The genetic analysis of herpesviruses has been a constant challenge, due to the large, complex genomes of herpesviruses and mutagenesis of viral genes by conventional recombination methods in cell culture. Recently, a completely new approach for full-length infectious clones of herpesviruses based on bacterial artificial chromosomes (BACs) has been developed. This technique allows the maintenance, propagation and genetic modification of the viral genome as a BAC plasmid in E.coli, thus making the procedures fast, safe and effective in prokaryotic cells. This technique also makes it possible for the reconstitution of viral progeny or mutants by transfection of the BAC plasmid into eukaryotic cells, thereby facilitating the analysis of viral gene functions in the context of genome. In this presentation, Epstein-Barr virus was used as an example to describe the principle, establishment of the technique and mutation introduction into the BAC plasmid, and to discuss the perspective in the use of BAC-cloned herpesviruses.

To investigate the impact of exercise on the expression of adiponectin and GLUT4 mR NA in type 2 diabetic rats, type 2 diabetic rat model was made. The diabetic rats were treated with swimming training for 8 weeks. The expression of adiponectin mRNA in perirenal fat and GLUT4mRNA in skeletal muscles were assessed by reverse transcription polymerase chain reaction (RT PCR) and the levels of blood glucose, serum insulin, and blood lipid were measured. Our results showed that the expression of adiponectin mRNA and GLUT4 mRNA in diabetic model group was decreased by 45 % (P＜0.01), 43 % (P＜0.01) respectively. The gene expression of adiponectin and GLUT4 was increased significantly in swimming group (P＜0.05 and P＜0.01, respectively).Compared with the model group, fasting insulin, TG, TC and FFA were decreased significantly in the training group (P＜0.05 or P＜0.01) as compared with model group. It is concluded that exercise can promote the expression of adiponectin mRNA and GLUT4 mRNA in type 2 diabetic rats,which may be one of the mechanisms responsible for the amelioration of insulin resistance in the rats.

This study was aimed to investigate the immunological effect of modified dendritic cells (DC) which inducing cytotoxic T cells (CTL) against lymphoma cells. The DC were isolated from the lymph node and peripheral blood of patients with diffuse large B cell lymphoma (DLBCL). DC were transfected with recombinant adenovirus vector carrying human p53 gene (rAd-p53-DC). The expression of p53 gene was detected by flow cytometry. Western-blot was used to detect the expression of P53. ELISA was used to detect IL-12 level in supernatant. The mixed lymphocyte reaction (MLR) was used to detect the proliferative ability of auto-lymphocyte stimulated by DC. The lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTL. The results indicates that the expressions of DC surface molecule (except for CD1a) such as CD83, CD80, CD86 and HLA-DR were significantly higher in experiment group than that in control group and blank control group. The secretion of IL-12 in supernatant was higher in experiment group than that in control group. The autologous T lymphocyte proliferation and cytotoxic activity against the same kind of DLBL-cells increased in experiment group as compared with control group and blank control group (P < 0.05). The ability to stimulate T lymphocyte proliferation increased with the rising of the ratio of DC and T lymphocyte. However, there was statistically significant difference between rAd-p53-DC derived from Lymph node and peripheral blood (P < 0.05). It is concluded that rAd-p53-transfected DC can induce CTL response in vitro against lymphoma cells.
Adenoviridae ; Cell Line, Tumor ; Dendritic Cells ; cytology ; immunology ; Genes, p53 ; Genetic Vectors ; Humans ; Lymphocyte Activation ; Lymphocyte Culture Test, Mixed ; Lymphoma, Large B-Cell, Diffuse ; blood ; immunology ; Transfection

Objective:To preliminarily verify the cross talk between tissue factor/active coagulation factor Ⅶ (TF/FⅦa) and epidermal growth factor receptor (EGFR) pathways in human colon cancer cells in culture.Methods:FⅦa was treated to HT-29 (KRAS-wild type) and LoVo (KRAS-mutant) colon cancer cells to activate TF/F Ⅶa pathway,qRT-PCR and Western blot were used to detect the expressions of amphiregulin (AREG) and epiregulin (EREG),ligands of EGFR on mRNA and protein levels,respectively.After knocking down expression of TF by TF-targeted siRNA transfection,FⅦa was treated and mRNA expressions of AREG and EREG were detected to see whether the FⅦa-induced effects were dependent on TF.Expressions of mRNA of TF and FⅦwere detected by qRT-PCR following the activation of EGFR pathway by treatment with epidermal growth factor (EGF) to HT-29 and LoVo cells.Results:After TF/FⅦa pathway was activated,for HT-29 cells,expressions of AREG (on mRNA level) and EREG (both on mRNA and protein level) were significantly down-regulated versus those of control group,gene expressions of AREG and EREG were 0.55 ± 0.09 vs.0.99 ± 0.09,0.67 ± 0.10 vs.1.02 ± 0.02,protein expressions of EREG were 0.54 ± 0.09 vs.1.04 ± 0.13,all P ＜ 0.05.For LoVo cells,expressions of AREG (both on mRNA and protein level) and EREG (on protein level) were significantly up-regulated versus those of control group,gene expression of AREG were 1.87 ± 0.39 vs.0.93 ± 0.23,protein expressions of AREG and EREG were 3.09 ±0.73 vs.1.11 ±0.21,1.53 ±0.19 vs.0.97 ± 0.23,all P ＜0.05.The regulating effect of AREG and EREG mRNA expression by FⅦa in HT-29 and LoVo cells could both be partly blocked by knocking down TF expression.For HT-29 cells,activation of EGFR pathway induced no significant TF mRNA expression,F Ⅶ mRNA expression was not detected.However,for LoVo cells,activation of EGFR pathway induced significantly higher mRNA expressions of both TF and FⅦ,expressions were 1.53 ± 0.23 vs.1.00 ± 0.23,53.20 ± 6.08 vs.1.00 ± 0.15,all P ＜0.05.Conclusion:In colon cancer cell LoVo,when activated,TF/FⅦa pathway and EGFR pathway could interact through upregulating the other pathway's effectors,and mutant KRAS might play a critical role in the two pathways'cross talk.

Adult ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Odontoid Process ; injuries ; Perioperative Nursing ; Postoperative Care ; Spinal Fractures ; surgery

The aim of this study is to investigate the effect of monoammonium glycyrrhizinate (MAG) on lipopolysaccharide (LPS) -induced acute lung injury (ALI) and its anti-inflammatory mechanism in mice. All male ICR mice were randomly divided into six groups: LPS group; control group; MAG 3, 10, and 30 mg x kg(-1) groups; and dexamethasone (DXM) 5 mg x kg(-1) group. Lung dry weight and wet weight percentage and permeability were detected. Neutrophil infiltration in bronchoalveolar lavage fluid (BALF) and lung tissues was detected by cell count and morphological analysis. The levels of TNF-alpha and IL-10 in lung were detected by ELISA. MPO activity was determined followed the specification. MAG induced a decrease in lung wet weight/dry weight ratio, and significantly decreased in total leucocyte number and neutrophil percentage in the BALF, and MPO activity of lung in a dose-dependent manner. Importantly, It could up-regulate the IL-10 level and down-regulate the TNF-alpha level in the lung tissue of ALI mice. These results suggested that the protective effect of MAG in mice on LPS induced ALI was associated with the regulation of TNF-alpha/IL-10 balance, and MAG maybe a potentially treatment for ALI/ARDS.
Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Bronchoalveolar Lavage Fluid ; cytology ; Glycyrrhizic Acid ; pharmacology ; Interleukin-10 ; metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung ; pathology ; Male ; Mice ; Mice, Inbred ICR ; Neutrophils ; pathology ; Organ Size ; drug effects ; Peroxidase ; metabolism ; Protective Agents ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

Objective To explore the relationship between HBV infection and the genotypes and allele frequencies of CⅡTA G-944C gene polymorphism in three minority populations(Jinuo,Dai and Aini population)in Xishuangbanna district,Yunnan province.Methods Polymerase chain reaction and sequencing method were used to study the genotypes and allele frequencies distributions of CⅡTA G-944C gene polymorphism in those three populations.Relationship between the genotypes distribution and HBV infection results were also analyzed.Results The rates on HBV infection and HBsAg carrier status in Aini minority population were 89.2% and 16.3%,which were significantly higher than in Jinuo(27.9% and 3.9%,χ~2=135.196 and 10.361,P=0.000 and 0.001)and Dai population(44.9% and 6.6% χ~2=96.783 and 8.748,P=0.000 and 0.003)while among Aini population it was significantly different with the other two minority populations.The CC genotype and C allele frequencies were more distributed in Aini population than in the other two minority populations.In contrast,the GG genotype and G allele frequencies were lower than the other two minority populations,with χ~2 rates between Aini and Jinuo population were 11.841 and 12.208 and the P as 0.003 and 0.000 respectively while the χ~2 rates between Ami and Dai population were 23.902 and 20.220 with P value as 0.000 and 0.000.The genotypes frequencies of CⅡTA G-944C was significantly different in the infected individuals(IF)group and health control(HC)group in Jinuo population(χ~2=6.150 and 4.911,P=0.046 and 0.027).When compared with HBsAg+ group and HBsAg~- group,the genotypes and allele frequencies were different in Aini population and the total three minority populations(χ~2 rates in Jinuo minority were 8.650 and 5.034 with P values as 0.013 and 0.025).However,the χ~2 rates in the whole population were 13.047 and 9.416 with P values as 0.001 and 0.002,respectively.The distribution of CC genotype and C allele gene in HBsAg~+ group was increasing.Data from non-condition logistic regression analysis and adjusting for confounding factors,the HBsAg~+ group had a significantly increase of HBsAg~- group under the C allele Recessive Model(P=0.000;OR=2.964;95% CI:1.609-5.460).Conclusion The genotypes and allele frequencies distribution of CⅡTA G-944C were different in the three ethnic populations.Polymorphism of this gene was closely associated with HBsAg carrier.The CC genotype patients were more easily to become HBsAg carrier.

Objective To explore the relationship between gray matter heterotopia and epilepsy and assess the therapeutic effect of surgical intervention. Methods Six cases of gray matter heterotopia-induced epilepsy treated in our department between May, 2004 and May, 2006 were analyzed retrospectively for the clinical characteristics, surgical approaches, and the outcomes in the 2- to 4-year-long follow-up. Results All the patients received surgical interventions through different approaches, including resection of the heterotopic gray matter plus bipolar coagulation of the cortexes in 4 cases, heterotopic gray matter resection with bipolar coagulation of the cortexes and temporal lobectomy in 1 case, and exclusive bipolar coagulation of the cortexes in 1 case. Five patients were free of seizure attach and 1 patient showed significantly reduced seizure attack after the operation. Conclusion Surgical intervention can be effective for treatment of intractable epilepsy induced by gray matter heterotopia.

OBJECTIVE: To explore the effect of LRRC4 on the mobility and invasion of glioblastomas U251 cells through the SDF-1alpha/CXCR4 axis. METHODS: RT-PCR, transfilter cell invasion assay, adhesion assay, scraping test, scrape loading, and dye transfer assay were used to determine the effect of LRRC4 on U251 cells. RESULTS: SDF-1 alpha could increase the invasion in U251 which expressed CXCR4. The reintroduction of LRRC4 in U251 cells could inhibit the expression of CXCR4. LRRC4 also inhibited the adhesion ability of U251 to ECV304 as well as the mobility and invasion ability in vitro, which was mediated by the SDF-1alpha/CXCR4 axis. Furthermore, LRRC4 could greatly enhance the gap junctional intercellular communication of U251 cells. CONCLUSION The reintroduction of LRRC4 in U251 cells can inhibit the expression of CXCR4 and the SDF-1alpha/CXCR4 axis-mediated cell invasion in vitro.
Cell Adhesion ; Cell Line, Tumor ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Glioblastoma ; pathology ; Humans ; Neoplasm Invasiveness ; Nerve Tissue Proteins ; genetics ; Receptors, CXCR4 ; metabolism

OBJECTIVE: To examine the expression absence of LRRC4 gene in glioblastoma cell lines. METHODS: RT-PCR and Northern blot were used to detect the expression of LRRC4 gene in 6 glioblastomas cells lines. Polymerase chain reaction and DNA sequencing were used to screen the LRRC4 gene mutation, while bioinformation assay was used to search for the reason of LRRC4 gene absence in U251 cell lines. RESULTS: The expression of LRRC4 was absent in 6 malignant glioma cell lines (U251, U87, BT325, SF126, SF767 and M17), which were examined by Northern-blot and RT-PCR assay. All sequencing of PCR products from gDNA of SF126, SF767, and M17 cell lines contained the point mutation at the same position ( LRRC4 geneT977A) (3/5), which was a synonymous mutation. However, PCR products from gDNA of U251 and U87 cell lines (2/5) were not obtained. The expression absence of LRRC4 was ascribed to the loss of homozygosity of 7q32-ter in U251 cell lines. CONCLUSION The expression of LRRC4 gene is absent in glioblastoma cell lines, and it offers the important experiment proof for LRRC4 to act as a new candidate of brain tumor suppressor gene from glioma. The loss of homozygosity of 7q32-ter contributed to the expression absence of LRRC4 in U251 cell lines.
Base Sequence ; Blotting, Northern ; Brain Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; DNA Mutational Analysis ; Gene Expression Regulation, Neoplastic ; Glioblastoma ; genetics ; pathology ; Humans ; Nerve Tissue Proteins ; genetics ; Point Mutation ; Reverse Transcriptase Polymerase Chain Reaction