Objective To screen for Kaposi sarcoma (KS)-related genes. Methods Tissue samples were obtained from the lesion and normal skin of a patient with KS in Xinjiang Uygur Autonomous Region,and total RNA was extracted from these samples and reverse transcribed into cDNA. Real-time fluorescent quantitative reverse transcription PCR (RT-qPCR) was performed to determine the expression of K8.1, K2 and ORF50 in these samples. The cDNA was labeled with fluorescein and hybridized to a human 35K genome array containing 25 100 genes. Subsequently, the signal images were scanned by a laser scanner and acquired images were analyzed by software. Results RT-qPCR revealed the mRNA expression of K8.1, K2 and ORF50 in the KS tissues but not in the normal skin tissues, indicating that there was no crossed contamination in these specimens. Among the 25 100 genes, 1313 genes were identified to be differentially expressed between KS and normal skin tissues, including 756 up-regulated genes and 557 down-regulated genes. These differentially expressed genes, such as myeloid cell leukemia-1 gene (MCI-1), annexins (ANX) and serine proteinase inhibitor Kazal type 5 (SPINK5), were associated with apoptosis, angiogenesis, cell signaling, protein processing, cell cycle regulation, and so on. Conclusion The differentially expressed genes such as MCI-1 and SPINK5 may be associated with the development of KS.

Objective To investigate the ecological structure of intestinal fungal flora of patients with chronic hepatitis B infection. Methods HBV-liver cirrhosis patients, chronic hepatitis B patients, HBV carriers, and healthy volunteers were selected as research subjects to extract the total DNA from stool of each subject, 18S rRNA genes of intestinal fungi was cloned and sequence was analyzed for the construction of 18S rDNA clone libraries and phylogenetic tree, diversity and structural characteristics of intestinal fungal flora in each group was then analyzed. Results A total of 29 operational taxonomic units (OTUs) of intestinal fungal flora in all research subjects was acquired. All positive clones belonged to three fungal taxa: Zygomycetes (3.4%) , Ascomycetes (82. 8%) and Basidiomycetes (13. 8%) . The dominant fungal community was Candida spp. , uncultured fungus and Saccharomyces spp. , which accounted for 29. 2% , 15. 9%, 15.0% respectively. The OTUs which belonged to HBV-liver cirrhosis patients, chronic hepatitis B patients, HBV carriers, and healthy volunteers was 20,16,12,14 respectively. Conclusion There is an abundant fungal flora in the human intestine. Ecological structure of intestinal fungal flora appears to be various in patients with chronic hepatitis B infection, which indicate the close relationship between the alteration of ecological structure of fungal flora and the stage of chronic hepatitis B infection.

ObjectiveTo investigate the expression of programmed death receptor ligand 1 ( PD-L1 ) of peripheral blood mononuclear cells (PBMCs) in patients with hepatic cystic echincccccosis (HCE) and its relation with interferon-γ.MethodsThe clinical data of 63 patients with HCE who were admitted to the First Affiliated Hospital of Xinjiang Medical University from June 2010 to February 2011 were retrospectively analyzed.All patients were divided into HCE active group (38 patients) and HCE non-active group (25 patients) according to the system established by the World Health Organization's Informal Working Group on Echinocoecosis.Twenty patients with hepatic hemangioma or healthy individuals were recruited in normal control group.The positive rate of PD-L1 expression was detected by flow cytometry and immunocytochemistry.The expression of interferon-γ was detected by enzyme-linked immtmosorbent assay (ELISA).All data were analyzed by the t test,one-way analysis of variance,LSD test and chi-square test.The relationship between the expression of interferon-γ and positive rate of PD-L1 expression was analyzed by the Pearson test.ResultsThe results of flow cytometry showed that the positive rates of PD-L1 expression in the HCE active group,HCE non-active group and normal control group were 12.1％±3.8％,10.9％ ± 2.5％ and 9.1％ ±2.5％,respectively.There was a significant difference in the positive rate of PD-L1 expression between the HCE active group and normal control group (t =3.327,P ＜ 0.05 ).The results of immunohistochemistry showed that the positive rates of PD-LI expression in the HCE active group,HCE non-active group and normal control group were 11.9％ ± 3.4％,i0.6％ ± 2.9％ and 9.5％ ± 3.6％,respectively.There was a significant difference in the positive rate of PD-L1 expression between the HCE active group and normal control group (t =2.470,P ＜ 0.05 ).The expressions of intefferon-γ in the HCE active group,HCE non-active group and normal control group were ( 141 ± 38 ) μμg/L,( 124 ± 32 ) μg/L and ( 105 ± 42 ) μg/L.There wasasignificant difference in the expression of interferon-γ between the HCE active group and normal control group ( t =3.280,P ＜ 0.05).The results of flow cytometry and immunohistochemistry revealed that the positive rate of PD-L1 expression was positively correlated with the expression of interferon-γ( r =0.59,0.61,P ＜ 0.05 ).Conclusion With the help of interferon-γ,PD-L1 may play an important role in promoting the immune.evasion of echinococcus.

h Kaposi' s sarcoma in Xingjiang, which have a high homology with those strains from Africa and Europe. A5 and C7 genotypes of HHV-8 have been first isolated in China.

Objective To perform molecular cloning and sequencing, bioinformatics analysis,protein expression and function of extracellular signal regulated kinase (EgERK1) of Echinococcus granulosus in Xinjiang. Methods The specific primers of EgERK1 were designed and total RNA was extracted from Echinococcus granulosus in Xinjiang. EgERK1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and prokaryotic expression plasmid pET28a-EgERK1 was constructed and sequenced. The sequences were analyzed by DNA sequencing and bioinformatics technology. The recombinant EgERK1 protein was induced and expressed. The biological function was detected using sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot. Results The sequence of RT-PCR product was 1125 bp, encoding 374 amino acids with isoelectric point of 6.34.This gene was a new ERK-homologues gene indicated by BLAST, named EgERK1(EU701008).Homology comparisons indicated that the homology of EgERK1 and EmMPK1from Echinococcus multilocularis was 95.45%, and was 43.04%-61.88% to ERK from Caenorhabditis elegans, S. cerevisiae, D. melanogaster and human. Phylogenetic analysis showed that EgERK1 clustered with EmMPK1. Bioinformatics analysis predicted that EgERK1 contained a highly conserved T-X-Y motif and activation loop segment of ERK-like kinase.Western blot results showed the EgERK1 recombinant protein could reacted specifically with anti-human ERK monoclonal antibody. Conclusion A new EgERK1 gene of Echinococcus granulosus is successfully cloned and its recombinant protein could reacted specifically with ERK1/2 antibody, which provides the basis for further study of EgERK1 function in the host-parasite interaction.

Objective To establish a real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) for detection of human Herpesvirus-8 (HHV-8) viral load. Methods pMD19-T recombinant vectors inserted with an open reading frame (ORF) 26 of HHV-8 or β-actin gene were constructed respectively. A sensitive RT-qPCR method was established and optimized. The effectivity of the method was evaluated by determining the HHV-8 viral loads in 30 (formalin fixed, paraffinised)biopsy samples of Kaposi's sarcoma. Results The key factors for optimizing the method included anneal temperature and extension. The standard curve showed that the Ct value of ORF26 and β-actin had a good linear relationship (r2 ＞0.990) with the standard samples. The melt curve and electrophoresis showed the specificity of our study. The sensitivity of this method was very high and the detection rate could reach 100%. The viral loads were significantly higher in patients with classic Kaposi's sarcoma compared to patients with acquired immunodeficiency syndrome-associated Kaposi's sarcoma(69.18 va 8. 63, x2 =7.950,P=0.005).Conclusions The established RT-qPCR method is highly sensitive, which can be used as a routine assay for detecting HHV-8.This system offers a good platform for diagnosing other causative organism.

Objective To explore the expression of TLR2,4,7 mRNA on peripheral blood mono-nuclear cells (PBMCs) in patients with chronic cystic echinococcosis(CE) infection, and the level of serum IL-10. Methods The expression level of TLR2,4,7 mRNA on peripheral blood mononuclear were tested in 42 chronic CE cases and 28 normal controls (NC) by real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) method. GAPDH was selected as the internal control. The level of serum IL-10 was determined in ELISA. The subjects were determined by t test. The correlations between TLR2, TLR4, TLR7 and IL-10 were determined by differences of expression of TLR2, TLR4, TLR7 on PBMCs and serum IL-10 in two groups of study linear correlation test. Results The expressions of TLR2, TLR4,TLR7 mRNA in chronic CE group were higher than those of in NC group. Compared with the NC group, the expressions of TLR2, TLR4 and TLR7 mRNA increased more than 7.3-, 3.6-, 3.6-fold, respectively. In chronic CE group, TLR2, TLR4 and TLR7 mRNA expressions were 1.0729 ±0.4006, 5.0976 ±1.6682, 0. 6481 ±0. 2574, respectively. TLR2, TLR4 and TLR7 mRNA expressions were 0. 1468 ± 0.0435, 1.4067 ±0. 3279, 0. 1804 ±0. 0568 in NC group, respectively. Compared with NC group, the differences of TLR2 and TLR4 mRNA expression were significant (P = 0.0287, 0. 033), while the expression of TLR7 mRNA was not difference (P =0.0862). Moreover, in chronic CE group, the level of serum IL-10 was higher than that of in NC group. In chronic CE group and NC group, the level of serum IL-10 was (17.6770±1.6298) pg/ml, (9.4898 ±0.7049) pg/ml. Compared with NC group, there was significant difference in chronic group (P<0.01). Significant positive correlation between TLR2 and TLR4 was found in chronic CE group, r = 0. 1135, P =0.036. Others were not correlations. Conclusion In the development of chronic CE, TLR2 and TLR4 participate in this progression. As the receptors of antigen of cystic echinococcus, TLR2 and TLR4 can regulate the immune response through interacting with different antigens from cystic echinococcus. Meanwhile, under the participation of TLR2, TLR4 and increased serum IL-10, they will approach to Th2 immune reaction, which play an important role in chronic CE that can induce immune evasion.

Objective To construct a colon cancer chemotherapy-resistant cell line COLO,and study its characteristics and its relationship with tumor stem cells.Methods We constructed two 5-fluorouraci (5-FU)-resistant colon cancer cell line COLO/5-FU-1 and COLO/5-FU-2, which were resistant to 0.1 0 μmol/ml and 0.20 μmol/ml 5-FU respectively through gradiently increased drug concentration.The cha-racteristics of 5-FU-resistant cell lines were compared with parental colon cancer cell line COLO related to proli-feration,colony forming ability,migration and invasion,sphere forming ability,expression of stemness genes and cross drug-resistance.Results In the cell viability assay,4 days after regular training,the absorbancy of colon cancer 5-FU-resistant cell lines COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were 0.61 ±0.1 3,0.54 ±0.07 and 0.41 ±0.09 respectively,with significant difference (F =63.43,P =0.033).With the increased concentration of 5-FU,5-FU-resistant cell lines presented increasing clonality. The cloning efficiency of COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were (87.6 ±1 2.7)%,(65.3 ±9.7)% and (38.5 ±7.6)% respectively,with significant difference (F =33.64, P =0.01 7).In each high power field of vision,the cell numbers of migration through the basement membrane of COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were 482 ±39,434 ±45 and 373 ±38 respectively;and the cell numbers of invasion through the basement membrane were 1 74 ±42,1 1 2 ± 31 and 87 ±29 respectively,with significant differences (F =1 09.61 ,P =0.009;F =67.31 ,P =0.032). Compared with parental colon cancer cell line COLO,5-FU-resistant cell lines had higher expression of stem-ness genes (F =47.31 ,P =0.042).5-FU-resistant cell lines were cross-resistant to other chemotherapeutic drugs such as mitoxantrone.For example,after incubation for 96 hours,inhibition rate of mitoxantrone to parent colon cancer cell line COLO was higher significantly than COLO/5-FU-1 and COLO/5-FU-2 (0.749 ± 0.042,0.423 ±0.024,0.342 ±0.01 8),with significant difference (F =1 2.61 ,P =0.028).The micro-sphere forming rates of COLO/5-FU-2,COLO/5-FU-1 and parental colon cancer cell line COLO were (8.90 ± 0.97)%,(6.20 ±0.75)% and (3.90 ±0.32)% respectively,with significant difference (F =1 64.32,P =0.006).Conclusion Colon cancer drug-resistant cell line COLO possess tumor stem cell-like characteristics, which are enriched in cancer stem cells.

The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.
Animals ; Ducks/virology ; Genes, Viral/genetics ; Mardivirus/*genetics ; Membrane Glycoproteins/*genetics ; Microscopy, Fluorescence ; Phylogeny ; Polymerase Chain Reaction/veterinary ; Viral Envelope Proteins/*genetics

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.
*Adenoviridae/classification/pathogenicity ; Adenoviridae Infections/pathology/*veterinary/virology ; Animals ; *Anseriformes ; *Apoptosis ; Bird Diseases/*virology ; DNA Fragmentation ; Enteritis/*veterinary/virology ; Epithelial Cells/cytology/virology ; In Situ Nick-End Labeling ; Intestines/cytology/virology ; Leukocytes/cytology/virology ; Lymphoid Tissue/cytology/virology ; Macrophages ; Microscopy, Electron, Transmission