Objective To construct RNAi combinant adenoviral expressive vectors specific to p65 subunit of NF-?B and to observe their gene silencing effect on p65 subunit.Methods Three pairs of complementary. single-strand DNA oligos targeting three various sites of p65 mRNA were designed and synthesized.Annealling was used to generate double-strand oligos(ds-oligos),and then the ds-oligos were cloned into pENTR~TM/u6 to generate the entry clone named pENTR.Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT~TM- DEST was used to creat the adenovirus plasmid which contains the RNAi cassette.Then,the adenovirus plasmids digested with PacI were transfected into HEK293A cells to product adenovirus,and latter infected the HEK293A cells to amplify the adenoviral stock.Plaque forming assay was used to titer the adenoviral stock.The p65 gene silencing effect induced by the RNAi adenovirus was detected by Western blot and immunocytochemistry assay in ECV304 cells.Results The RNAi adenovirus specific to p65 subunit of NF-?B were produced with titer of 3.0 x 10~9pfu/ml to 2.5?10~10pfu/ml.The expression of p65 protein in ECV304 cells could be down-regulated efficiently by the RNAi adenovirus 48-72 h after infection,which would last for more than 6 days after infection.Conclusion RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently.

OBJECTIVETo screen the carcinogenesis associated genes in gastric carcinoma by gene chip.

METHODSU133A (Affymetrix Santa Clara, CA) gene chip was used to detect differentially expressed genes in tumor tissues, paratumor mucosa and normal mucosa. Bioinformatics was used to analyze the screened results.

RESULTSA total of 150 genes were detected with a difference of expression levels more than 3 times in paratumor mucosa compared with normal gastric mucosa, 130 of which were up-regulated and 20 down-regulated. According to the function classifications of the differentially expressed genes, the most common ones were enzyme and enzyme regulon activity associated genes(28, 18.7% ). The frequencies of nuclei acid binding activity associated genes,signal transduction associated genes and protein binding associated genes were 11.3%, 10%, and 8.7% respectively. Seventy-one differentially expressed genes were detected both in tumor tissues and paratumor mucosa compared with normal mucosa, 61 of which were up-regulated and 10 down-regulated. Among these 71 genes,e leven genes were localized on chromosome 19, 6 on chromosome 1, 2, 16, 17 respectively. No abnormal differentially expressed gene were detected on chromosome 5, 14, 22 and Y.

CONCLUSIONSThese 71 genes differentially expressed both in tumor tissues and paratumor mucosa may be associated with carcinogenesis of gastric carcinoma. The four kinds of genes associated with enzyme and enzyme regulon activity, nuclei acid binding activity, signal transduction, and protein binding should be the main genes for the study of carcinogenesis in gastric carcinoma.

Gastric Mucosa ; pathology ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Oligonucleotide Array Sequence Analysis ; Stomach Neoplasms ; genetics ; pathology

OBJECTIVETo study the difference of gene expression profiles in gastric cancer (T), pericancerous mucosa (P) and the gastric mucosa from distant cutting margin (C), and to screen an associated novel gene in early gastric carcinogenesis by oligonucleotide microarray.

METHODSU133A (Affymetrix, Santa Clara, CA) gene chip was used to detect the gene expression profile difference in T, P and C, respectively. Bioinformatics was used to analyze the detected results.

RESULTSWhen gastric cancer was compared with normal gastric mucosa, 766 genes were found,with a difference of more than four times in expression levels, including 530 up-regulated [Signal Log Ratio (SLR) > 2], and 236 down-regulated (SLR< -2). When P was compared with C, 64 genes were found, with a difference of more than four times in expression levels, including 50 up-regulated (SLR > 2), and 14 down-regulated (SLR< -2). Compared with C, a total of 143 genes with a difference of more than four times in expression levels both in T and P tissues. Of the 143 genes, 108 were up-regulated (SLR > 2), and 35 were down-regulated (SLR< -2).

CONCLUSIONSGene chip can reveal 143 same genes both in pericancerous mucosa and gastric mucosa. These genes may be related to the carcinogenesis and development of early gastric cancer.

Female ; Gastric Mucosa ; pathology ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; methods ; Precancerous Conditions ; Stomach Neoplasms ; genetics ; pathology

OBJECTIVETo investigate the expression of 5-hydroxytamine receptors in hepatic stellate cells HSCs and action of 5-hydroxytamine on biological characteristics of HSC.

METHODSLiver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate hepatic stellate cell. RT-PCR was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. HSCs were cultured on silicone membrane. The effect of 5-hydroxytamine, ketanserin and ondanosetron on cell contraction were studied.

RESULTSHSC expressed 5-hydroxytamine receptors subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P < 0.05). This was antagonized by ketanserin, not by ondanosetron. 5-hydroxytamine induced cell contraction in a dose-dependant manner. Ketanserin antagonized this action, but ondanosetron did not.

CONCLUSIONSHSCs express 5-hydroxytamine receptors. 5-hydroxytamine could affect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.

Animals ; Cells, Cultured ; Hypertension, Portal ; etiology ; Liver ; chemistry ; cytology ; Liver Cirrhosis ; etiology ; Male ; Rats ; Rats, Wistar ; Receptors, Serotonin ; analysis ; physiology ; Serotonin ; pharmacology ; Serotonin Antagonists ; pharmacology ; Transforming Growth Factor beta ; physiology ; Transforming Growth Factor beta1

Objective To analyze the feasibility of local LINGO-1 polyclonal antibody administration for treatment of spinal cord injury in adult rats. Methods Twenty-four adult female SD rats were randomized into sham-operated group, rabbit IgG group and LINGO-1 antibody group. In the latter two groups, partial transaction of the T9 segment of the spinal cord was performed to completely sever the dorsal eorticospinal tract, followed immediately by administration of rabbit IgG and anti-LINGO polyclonal antibody via a mini-osmotic pump, respectively. At 3 and 28 days after the operation, the T8～10 segments of the spinal cord were harvested to prepare cryosections, and immunofluorescence staining was used to analyze the penetration of LINGO-1 polyclonal antibody into the spinal cord tissue and its specific binding to LINGO-1 molecules. Results In LINGO-1 antibody group, the presence of rabbit antibodies was detected at the injured sites of the spinal cord at 3 and 28 days after the operation. The mean immunofluorescence density was significantly lower in L1NGO-1 antibody group than in rabbit IgG group at 3 days after the operation (P<0.05). In rabbit IgG group, the mean immunofluorescence density for LINGO-1 in the crysections pre-treated with LINGO-1 polyclonal antibody was significantly lower than that in sections pre-treated with rabbit IgG(P<0.05). Conclusion Locally administered LINGO-1 polyclonal antibody can penetrate into the injured sites in the spinal cord in a wide time window and recognizes LINGO-1 molecule specifically, suggesting the feasibility of passive immunotherapy for spinal cord injury.

Objective To detect the immunogenicity of the recombinant DNA vaccine that encoded for neurite growth inhibitors: Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), tenascin-R (TN-R) and myelin-associated glycoprotein (MAG) after the nerve injury under the help of pAdEasy, a kind of adenovirus plasmid being the vector of the DNA. Methods Sixteen 5-w-old Lewis rats were randomized into DNA vaccination group (vaccine group) and pAdEasy group. Rats in the vaccine group were immunized once weekly for a consecutive 8 w by bilateral injection of the recombinant plasmid into the musculus tibialis. The immunized animals in the 2 groups were exsanguinated each time before the vaccination for sera collection, and the qualitation and quantitation of the antibodies in the serum were detected by Dot-blot analysis and ELISA. Results The vaccine group could produce fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R at the 6th w of vaccine injection, while pAdEasy group could not. The valency of antiserum was shown by ELISA as 1:1 000 000 at the 6th w of vaccine injection and kept this level stably. Conclusion The DNA vaccine exclusively induces the generation of the fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R in vivo, which controls the favorable immunogenicity.

Objective To explore whether aggressive treatment of primary focus can benefit nonsmall cell lung cancer(NSCLC)patients with synchronous brain metastasis,and search the appropriate treatment protocols.Methods The clinical data of 19 NSCLC patients with synchronous brain metastasis,received treatment at our Cancer Center from January 2000 to January 2009,were reviewed; their treatments and survival statuses were analyzed.Results Median survival time of these patients was 14.5 months; the 1-year survival rate was 52.6％,and 2-year survival rate was 17.5％.Patients had different survival rates when different treatments were given to the primary focus,and significant difference was noted(x2=10.532,P=0.005); after neurosurgical intervention,patients underwent thoracic operation and chemotherapy(24.9 months)had a significantly longer survival time than those underwent chemotherapy(14.5 months)or palliative therapy(5.4 months,P＜0.05).The survival time of patients with single metastases was 16.3 months,and that of those with multiple metastases was 5.4 months; and significant difference was noted between them(P＜0.05).Conclusion Aggressive therapy including neurosurgical intervention,pulmonary resection and chemotherapy should be recommended for NSCLC patients with synchronous brain metastasis,especially those with single brain metastasis.

ObjectiveTo explore the carcinogenic mechanism of Campylobacter jejuni. MethodsEighteen female C57BL/6 Apc^Min/+ mice were randomly divided into the C. jejuni-infected group and the non-infection control group， each group with nine mice. Colorectal cancer of Apc^Min/+ mice was induced by dextran sulfate sodium and gavage of C. jejuni （or PBS as a control）. At the end of the experiment， the number of tumors in colorectal tissues of mice in each group was counted， and RNA was extracted from colorectal tumors， along with para-cancer tissues as controls. Transcriptome sequencing was performed by RNA-Seq technology， and data were analyzed for differentially expressed genes （DEGs）. Further， selected DEGs were subjected to GO （gene ontology） enrichment analysis and KEGG pathway enrichment analysis. ResultsCompared with that of the non-infection control group， the incidence of tumor in C. jejuni-infected group was significantly higher （P < 0.01）， which indicated the success of recreating the C. jejuni-induced CRC model. RNA-seq results showed that there were 394 and 501 DEGs （fold change > 4 and P < 0.05） in the C. jejuni-infected group compared with the two control groups， respectively. In GO enrichment analysis， DEGs were mainly enriched in immune response regulation and activation pathways， multiple protein transport pathways and receptor binding pathways. Cancer-related pathways and metabolic pathways were significant enriched in KEGG pathway enrichment analysis. Among these DEGs， 17 genes were found in comparisons with both control groups. The 17 genes were further selected， resulting in 14 “core” DEGs. In further validation of qRT-PCR， 9 genes were significantly differentially expressed， among which 3 genes were up-regulated （Gm1987， Saxo1 and Plekhs1） and 6 were down-regulated （Lrp2， Serpina3c， Fabp4， Tmem52， Lrrn4 and Upk3b）. ConclusionThis study emphasizes 9 host genes that may play important and unique roles in the occurrence and development of colorectal cancer induced by C. jejuni， which provides new insights for further studies on the carcinogenic mechanism of C. jejuni.

Objective To investigate the photodynamic effect mediated with 5-aminolevulinic acid (5-ALA) on U251 human glioma cells. Methods Fluorescence microscope and confocal laser scanning microscope were used to detect the localization of Pp Ⅸ in U251 human glioma cells. The cells with/without 5-ALA were irradiated at the wavelength of 635 nm. MTT assay was used to measure the cell survival after laser irradiation. Results 5-ALA cocultured with U251 cells successfully produced endogenous Pp Ⅸthat was observed distributively in the cytoplasm, but not in nuclear region. The overall survival rates of the U251 glioma cells photodamaged by ALA-PDT decreased as the incubation time went by or the 5-ALA concentration increased, while peaked at the incubation time of 6 h and the 5-ALA concentration of 2.0mmol/L. Without one of 5-ALA and light irradiation, the survival rate of the cells had no significant difference compared with that of cells of the control group. Conclusion The 5-ALA-induced PDT appears to be a promising therapy for human glioma. The optimal incubation time may be 6 h and the optimal 5-ALA concentration be 2.0 mmol/L.

OBJECTIVETo evaluate the techniques and clinical applications of 16 multislice helical CT in colonic lesions.

METHODSEighty-one patients including 54 colorectal carcinomas, 5 adenomas, 1 non-Hodgkin's lymphoma, 6 inflammatory bowel diseases, and other 15 cases underwent volume scanning using 16 multislice helical CT. Four types of reconstruction included multiple planar reconstruction, shaded surface display, raysum, and CT virtual colonoscopy.

RESULTSComplete colon could be shown in all patients. The lesions' morphology, number, size, location, intestinal cavity, pericolonic changes, and other abdominal organs were satisfactorily shown by CT.

CONCLUSIONSSixteen multislice helical CT colonography is a valuable imaging technique for detecting colonic diseases. It is effective in diagnosis and treatment planning. It can display the portions of colon that is inaccessible at colonoscopy.

Adenocarcinoma ; diagnostic imaging ; Adenoma ; diagnostic imaging ; Adult ; Aged ; Aged, 80 and over ; Colonography, Computed Tomographic ; methods ; Colonoscopy ; Colorectal Neoplasms ; diagnostic imaging ; Female ; Humans ; Inflammatory Bowel Diseases ; diagnostic imaging ; Male ; Middle Aged ; Tomography, Spiral Computed ; methods