Association of HLA-DQA1 gene rs9272346 polymorphism with clinical outcome of hepatitis B virus infection in ethnic Han population from Hubei / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the association of rs9272346 polymorphism of HLA-DQA1 gene with clinical outcome of hepatitis B virus (HBV) infection in ethnic Han population from Hubei, China.</p><p><b>METHODS</b>A case-control study was conducted, which have involved 1028 unrelated subjects including 238 asymptomatic HBV carriers (AHC), 173 acute liver failure (ALF), 292 liver cirrhosis (LC) and 325 hepatocellular carcinoma (HCC). Genotypes of rs9272346 were determined by real-time polymerase chain reaction with a TaqMan MGB probe. Statistical results were analyzed using Chi square test, student's t test and unconditional logistic regression.</p><p><b>RESULTS</b>No significant differences were detected in the frequencies of G allele between ALF, LC, HCC and AHC groups (P= 0.312, 0.314, 0.264). Compared with the AA genotype, the GG and GA genotypes were not associated with the patients groups under the dominant model: ALF group vs. AHC group (adjusted OR= 1.08, 95%CI: 0.7-1.68), LC group vs. AHC group (adjusted OR= 1.11, 95%CI: 0.87-1.26), HCC group vs. AHC group (adjusted OR= 0.93, 95%CI: 0.65-1.33). For women, the GG and GA genotypes have conferred a protective effect for the outcome of ALF (OR= 0.30, 95%CI: 0.1-1.87).</p><p><b>CONCLUSION</b>Our results suggested that rs9272346 polymorphism of HLA-DQA1 may not independently influence the outcome of HBV infection in ethnic Han Chinese in Hubei, while the GG and GA genotypes may confer a protective effect against ALF in women.</p>

Inhibition of Hepatic Stellate Cell Proliferation and Extracellular Ca2+ Influx by Leptin-siRNA / 华中科技大学学报(医学版)

Objective To evaluate the effect of silencing leptin by small interfering RNA(siRNA)on the expression of lep‐tin ,and apoptosis ,proliferation and intracellular Ca2+ concentration([Ca2+ ]i )of hepatic stellate cells(HSCs)and to provide evi‐dence for liver fibrosis gene therapy.Methods HSCs were divided into normal control group ,blank vector group ,siRNA nega‐tive control group and leptin‐siRNA group.After transfection of the leptin‐siRNAs into HSCs ,cell proliferation was measured by MTT assay.Cell cycle and apoptosis were measured by flow cytometry.Expression of leptin was detected by immunocyto‐chemistry and Western blot. [Ca2+ ]i was measured by Fura‐2/AM loading.Results Compared with the normal control group , the blank vector group and the siRNA control group ,the protein expression of leptin and the cell growth were significantly in‐hibited in the leptin‐siRNA group(P<0.05). The proliferation rate of HSCs was significantly different at different time points (24 ,48 and 72 h)(P<0.05).The cell apoptosis rate was increased significantly in the leptin‐siRNA group(P<0.01).At the same time ,Leptin‐siRNA‐induced [Ca2+ ]i was also significantly reduced(P<0.05).Conclusion The leptin gene may play an important role in liver fibrosis progression and is potentially a novel predictive and prognostic marker for liver fibrosis.

Effects of variant rs346473 in ARHGAP24 gene on disease progression of HBV infection in han Chinese population / 华中科技大学学报（医学）（英德文版）

Host genetic, environmental and viral factors are classified as three categories that determine clinical outcomes of hepatitis B virus (HBV) infection. The objective of this study was to detect the associations between polymorphisms rs346473 and rs346482 in Rho GTPase-activating protein 24 (ARHGAP24) gene and disease progression of HBV infection in Han Chinese population. These two SNPs were found by our DNA pooling using Affymetrix Genome-Wide Human Mapping SNP6.0 Array in HBV carriers, and verified by using TaqMan 7900HT Sequence Detection System with 758 progressed HBV carriers versus 300 asymptomatic HBV carriers (AsC) in a discovery phase and 971 progressed HBV carriers versus 328 AsC in a replication phase. Multivariable logistic regression revealed that individuals with genotype TT at variant rs346473 displayed remarkable correlations with disease progression of HBV infection both in the discovery phase (OR, 2.693; 95% CI, 1.928-3.760; P=6.2×10(-9); additive model) and the replication phase (OR, 1.490; 95% CI, 1.104-2.012; P=9.0×10(-3); additive model). These two SNPs were in strong linkage disequilibrium with D'=0.99 and r (2)=0.951, and haplotype TT disclosed an increased susceptibility to HBV progression (OR, 1.980; 95% CI, 1.538-2.545; P=8.1×10(-8)). These findings suggest that polymorphism rs346473 in the ARHGAP24 gene might be a part of the genetic variants underlying the susceptibility of HBV carriers to disease progression.

Relationship between-844T/C polymorphisms of Fas ligand gene promoter and fulminant hepatitis B / 中华传染病杂志

Objective To investigate the relationship between single nucleotide polymorphism (SNP) of Fas ligand (FasL) and fulminant hepatitis B in Han Chinese. Methods HBV infected subjects were enrolled in this case-control study, including 233 cases of inactive HBsAg carrier, 68 patients with fulminant hepatitis B,100 cases of spontaneous hepatitis B clearance, 102 patients with hepatitis B virus (HBV) related cirrhosis and 112 patients with HBV related primary hepatocellular carcinoma. The blood samples and clinical data were collected. FasL-844T/C polymorphisms of enrolled subjects were examined by TaqMan real time fluorescent genotyping polymerase chain reaction (RT-PCR). A adjusted odds ratios (OR)and 95% confidence intervals (CI)were calculated using the Logistic regression model. Results After adjusting the factors of gender and age, binary Logistic regression analyses indicated that the genotype frequencies of FasL-844 CC,CT,TT in inactive HBsAg carriers were 50. 64% ,39. 91% and 9. 44% respectively, and those in cases of fulminant hepatitis B were 79. 41%, 17. 65% and 2. 94%, respectively. The analysis also revealed that FasL-844CC genotype in inactive HBsAg carriers was high risk factor of developing fulminant hepatitis B (OR =4. 729,95%CI:0. 510 - 21. 282,P = 0. 043), while there were no statistic significances in other cases (P>0. 05). Conclusion The inactive HBsAg carriers harboring FasL-844CC may have greater susceptibility to fulminant hepatitis B, which need arouse high attention.

MCM3AP, a novel HBV integration site in hepatocellular carcinoma and its implication in hepatocarcinogenesis / 华中科技大学学报（医学）（英德文版）

A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.

DNA double-strand breaks, potential targets for HBV integration / 华中科技大学学报（医学）（英德文版）

Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is one of the most frequently occurring cancers. Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis. More and more researches were designed to find the relationship of the two. In this study, we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks (DSBs), one of the most detrimental DNA damage. An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection, then cells were incubated in patients' serum with high HBV DNA copies and at the same time, DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. By using nest PCR, the viral DNA was detected at the sites of the break. It appeared that integration occurred between part of HBV x gene and the I-SceI induced breaks. The results suggested that DSBs, as the DNA damages, may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily. It provided a new site to investigate the integration.

Preparation of Rabbit Polyclonal Antibody against ELF3 and Its Immunolocalization in Mice / 华中科技大学学报(医学版)

Objective To prepare and identify rabbit polyclonal antibody against embryonic liver fordrin 3(ELF3),and investigate the distribution of ELF3 in mice tissue.Methods ELF3 specific N-terminal peptide was synthesized,and conjugated to Keyhole limpet hemocyanin(KLH)as immunogen.The ELF3-KLH complex was injected into rabbits subcutaneously,and then ELF3 antibody was purified using affinity chromatography.The titer of the antibody was evaluated by ELISA.The specificity of antibody against ELF3 and immunolocalization of ELF3 were evaluated by using Western blot and immunohistochemistry.Results Rabbit polyclonal antibody against ELF3 was prepared by the immunization of ELF3-KLH complex.ELISA and Western blot results showed the antibody against ELF3 had high titer and specificity.Western blot and immunohistochemical studies demonstrated ELF3 was expressed in the mouse heart,liver,brain and kidney tissue,particular on the cell membrane.Conclusion The preparation of polyclonal antibody against ELF3 was successful due to its high titer and specificity;ELF3 was expressed in the mice heart,liver,and kidney,particular on the cell membrane.It will provide an excellent tool for further study on the ELF3 function.

Suppressive effects of genomic imprinted gene PEG10 on hydrogen peroxide-induced apoptosis in L0(2) cells / 华中科技大学学报（医学）（英德文版）

The effects of PEG10 on hydrogen peroxide (H(2)O(2))-induced apoptosis in human normal liver cell line L0(2) were investigated. The PEG10 gene was transfected into L0(2) cells by lipofectamine, the positive clone was screened by G418 and defined as L0(2)/PEG10, while the cell transfected with empty expression vector (pEGFP-N1) was defined as L0(2)/vector. L0(2)/vector and parental L0(2) cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H(2)O(2) (50-400 mmol/L) was administered to induce the apoptosis of L0(2) cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L0(2)/PEG10 cells were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H(2)O(2) for 24 h, the cellular growth inhibition rate of L0(2)/PEG10 cells was significantly lower (58.2%) than that of L0(2) (92.5%) and L0(2)/vector (88%). Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L0(2)/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L0(2) and L0(2)/vector cell lines, but not in L0(2)/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L0(2) by antagonizing H(2)O(2)-induced apoptosis.

Advances in Research on Hepatitis B Virus DNA Integration / 中国病毒学·英文版

Since HBV DNA integration was discovered for the first time in 1980, various methods have been used to detect and study it, such as Southern Blot, in situ hybridization, polymerase chain reaction and so on. HBV DNA integration is thought to be random on the whole although some hot spots of integration were described by some researchers, one of which might be the repetitive sequences of the genomic DNA. Besides, DNA damage, especially double-strand breaks could promote HBV DNA integration into host genome. HBV DNA integration into cells may damage the stability of the genome, cause DNA rearrangement, promote DNA deletion and induce the formation of HCC.

Effect of ATRA on Contents of liver Retinoids, Oxidative Stress and Hepatic Injury in Rat Model of Extrahepatic Cholestasis / 华中科技大学学报（医学）（英德文版）

The effects of all-trans-retinoic acid (ATRA) administration on the concentration of retinoids (RA and vitamin A) in liver, oxidative stress and the hepatic injury in a rat model of com-mon bile duct ligation (CBDL)-induced liver injury were investigated. Female rats were subjected to a sham (n=5) or CBDL (n=48). Two weeks after operation, rats undergoing CBDL were randomized to receive treatment with either ATRA at three different doses (0.1, 1.5, 7.5 mg/kg) dissolved in bean oil or only bean oil every day over a 4-week experimental period. Rats were killed and blood samples were collected from the heart for determination of the serum transaminase. The contents of retinoids in rat liver were detected by using HPLC. Malondialdehyde (MDA), glutathione (GSH) and superox-ide dismutase (SOD) levels in liver were determined by a spectrophotometric method according to the instruction of the kits. Liver pathologic changes were observed under the light microscopy and electron microscopy. The results showed that compared with sham-operated group, the levels of reti-noids in the liver tissue were significantly decreased in the CBDL group (P<0.01). ATRA (0.1 mg/kg) administration in CBDL rats partially restored the contents of retinoids (P<0.05). Liver RA and vita-min A contents in CBDL group were significantly increased after ATRA (1.5 and 7.5 mg/kg) sup-plementation as compared with sham-operated group (P<0.05). However, in ATRA-treated CBDL group, hepatic GSH level and SOD activity, depressed by CBDL, and hepatic MDA level, increased by CBDL were returned to those in sham-operated group (P<0.05). The histologic observation of liver tissues indicated that ATRA treatment notably alleviated hepatocellular swelling, steatosis, the swelling of mitochondria and proliferation of smooth endoplasmic reticulum (SER). Treatment with ATRA could reduce levels of serum transaminase as compared with sham-operated group, more greatly in 1.5 and 7.5 mg/kg ATRA-treated groups than in 0.1 mg/kg ATRA-treated group. It was concluded that ATRA treatment can recover MDA and GSH levels and SOD activity in CBDL rat liver through restoring RA and vitamin A contents, and eventually ameliorate liver injury.

High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System / 华中科技大学学报（医学）（英德文版）

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

High-level production of a functional recombinant hepatitis B virus polymerase in insect cells with a baculovirus expression system / 华中科技大学学报（医学）（英德文版）

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerase gene to express the target protein. HBV polymerase expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

Low-dose ATRA Supplementation Abolishes PRM Formation in Rat Liver and Ameliorates Ethanol-induced Liver Injury / 华中科技大学学报（医学）（英德文版）

The effects of all-trans-retinoic acid (ATRA) in low doses supplementation on concentrations of polar retinoid metabolites (PRM) and retinoids in the ethanol-fed rat liver, and on hepatocyte injury were investigated. The rat model of alcoholic liver disease (ALD) was induced by intragastric infusion of ethanol, and then the rats were administrated with ATRA in two different doses (150 μg/kg body weight and 1.5 mg/kg body weight) for 4 weeks. Concentrations of retinoids in rat liver and plasma were determined by using HPLC. Liver tissues pathologic changes were observed under the light microscopy and electron microscopy. The serum transaminases concentrations were measured. The results showed that the HPLC analysis of retinoids revealed that retinoids (vitamin A,RA, retinyl palmitate) concentrations in ethanol-fed rat liver and RA concentration in ethanol-fed rat plasma were markedly diminished (P＜0.01) after ethanol feeding for 12 weeks. Furthermore, obvious peaks of PRM were formed in livers of ethanol-fed rats. ATRA 150 μg/kg supplementation in ethanol-fed rats for 4 weeks raised RA concentration in both liver and plasma, and also raised vitamin A concentration in liver to control levels, partially restored retinyl palmitate concentration (P＜0.05) in liver. ATRA 1.5 mg/kg supplementation raised not only RA concentrations in liver and plasma but also retinyl palmitate concentrations in liver. However, the vitamin A concentration in liver of ATRA-supplemented rats (1.5 mg/kg) was higher than that of controls (P＜0.05). The histologic observation of liver tissues indicated that ATRA treatment notably alleviated hepatocellular swelling,steatosis, the swelling of mitochondria and proliferation of smooth endoplasmic reticulum (SER).ATRA treatment greatly decreased levels of serum transaminases as compared with the only ethanol-fed group (P＜0.05). It was concluded that low-dose ATRA treatment could restore retinoids concentrations and abolish the PRM formation in liver of ALD rats, and then ameliorate the injury of liver cells.

Screening of Efficient siRNA Target Sites Directed against Gatekeeper Genes for DNA Repair / 华中科技大学学报（医学）（英德文版）

To investigate the RNA interference (RNAi) effect induced by vector-derived small interfering RNA (siRNA) targeting the three gatekeeper genes (Rad52, Ku70, Ku80) and screen the more effective target sites from candidates for further research, by using siRNA design tools online,we selected 2 candidate sequences directed to every gatekeeper gene. According to the sequences, six vector-derived siRNAs (denoted psiRNA1-6) and one mocking psiRNA7 were constructed. Among them, psiRNA1 and psiRNA2 targeted Rad52, psiRNA3 and psiRNA4 to Ku70, psiRNA5 and psiRNA6 to Ku80. The mocking psiRNA7 was used as control. After sequence identification, the seven plasmids were transfected into HepG2 cell line. siRNA-induced silencing of gatekeeper genes was determined by using RT-PCR at RNA level and Western Blot at protein level. The results showed that the six plasmids specifically targeting the coding region of gatekeeper genes were successfully designed and constructed. To some extent, the six plasmids could reduce the expression of target gene.Comparatively, the plasmid-derived siRNA psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. The results suggest that the gene silencing efficiency of siRNA is different,depending on their targeted region, and siRNA may provide us with practical tools for further study on the three gatekeeper genes, i.e. Rad52, Ku70, Ku80.

Screening of efficient siRNA target sites directed against gatekeeper genes for DNA repair / 华中科技大学学报（医学）（英德文版）

To investigate the RNA interference (RNAi) effect induced by vector-derived small interfering RNA (siRNA) targeting the three gatekeeper genes (Rad52, Ku70, Ku80) and screen the more effective target sites from candidates for further research, by using siRNA design tools online, we selected 2 candidate sequences directed to every gatekeeper gene. According to the sequences, six vector-derived siRNAs (denoted psiRNA1-6) and one mocking psiRNA7 were constructed. Among them, psiRNA1 and psiRNA2 targeted Rad52, psiRNA3 and psiRNA4 to Ku70, psiRNA5 and psiRNA6 to Ku80. The mocking psiRNA7 was used as control. After sequence identification, the seven plasmids were transfected into HepG2 cell line. siRNA-induced silencing of gatekeeper genes was determined by using RT-PCR at RNA level and Western Blot at protein level. The results showed that the six plasmids specifically targeting the coding region of gatekeeper genes were successfully designed and constructed. To some extent, the six plasmids could reduce the expression of target gene. Comparatively, the plasmid-derived siRNA psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. The results suggest that the gene silencing efficiency of siRNA is different, depending on their targeted region, and siRNA may provide us with practical tools for further study on the three gatekeeper genes, i.e. Rad52, Ku70, Ku80.

Low-dose ATRA supplementation abolishes PRM formation in rat liver and ameliorates ethanol-induced liver injury / 华中科技大学学报（医学）（英德文版）

The effects of all-trans-retinoic acid (ATRA) in low doses supplementation on concentrations of polar retinoid metabolites (PRM) and retinoids in the ethanol-fed rat liver, and on hepatocyte injury were investigated. The rat model of alcoholic liver disease (ALD) was induced by intragastric infusion of ethanol, and then the rats were administrated with ATRA in two different doses (150 microg/kg body weight and 1.5 mg/kg body weight) for 4 weeks. Concentrations of retinoids in rat liver and plasma were determined by using HPLC. Liver tissues pathologic changes were observed under the light microscopy and electron microscopy. The serum transaminases concentrations were measured. The results showed that the HPLC analysis of retinoids revealed that retinoids (vitamin A, RA, retinyl palmitate) concentrations in ethanol-fed rat liver and RA concentration in ethanol-fed rat plasma were markedly diminished (P<0.01) after ethanol feeding for 12 weeks. Furthermore, obvious peaks of PRM were formed in livers of ethanol-fed rats. ATRA 150 microg/kg supplementation in ethanol-fed rats for 4 weeks raised RA concentration in both liver and plasma, and also raised vitamin A concentration in liver to control levels, partially restored retinyl palmitate concentration (P<0.05) in liver. ATRA 1.5 mg/kg supplementation raised not only RA concentrations in liver and plasma but also retinyl palmitate concentrations in liver. However, the vitamin A concentration in liver of ATRA-supplemented rats (1.5 mg/kg) was higher than that of controls (P<0.05). The histologic observation of liver tissues indicated that ATRA treatment notably alleviated hepatocellular swelling, steatosis, the swelling of mitochondria and proliferation of smooth endoplasmic reticulum (SER). ATRA treatment greatly decreased levels of serum transaminases as compared with the only ethanol-fed group (P<0.05). It was concluded that low-dose ATRA treatment could restore retinoids concentrations and abolish the PRM formation in liver of ALD rats, and then ameliorate the injury of liver cells.

Cloning and expression of human keratinocyte growth factor-2 and the purification and identification of its products / 中国组织工程研究

BACKGROUND: Human keratinocyte growth factor-2 (hKGF-2) has extensive physiological functions, which plays an important role in embryonic development, tissue-repairing, nervous regeneration, vascularization and development of tumor.OBJECTIVE: To clone hKGF-2 gene, obtain the expression of hKGF-2 in Escherichia coli(E.coli) and determine its bioactivity, so as to provide experimental basis for further investigation.DESIGN: Open experiment.SETTING: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.MATERIALS: The experiment was conducted in State Key Laboratory of Viral Genetic Engineering, Institute for Viral Disease Control and Prevention of Chinese Center for Disease Control and Prevention. The temperature control expression vector pBV220 was constructed by State Key Laboratory of Viral Genetic Engineering; EcoR Ⅰ , BamH Ⅰ , T4 DNA ligase (Promega Co., Ltd.); The specific polymerase chain reaction (PCR) of hKGF-2 (Manufactured by Shanghai Boya Biotechnology Co., Ltd.); Heparin-Sepharose CL-6B (Pharmacia Company); PCR rapid purification kit,Trizol kits for total RNA extract, Kits for RT-PCR (GIBCO Co., Ltd.); Kits for rapid extraction of plasmid DNA (Boda Company); BL-21-codon plus compent cells (Stratagene Co., Ltd.).METHODS: High-expression strain BL-21 codon plus competent cells was used to express and purify initially recombinant hKGF-2 protein, and its activity was detected. RT-PCR was adopted to obtain hKGF-2 cDNA from lung tissues of naturally aborted fetus and clone it into pBV220 carri er plasmid. The hKGF-2 protein expressed in BL-21 codon plus competent cells of E.coli. Affinity chromatography and ion exchange chromatography were applied in isolation and purification, and the bioactivity of expression protein was determined in cell proliferation test.MAIN OUTCOME MEASURES: The length and sequence of cDNA segment in hKGF-2, the expression of hKGF-2 gene inE.coli and the purification of hKGF-2 activity.RESULTS: The segment of hKGF-2 cNDA was about 500 bp, and hKGF-2 protein highly expressed in BL-21, which had soluble expression in the supernatant. SDS-PAGE showed that the relative molecular mass was about 20000, and hKGF-2 protein could significantly promote the mitotic activity of NIH3T3 cells. The A value (490 nm) of hKGF-2 in the 1 μg/L, 5 μg/L and 10 μg/L groupswere higher than that in the blank control group, and the differences were significant (which were 0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004 respectively,P ＜ 0.001).CONCLUSION: hKGF-2 gene is successfully cloned, which highly expresses in BL-21 of the E.coli. Purified hKGF-2 protein can stimulate the proliferation of NIH3T3 cells and significantly promote its mitotic activity.

RNA Interference Targeting Leptin Gene Effect on Hepatic Stellate Cells / 华中科技大学学报（医学）（英德文版）

To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte , and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 μg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.

Experimental Studies on PNP Suicide Gene Therapy of Hepatoma / 华中科技大学学报（医学）（英德文版）

To investigate the killing effect of PNP/MeP-dR suicide gene system on hepatoma cells,pcDNA3. 0/PNP, an eukaryotic expression vector harboring E. coli PNP gene, was transfected into human hepatoma HepG2 cells by liposome-mediated method. A HepG2 cell line with stable PNP gene expression, HepG2/PNP, was established with presence of G418 selection. The cell growth curves were determined with trypan blue staining. The sensitivity of HepG2/PNP to MePdR and bystander effects were assayed by MTT and FCM methods. The enzymatic activity of the product of PNP gene was determined by HPLC method. The cytotoxic effects of MeP-dR on HepG2/PNP cells were obvious (IC50 =4.5μmol/L) and all HepG2/PNP cells were killed 4 days after the treatment with 100μmol/L MeP～dR. In mixed cultures containing increasing percentages of HepG2/PNP cells, total population killing was demonstrated when HepG2/PNP cells accounted for as few as 5% of all HepG2 cells 8 days after the treatment with 100μmol MeP-dR. Highpressure liquid chromatography (HPLC) demonstrated that the PNP enzyme could convert MePdR into 6-MP. PNP/MeP-dR suicide gene system had an advantage over traditional suicide gene systems for hepatoma gene therapy. Our e results suggest that high-level bystander effects of this system result in significant anti-tumor responses to hepatoma gene therapy, especially in vivo.

Expression of DNA Repair Enzyme hMTH1 mRNA and Protein in Hepatocellular Carcinoma / 华中科技大学学报（医学）（英德文版）

To study the expression of DNA repair enzyme hMTH1 mRNA and protein in hepatocellular carcinoma (HCC) tissues, tissues adjacent to the cancers, normal liver cells and hepatoma cell lines, and to investigate their function in the progress of HCC, semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was employed to examine the expression of hMTH1 mRNA in matched HCC tissues (HT)/surrounding tissues (HST) of HCC, normal liver cell L02 and hepatoma cell lines SMMC7721, HepG2. hMTH1 protein was detected in corresponding HT as well as their HST by immunohistochemistry. Our results showed that the expression level of hMTH1 mRNA in HT was higher than that in HST (t=2.424, P <0.05). The expression level of hMTH1 mRNA in two hepatoma cell lines was higher than that in normal liver cell line (F=6.810, P <0.01). The expression of hMTH1 mRNA in SMMC7721 was similar to that in HepG2. hMTH1 protein was 88.2 % (15 of 17) positive in HT and 82.4 % (14 of 17) in HST. The protein level of hMTH1 in HT was correspondingly higher than in their HST (t=2.618,P<0.05). It is concluded that hMTH1 mRNA and protein were over-expressed in HCC and hepatoma cell lines. It may be one of the key events during the carcinogenesis,progression of HCC and may promote the malignant growth. These results suggest that hMTH1 plays a role in HCC and may be a candidate marker for the diagnosis of HCC.