Preliminary identification of the cloning， expression， and function of Marmota himalayana type I interferon receptor β subunit / 临床肝胆病杂志

ObjectiveTo clone the gene of Marmota himalayana type ‍Ⅰ interferon receptor β subunit （mhIFNAR2）， and to perform antibody preparation and functional identification. MethodsRT-PCR was used for amplification in the spleen tissue of Marmota himalayana to obtain the sequence， which was cloned to the prokaryotic expression vector pRSET-B to express the recombinant protein. Electrophoresis and Western blot were used for identification. BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody of its extracellular domain； immunohistochemistry， immunofluorescence assay， and Western Blot were used for identification， and the method of siRNA blockade was used to investigate its function. An analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for comparison between two groups. ResultsA fragment of mhIFNAR2 （149‍ ‍—‍ ‍1 ‍300 bp） was obtained from spleen tissue， which showed the highest homology of 98.05% in marmot. A prokaryotic expression plasmid was successfully constructed for expression of the extracellular domain of the mhIFNAR2（50-181aa） and was named pRSET-B.mhIFNAR2， and the recombinant protein expressed by this plasmid had a molecular weight of 27 kD， a purity of about 95% after purification， and a concentration of 160 μg/mL. After BALB/c mice were immunized with the purified recombinant protein， 1∶1 000 specific polyclonal antibodies were obtained， and immunohistochemistry and immunofluorescence assay showed the expression in cell membrane and cytoplasm. Among the three siRNAs synthesized， the siRNA starting from the 277 locus （siRNA277） could silence the expression of target genes and weaken the interferon signaling pathway compared with the blank control group and the negative control group （both P<0.05）. ConclusionThe fragment of mhIFNAR2 is obtained， and the polyclonal antibody for the extracellular domain of mhIFNAR2 is successfully prepared， with relatively high titer and specificity， and can be used for immunohistochemistry， immunofluorescence assay， and Western blot.

Research advances in the role of gut microbiota in chronic hepatitis B, chronic hepatitis C, and related liver diseases / 临床肝胆病杂志

Hepatitis B virus infection and hepatitis C virus infection often progress to end-stage liver diseases such as liver cirrhosis, liver failure, and hepatocellular carcinoma, which endanger the life of patients. Recent studies have shown that gut microbiota are closely associated with chronic viral liver diseases. This article reviews the association of gut microbiota with chronic hepatitis B (CHB), chronic hepatitis C (CHC), and their related liver diseases and the research advances in therapies targeting gut microbiota against CHB and its related liver diseases, in order to provide more ideas for the clinical treatment of CHB, CHC, and their related liver diseases.

Genetic Diversity of Echinococcus granulosus Genotype G1 in Xinjiang, Northwest of China

Cystic echinococcosis (CE) caused by E. granulosus is a serious helminthic zoonosis in humans, livestock and wildlife. Xinjiang is one of high endemic province for CE in China. A total of 55 sheep and cattle livers containing echinococcal cysts were collected from slaughterhouses in Changji and Yining City, northern region of Xinjiang. PCR was employed for cloning 2 gene fragments, 12S rRNA and CO1 for analysis of phylogenetic diversity of E. granulosus. The results showed that all the samples collected were identified as G1 genotype of E. granulosus. Interestingly, YL5 and CJ75 strains were the older branches compared to those strains from France, Argentina, Australia. CO1 gene fragment showed 20 new genotype haploids and 5 new genotype haplogroups (H1-H5) by the analysis of Network 5.0 software, and the YLY17 strain was identified as the most ancestral haplotype. The major haplotypes, such as CJ75 and YL5 strains, showed identical to the isolates from Middle East. The international and domestic trade of livestock might contribute to the dispersal of different haplotypes for E. granulosus evolution.

Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR / 华中科技大学学报（医学）（英德文版）

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

Silencing of UBP43 by shRNA Enhances the Antiviral Activity of Interferon against Hepatitis B Virus / 中国病毒学·英文版

Previous studies have shown that expression of the interferon-sensitive gene (ISG)I5 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by Hi (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by HI (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan / 华中科技大学学报（医学）（英德文版）

The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E. coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

Frequency of loss expression of the DPC4 protein in various locations of biliary tract carcinoma / 中华预防医学杂志

<p><b>OBJECTIVE</b>To clarify the relationship between the loss of expression of the deleted in pancreatic carcinoma locus 4 (DPC4) proteins and the pathogenesis of biliary tract carcinoma.</p><p><b>METHODS</b>71 primary biliary tract carcinoma (BTCa), including 38 common bile duct (CBD) carcinomas, 18 gallbladder carcinomas, 15 hilar bile ducts (HBD) carcinomas were examined by immunohistochemical staining. In addition, the CBD carcinomas were divided into two groups: tumors with metastasis (M(+) group, 27 cases) and tumors without metastasis (M(-) group, 11 cases).</p><p><b>RESULTS</b>The frequency of loss of the expression of DPC4 protein was 32.8% in BTCa, 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, 13% in HBD carcinoma. Comparison of the frequency of loss expression of DPC4 was significant statistical difference in CBD carcinoma versus gallbladder carcinoma and HBD carcinoma (P < 0.01). The frequency of loss expression of DPC4 was 48.1% in the M(+) group and 45.4% in the M(-) group.</p><p><b>CONCLUSION</b>There are a close relationship between pathogenesis of BTCa and inactivation of DPC4 and different frequencies of DPC4 gene alternation in various locations of the biliary tract, which are not significantly increased with tumor metastasis in BTCa.</p>

Expression of heme oxygenase-1 mRNA in portal hypertension rats / 中国普通外科杂志

Objective To study the relationship between portal hypertension(PHT) and heme oxygenase-1(HO-1)mRNA expression in rats.Method An animal model of PHT induced by partial portal vein constriction was established to detect the expression of HO-1mRNA in the liver,spleen, and splenic vein of rats by situ hybridization method. Result HO-1mRNA was not detected in the liver, spleen, and splenic vein in the control group. In PHT rats, HO-1mRNA was not detected in the liver;but in the spleen,the expression of HO-1mRNA was 83.3%;in the splenic vein,the expression was 55.6%. Conclusions The PHT rat is in stress condition.The expression of HO-1mRNA in the spleen and splenic vein of PHT ratsare increased and the metabolic product of HO-1 may exacerbate the PHT.