Tumor Cells, Cultured

In order to explore the relationship between human papilloma virus ( HPV) and upper gastrointestinal cancer(esophageal cancer), An esophageal squamous cell carcinoma(ESCC) tissue was obtained from a 76 year old Chinese female patient from Anyang city, a high-incidence area for esophageal cancer, in China. Transplanted tumor was formed through direct SCID mouse tumorigenicity experiment and cultured monolayer cells were obtained after several passages and screenings Immunofluorescence test, cell growth curve, soft agar assay, chromosome analysis and tissues HE staining were also performed to confirm the epithelial cell origin. Cell DNA STR typing results showed that no three alleles was observed,indicating no contamination of human cells. DNA analysis revealed the presence of HPV type 18 DNA in this cell line. DOLINK test found the E6 protein expression of HPV virus. We concluded that the established cell line is a new esophageal squamous cell-origincarcinoma cell line with HPV DNA positive and expression of viral oncoprotein. It provides new cytologic material for performing etiology studies on the occurrence and development of esophageal cancer.
Aged ; Animals ; Carcinoma, Squamous Cell ; virology ; Cell Proliferation ; China ; Esophageal Neoplasms ; virology ; Female ; Human papillomavirus 18 ; genetics ; isolation & purification ; Humans ; Male ; Mice ; Mice, SCID ; Papillomavirus Infections ; virology ; Tumor Cells, Cultured ; cytology ; virology ; Tumor Virus Infections ; virology

OBJECTIVETo explore hepatitis C virus (HCV) non-structural protein 5A (NS5A)'s influence on inhibition of AFP expression executed by p53 protein and its possible molecular mechanism.

METHODSPlasmid transfection and MEIA were employed to observe p53's inhibitive effect on AFP expression of Huh7 cells and the HCV NS5A's influence on p53 function. Western blot was employed to find out if HCV NS5A affects p53 protein expression and GST pull down assay was applied to examine the interaction between HCV NS5A and p53.

RESULTSThe AFP concentration in the supernatant of the culture of the Huh7 cells transfected with pRc/CMV was (14322+/-2412) ng/ml, and that of the Huh7 cells transfected with pCNS5A was (13843+/-3218) ng/ml; no significant difference existed between these two groups (t = 1.42, P > 0.05). After transfection with pC53-NS3, the AFP level was decreased to (10 241+/-1326) ng/ml, and in comparison to the above two groups it had a statistically significant difference (t values were 2.41 and 2.38, P < 0.05). When co-transfected with pCNS5A and pC53-NS3, the AFP expression (14582+/-1238) ng/ml returned to the level of pRc/CMV transfected, and there was a remarkably significant difference between this and that of the pC53-NS3 transfected cells (t = 3.12, P < 0.01). HCV NS5A had no function on the p53 protein expression with Western blot experiment. In the GST pull down assay, an HCV NS5A protein band was found after GST-p53 was added, but not detected with GST only.

CONCLUSIONWe found that p53 has an inhibitive function on the AFP expression in Huh7 cells and HCV NS5A minimized this p53 function. HCV NS5A did not affect p53 protein expression, but was able to form a complex with p53, by which HCV NS5A inactivated this p53 function.

Carcinoma, Hepatocellular ; metabolism ; virology ; Hepacivirus ; genetics ; Humans ; Liver Neoplasms ; metabolism ; virology ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; genetics ; pharmacology ; Viral Nonstructural Proteins ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that targets HBV core gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro.

METHODSHepG2 2.2.15 was used as target cells. The plasmid and liposome metafectene were cotransfected into the cultured cells, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR.

RESULTSThe plasmid expressing siRNA was successfully constructed. The two constructed siRNAs could effectively inhibit HBV replication, and their inhibitive effect on HBV was dose-dependent.

CONCLUSIONThese results showed that siRNA could substantially inhibit HBV replication in the infected cells

Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; virology ; RNA Interference ; Tumor Cells, Cultured ; Virus Replication ; genetics

OBJECTIVETo identify the siRNA interference ability for the replication of HBV.

METHODSBased on the sequence of HBV in HepG2 2.2.15 cells in GenBank, one sequence targeting the C antigen of HBV was cloned into the RNA polymerase III based expression vector pSuper. This recombinant was electroporated into HepG2 2.2.15 cells and the expression of HBsAg and HBeAg was assayed using ELISA.

RESULTSThe construction of the recombinant expression vector pSuper-C and its control vector pSuper was successfully confirmed by the results of enzyme digestion, electrophoresis and sequencing. However, there was no difference between the expression of HBsAg and HBeAg in the supernatant of HepG2 2.2.15 cell culture in the experimental and control groups.

CONCLUSIONSThe constructed pSuper-C did not show an interfering effect on the replication of HBV in HepG2 2.2.15 cells. In order to display this effect, further study is needed

Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; Tumor Cells, Cultured ; Virus Replication ; genetics

OBJECTIVETo establish a method for efficient induction and expansion of Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) in vitro and evaluate the possibility of using this strategy for treatment of nasopharyngeal carcinoma (NPC).

METHODSEBV-transformed B lymphoblastoid cells (BLCLs) were used as the antigen stimuli and antigen-presenting cells. EBV-specific CTL was induced by co-culture of the autologous peripheral blood mononuclear cells (PBMCs) and the irradiated BLCLs, and expanded with a cocktail method consisting of OKT-3, irradiated homologous PBMC, and IL-2. The specific activity of the CTL against the NPC cells was measured with MTT assay.

RESULTSEBV-specific CTL was successfully induced and expanded by 600 folds. The killing efficiency of the CTL was 76% for autologous BLCLs, 13% for homologous BLCLs, 51% for autologous NPC cells, and 27% for homologous CNE cell line, and after expansion, the corresponding killing efficiencies were 63%, 25%, 49%, and 33%, respectively. The non-specific killing only slightly increased after the expansion.

CONCLUSIONEBV-specific CTL can be successfully induced and expanded in vitro for specific killing of autologous NPC cells, suggesting the potential of this strategy in the treatment of NPC.

Antigen-Presenting Cells ; cytology ; immunology ; Antigens, Viral ; immunology ; B-Lymphocytes ; cytology ; immunology ; virology ; Cells, Cultured ; Coculture Techniques ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; virology ; Tumor Cells, Cultured

Apoptosis ; drug effects ; Hepatocytes ; pathology ; virology ; Humans ; Liver Neoplasms ; pathology ; virology ; Mutation ; Plasmids ; genetics ; Trans-Activators ; genetics ; pharmacology ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo establish a method for efficient transfer of 1.3-fold HBV/C genome into HepG2 cell line using adenoviral vector system for studying the replication and antigen expression of HBV.

METHODSThe 1.3-copy overlength genome of HBV genotype C was constructed and cloned into the shuttle vector pAdTrack. After confirmation of the constructed HBV genome by sequencing, the resultant plasmid linearized by digestion with Pme I was transformed into competent E.coli Adeasier-1 cells. The recombinants of pAdEasy-HBV/C were linearized by digestion with Pac I and transfected into the packaging cells (293 cells) via liposome. HepG2 cells were then infected with a proper quantity of the recombinant adenoviruses. The HBV DNA level and HBeAg and HBsAg titers were detected in the cell medium.

RESULTSThe 1.3-fold overlength HBV/C genome was efficiently transferred into HepG2 cells via the adenoviral vector system, which resulted in initiation of the virus replication and protein expression in the cells using the viral replication mechanism.

CONCLUSIONThe adenovirus vector system (AdEasy) allows convenient and effective transfer of HBV genome into HepG2 cells, and provides a convenient means for screening therapeutic drugs against HBV.

Adenoviridae ; genetics ; metabolism ; Carcinoma, Hepatocellular ; genetics ; virology ; Genetic Vectors ; genetics ; Genome, Viral ; genetics ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; genetics ; virology ; Transduction, Genetic ; Tumor Cells, Cultured

OBJECTIVETo study the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma.

METHODSRecombinant plasmid of HCV-C gene constructed by molecular cloning technique was identified with restricting enzyme map. Then, it was transfected into QBC939 cells with lipofectin. After selection with G418, the resistant colonies were obtained and analysed by immunocytochemistry and Western blotting. Their morphology was observed by transmission electron microscopy (TEM). The expression of NF-kappa B was detected by immunocytochemistry.

RESULTSThe results suggested that the recombinant plasmid was proved to carry the target gene by restricting enzyme map. Moreover, it could express HCV-C protein efficiently in QBC939 cells. The HCV-like particles were found in the cytoplasm by TEM, which were spherical with diameter of 50-80 nm possessing an outer membrane. Moreover, NF-kappa B activation was shown in HCV core gene-transfected cells.

CONCLUSIONBecause HCV-C gene could express steadily in cholangiocarcinoma cells, the transfected tumor cells (QBC939-HCVc) are an experimental model for studying the effect of HCV on the development of cholangiocarcinoma. The activation of NF-kappa B may be related to escape from immune surveillance and carcinogenesis of cholangiocarcinoma.

Cholangiocarcinoma ; genetics ; virology ; Gene Expression ; Hepacivirus ; genetics ; Humans ; NF-kappa B ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; virology ; Viral Core Proteins ; genetics ; pharmacology

Carcinoma, Hepatocellular ; pathology ; virology ; Cisplatin ; pharmacology ; Drug Synergism ; Flavonoids ; pharmacology ; Humans ; Liver Neoplasms ; pathology ; virology ; Trans-Activators ; Tumor Cells, Cultured ; Viral Regulatory and Accessory Proteins ; drug effects