Abstract:

Abstract:Objective：To evaluate the efficacy and safety of radiofrequency ablation（RFA） plus transcatheter arterial chemoemboliza－tion（TACE） in the treatment of hepatocellular carcinoma（HCC）. Methods：PubMed（1978-2013），Cochrane library（1978-2013），EMBase（1978-2013） and CNKI（1978-2013） were searched for randomized controlled trials comparing efficacy of RFA plus TACE and RFA alone for HCC. Results：According to the inclusion criteria，nine studies were selected. Meta analysis data revealed that RFA plus TACE treatment had significantly better effectiveness on 1，3，5-year overall survival rate（OR1 year=2.34，95%CI=1.54 to 3.57，P=0.000；OR3 years=1.99，95%CI=1.43 to 2.77，P=0.000；OR5 years=2.26，95%CI=1.03 to 4.95，P=0.040） and 1，3-year recurrence free sur－vival rate（OR1 year=1.74，95%CI=1.16 to 2.63，P=0.008；OR3 years=2.47，95%CI=1.61 to 3.80，P=0.000） than those of RFA alone treatment. There was no difference in terms of major complications（OR=1.24，95%CI=0.79 to 1.95，P=0.360）. Subgroup analyses by tumor size showed that 1，3-year overall survival rate was significantly improved in RFA plus TACE treatment than in RFA alone treatment in patients with HCC between 3 cm and 5 cm. Conclusion：Combination of RFA with TACE can significantly improve the recurrence free survival and overall survival of patients with HCC，especially for those with HCC between 3 cm and 5 cm.

Abstract:Objective：To examine the mRNA expression characteristics of hepatic energy metabolism-related transcription factors：peroxisome proliferator activated receptor γ coactivator-1α（PGC-1α），peroxisome proliferator activated receptor α （PPARα） and nuclear respiratory factor 1（NRF1） in ob/ob mice. Methods：Male ob/ob obese mice on a C57BL/6J genetic background were used as obese model（n=8） and normal male C56BL/6J mice as control（n=8）. At the age of 5 months，liver samples were obtained after sacri－ficing. Liver morphologic analysis was performed by HE staining and macrohoages were stained by immunohistochmistry method us－ing F4/80 antibody. RT-PCR was performed to determine the mRNA expression levels of interested genes. Results：Both body weight and liver weight of the ob/ob mice were higher than those of control group（t=9.66，P=0.000；t=9.98，P=0.000）. In the ob/ob group，HE showed lipid accumulation. Immunohistochmistry staining for F4/80 results showed more severe inflammatory cell infiltration in ob/ob group. Compared with those in control group，PGC-1α，PPARα，NRF1 mRNA expression levels increased significantly（t=3.02，P=0.009；t=5.64，P=0.000；t=2.93，P=0.011）. Conclusion：The ob/ob mice are obese than normal controls. There are significant hepatic lipid accumulation and inflammatory cell infiltration in the livers of ob/ob mice，which may related with the increased expressions of PGC-1α，PPARα and NRF1.

Abstract:Objective：To observe the expression of Smad7 protein in carbon tetrachloride（CCl4）-induced hepatic fibrosis rats treated with TIMP-1 short interference RNA（siRNA） and TIMP-2 siRNA. Methods：The hepatic tissues collected from hepatic fibrosis rats were divided into 5 groups：normal group，model group，negative control group，TIMP-1 siRNA 0.250 mg/kg group and TIMP-2 siRNA 0.250 mg/kg group. The expression of Smad7 was detected by real-time PCR，immunohistochemistry and Western blot. Results：Real-time PCR analysis showed that mRNA expression of Smad7 was significantly higher in TIMP-1 siRNA 0.250 mg/kg group（59.7±3.3）% and TIMP-2 siRNA 0.250 mg/kg group（60.2±3.4）% than in model group（28.0±1.6）% and negative control group（28.6±1.7）%（P=0.000）. Immunohistochemistry showed that protein expression of Smad7 was significantly higher in TIMP-1 siRNA 0.250 mg/kg group（8.55±0.67） and TIMP-2 siRNA 0.250 mg/kg group（8.23±0.85） than in model group（4.25±0.53） and negative control group（4.19±0.55）（P=0.000）. Western blot showed that protein expression of Smad7 was significantly higher in TIMP-1 siRNA 0.250 mg/kg group（0.706±0.039） and TIMP-2 siRNA 0.250 mg/kg group（0.698±0.038） than in the model group（0.278±0.013） and negative control group（0.285±0.014）（P=0.000）. Conclusion：Silencing the TIMP-1 and TIMP-2 with siRNA could be involved in the improvement of the hepatic fibrosis in rats.

Abstract:Objective：To investigate the effects of HBX on Gankyrin expression and role of Wnt/β-catenin signaling pathway in the regulation of HBX on Gankyrin. Methods：HBX adenovirus vector Ad-HBX-GFP was transfected into human hepatocellular carcinoma cell line HepG2，SMMC-7721 cells and Si-HBX molecules was transfected into HepG2 2.15 cells with LipofectamineTM 2000. Expres－sions of HBX and Gankyrin were detected by RT-PCR and Western blot. β-catenin adenovirus vector was transfected into HepG2 and SMMC-7721 cells，and expressions of β-catenin and Gankyrin were detected by Western blot. HBX was overexpressed in β-catenin silenced HepG2 and SMMC-7721 cells，and expressions of β-catenin and Gankyrin were detected by Western blot. Results：①Expres－sions of Gankyrin were higher in HepG2.2.15 cells than in HepG2 cells（P＜0.05）. ②mRNA and protein expressions of Gankyrin were increased significantly in Ad-HBX-GFP group than in Ad-GFP group in HepG2 and SMMC-7721 cell line（P＜0.01）. ③mRNA and protein expressions of Gankyrin were decreased significantly in Si-HBX group than in Si-NC group in HepG2.2.15 cells（P<0.05）. ④Protein expressions of β-catenin were increased significantly in Ad-HBX-GFP group than in Ad-GFP group after overexpressing HBX protein in HepG2 and SMMC-7721 cell line（P＜0.01）. ⑤Protein expressions of Gankyrin were increased significantly in Ad-HBX-GFP group than in Ad-GFP group after overexpressing β-cateninin HepG2 and SMMC-7721 cell line（P＜0.01）. ⑥Protein ex－pressions of Gankyrin were not increased in Ad-si-β-catenin-RFP group than in Ad-RFP group after silencing β-catenin in HepG2 and SMMC-7721 cell line and overexpressing HBX gene（P＜0.01）. Conclusion：HBX-Wnt/β-catenin-Gankyrin signal transduc－tion pathways are existed in hepatocarcinogenesis. Inhibiting the function of β-catenin can interrupt the up-regulation of Gankyrin by HBX，which may provide a new treatment method for hepa－tocellular carcinoma.

Abstract:Objective：To observe the inhibition of hepatitis B virus（HBV） transcription in vitro by Zinc finger protein（ZFP） which was designed specifically to target X gene promoter of HBV（HBX）. Methods：Recombinant plasmid pcDNA3.1-ZFP was transfected into HepG2.2.15 cells. Expression of HBX protein was detected by Western blot at 24 h after being transfected. Hepatitis B e antigen（HBeAg） and HBV DNA levels in supernatant of HepG2.2.15cells at 24 h after being transfected with pcDNA3.1-ZFP were detected by ELISA and real-time PCR. HBV mRNA was tested by RT-PCR. Results：ZFP could inhibit the expression of HBX in HepG2.2.15 cells. In the presence of ZFP，HBV DNA level and HBeAg was considerably reduced by 51.7%（t=23.079，P=0.000，９５％CI=44.98%-58.52%） and 33.8%（t=3.887，P=0.003，９５％CI=12.12%-55.48%） respectively，while HBV mRNA was sharply decreased（t=3.616，P=0.022）. Conclusion：ZFP protein designed to target X gene promoter of HBV can inhibit the transcription of HBV in HepG2.2.15 cells effectively.

Abstract:Objective：To study the identification the stem property of CD133+ liver cancer cells and inhibition effect of 131I-labeled monoclonal antibody（mAb）against CD133 on them in vitro and vivo. Methods：The monoclonal antibody CD133 labeled with 131I was prepared using the chloramines-T method and its stability was evaluated. Magnetic-activated cell sorting（MACS） was used to isolate CD133 and CD133- cells from HepG2 cells. Flow cytometry（FCM） was used to detect the expression of CD133 before and after the cell isolation. The stem cell properties of sorted CD133 cells were validated by sphere-forming assay，colony formation assay in vitro and tumorigenesis experiment in vivo. There were four groups including 131I-labeled anti-CD133 mAb group，131I group，CD133 mAb group and 131I CD133 mAb group. The inhibitory effects of different treatments on the proliferation of CD133 cells were measured by MTT assy. The animal model was established by subcutaneous inoculation of CD133 -HepG2 cells（5×104） to right front legs of BALB/c mice. After tumor model being established，12 mice were randomly divided into 4 groups. Drugs were injected into the tail vein of the nude mice at a frequency of one time/2 d（14 times in total）. The tumor size and volume were measured twice a week after the treatment and the tumor inhibitory rate was calculated. After 4 weeks，the mice were sacrificed for pathological examination. Results：The labeling ratio of the 131I-labeled anti-CD133 mAb was 89.34% and the radiochemical purity was 98.21%. The sorted CD133 cells showed a high purity（98.46±0.97）% compared with that before cell sorting（1.78±0.54）%. CD133 cells showed a high tumor sphere formation ability and turmorigenesis capacity compared with those of CD133- cells. The tumor inhibitory rate of CD133 cells was significantly higher in 131I-labeled anti-CD133 mAb group than in 131I group，CD133 mAb group and blank control group in vivo and vitro（P<0.05）. Conclusion：１31I-labeled anti-CD133 mAb can effectively inhibit the growth of CD133 -HepG2 cells in vivo and vitro.

Abstract:Objective：To investigate the effect of SIRTI inhibitor sirtinol，trichostatin A（TSA） on hepatitis B virus（HBV） replication and to discuss the role of histone deacetylase in the process of HBV replication. Methods：The tolerance degree of HepG2.2.15 cell to sirtinol and TSA was determined by MTS assay. The effects of sirtinol and TSA on the expression of HBV replicative intermediates were measured by real-time PCR and Southern blot. The effects of sirtinol and TSA on the expression of HBc，HBsAg and HBeAg were analyzed by Western blot and ELISA. Results：Sirtinol inhibited the expression of HBV replicative intermediates and HBc as well as the secretion of HBsAg and HBeAg. TSA promoted the HBV replication. Conclusion：SIRT1 inhibitor sirtinol may have anti-HBV potentiality.

Abstract:Objective：To detect adefovir-resistant mutants of hepatitis B virus（HBV） using peptide nucleic acid（PNA） probes. Methods：Specific PNA probes were designed for genotypes B，C D，which were then hybrized with biotinylated PCR products. Different hy－bridization factors such as probe concentration，hybridization temperature and time were investigated to establish the optimization conditions. Finally，72 hepatitis B virus（HBV） clinical samples with adefovir dipivoxil treatment were investigated using PNA hybriza－tion assay. Its usefulness was validated by comparing with sequencing data. Results：（1）PNA probes could be fixed on the membrane by cross link and used for reverse hybridization. （2）Complete concordance of PNA hybrization assays was 97.88% with direct sequenc－ing. Conclusion：We develop an assay using PNA probes to detect adefovir-resistant mutants of HBV.

Abstract:Objective：To construct the recombinant adenovirus vector of hepatitis B virus（HBV） and to provide a more effective and convenient method for HBV-relative research. Methods：HBV 1.3 fold genome was subcloned to shuttle vector pAdTrack-TO4，then pAdTrack-TO4-HBV1.3 was linearized by PmeⅠ and transfected into BJ5183 cells containing pAd-Easy1. The confirmed recombi－nant plasmid with PacⅠ restriction endonuclease was linearized and transfected into HEK-293 cells to generate recombinant aden－oviruses containing HBV1.3 fold genome. Efficiency of the infection was detected by fluorescence microscope and expression of HBV proteins was tested by ELISA and Western blot. Intravenous recombinant adenovirus HBV1.3 was injected into C57BL mouse and changes of HBs expression in mouse were observed. Results：Cellular efficiency of recombinant adenovirus HBV1.3 infection could reach above 95%. Expression of HBs and HBe in experimental group was much higher than that in control group according to ELISA test. High-level HBs expression was detected by Western blot after recombinant adenovirus HBV1.3 infection. Animal experiment showed that after being injected with the recombinant adenovirus HBV1.3，HBs began to express on the 3rd d，peaked on the 5th d and declined gradually on the 7th d. Conclusion：Recombinant adenovirus with high infective efficiency is constructed and can express HBV easily，providing a much more efficiency method for HBV-relative study.

Abstract:Objective：To evaluate the association between the susceptibility of knee osteoarthritis（KOA） and the gene polymorphisms of interleukin-16 in Han population. Methods：A case-control design was employed in this study. A total of 150 patients di－agnosed with primary KOA and 147 controls matched for age，sex and body mass index of Han ancestry were enrolled. Periph－eral blood samples were collected，and the polymerase chain re－action and restriction fragment length polymorphism analysis were used to check the three single nucleotide polymorphisms in the interleukin-16（IL-16）gene. The results were verified by gene sequencing. Results：The genotypes and polymorphisms were distributed in line with the Hardy-Weinberg equilibrium in the case and control groups. The results of non-conditional logistic regression analysis in codominant model showed that T/G genotype（rs11556218） may be a risk factor（OR=2.06；95%CI=1.26 to 3.35；P=0.005），G/G genotype（rs11556218） may be another risk factor（OR=2.52；95%CI=1.10 to 5.74；P=0.005），C/T genotype（rs4072111） may be a risk factor（OR=2.45；95%CI=1.42 to 4.22；P=0.000）and T/T genotype（rs4072111） also increased the risk of KOA（OR=4.04；95%CI=1.06 to 15.37；P=0.000）. Linkage disequilibrium was found between loci rs11556218 and rs4778889（D’=0.63，R2=0.236）. Further stratification analysis by SNPstats based on phenotype showed that TTT may be a risk factor（OR=3.70；95%CI=1.57 to 8.72；P=0.003） and GCC may be another risk factor（OR=6.22；95%CI=2.37 to 16.33；P=0.000）. Conclusion：The genetic polymorphisms of IL-16 gene are one of risk factors associated with the susceptibility of knee osteoarthritis in the Chinese Han population.

Abstract:Objective：To construct a lentiviral vector for RNA interference（RNAi） for augermenter of liver regeneration（ALR） gene and to detect its interference efficiency in human hepatoma cell line HepG2 and QGY. Methods：Effective sequence of siRNA target－ing ALR gene was confirmed. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed，synthesized and cloned into the HpaⅠ and XhoⅠ resriction sites of GV118 lentiviral vector. The resulting lentivirus vector containing ALR shRNA was named LV-ALR-shRNA. The competent E.coli cells were transformed and positive clone were screened，identified and sequenced. The recombinant lentivirus was packaged into mature lentivirus by 293T cells and the virus titer was measured by hole dilution method. The packaged lentivirus was used to infect HepG2 and QGY cells. The expression of ALR in the cells was detected by real-time PCR and Western blot. Results：PCR and sequencing verified that the recombinant lentiviral RNAi vector of ALR was constructed successfully. The titer of concentrated virus was 8E+8TU/ml.After being transfected by virus，ALR mRNA and protein levels were obviously reduced in both human hepatoma cell line HepG2 and QGY compared with those of nega－tive control and bland control cells（P<0.05）. Conclusion：The lentiviral RNAi expression vector targeting ALR gene is successfully constructed and it could effectively silence the expression of ALR in human hepatoma cell line HepG2 and QGY.

Abstract:Objective：To observe the changes in expressions of calpain2 and its inhibitor calpastatin cluring liver fibrosis and to explore their effects on apoptosis of hepatic cells. Methods：Forty male Wistar rats were randomly divided into four groups（4 weeks normal control group，8 weeks normal control group，4 weeks liver fibrosis group and 8 weeks liver fibrosis group），each group in－cluded 10 rats. Groups were induced by hypodermic injection of 40% CCl4 every 3 d for 4 weeks and 8 weeks respectively，the dosage of CCl4 is 0.3 ml/100 g body weight. Apoptosis of hepatic cells was detected by TUNEL. Additionally，protein expressions of calpain 2，calpastatin and activated caspase 3 were determined by immunohistochemistry and Western blot and mRNA expressions of cal－pain2 and calpastatin were determined by real-time PCR. Results：Results of immunohistochemistry and Western blot revealed that there was no difference in expressions of calpain 2 and calpastatin protein between 4 weeks fibrosis group and normal control group（P >0.05）. In 8 weeks fibrosis group，expressions of calpain2 were increased obviously and expressions of calpastatin were decreased with the increase of liver fibrosis degree. Additionally，real-time PCR results revealed that expressions of calpain2 mRNA in 4 weeks and 8 weeks fibrosis groups were elevated compared with those in normal control group（P＜0.01），while expressions of calpastatin mRNA were decreased in 8 weeks fibrosis group（P＜0.01）. Expressions of activated caspase3 protein in 4 weeks and 8 weeks fibrosis groups were increased compared with those in normal control group and the number of apoptotic cells were also elevated in fibrosis groups than in normal control group（P＜0.01）. Conclusion：Calpain2 and calpastatin may play important roles in the process of liver fibrosis. Their effects on fibrosis might be exerted through promoting the apoptosis of hepatic cells.

Abstract:Objectives：To examine the effects of curcumin on body weight，blood glucose，blood lipid，glucose tolerance and monocyte chemoattractant protein 1（MCP-1） and tumor necrosis factor-α（TNF-α） expression and secretion. Methods：Male SD rats were fed with either high fat diet（HFD） or control diet（CD）. HFD-induced obese rats were randomly divided into 4 groups：HFD with saline treatment，HFD plus 10 mg/kg，30 mg/kg，and 100mg/kg of curcumin，respectively，with 10 rats in each group. Curcumin was applied to obese rats for 3 weeks. Body weight in each group was monitored weekly，and glucose tolerance test was performed. Serum lipid and glucose levels were detected using an automated biochemistry analyzer. The transcriptional levels of MCP-1 and TNF-α in epidi-dymis white adipose tissues（WAT） were examined by real-time PCR. Results：Three weeks after the treatment with curcumin，body weight and the expression of MCP-1 and TNF-α mRNA of the obese rats were significantly decreased. The contents of serum lipids and glucose were also improved and high and middle dose groups were better than low dose group in improving HFD induced metabolic dysfunction. Conclusion：Curcumin treatment can improve metabolic dysfunction induced by HFD，possibly through an inhibitory effect on the expression of inflammatory cytokines in WAT in obese rats. In addition，high and middle doses of curcumin exhibit better effect than low dose of curcumin.

Abstract:Objective：To investigate whether inflammatory stress aggravates hepatosteatosis induced by carbon tetrachloride（CCl4） and the molecular mechanism. Methods：Male C57BL/6J mice of 6-8 weeks were randomly divided into control group（n=6） and inflam－mation group（n=6）. Inflammation was induced via subcutaneous casein injection of casein for 16 weeks after the treatment of CCl4 for 2 weeks. After 18 weeks，the liver indicator was measured to study the damage to the liver. Level of serum inflammatory factor inter－leukin-6（IL-6） was determined by ELISA. Oil red O staining was used to evaluate lipid accumulation in the liver. Lipid levels of liver tissues were measured by chemical enzymatic assay. mRNA expression of SREBP-1 and 3-hydroxy-3-methylglutaryl coenzyme A re－ductase（HMGCR） was measured by real-time PCR and the protein expression of SREBP-1 and HMGCR was analyzed by Western blot. Results：Expression of IL-6 was higher in inflammation group than in control group（t=8.434，P=0.000）. Expression of HMGCR and SREBP-1 mRNA was significantly increased in inflammation group than in control group（t=10.612，P=0.000；t=12.749，P=0.000）. Expressions of SREBP-1 and HMGCR protein were consistent with the mRNA levels based on Western blot. Hepatic lipid levels were significantly higher in inflammation group than in control group（t=6.148，P=0.000；t=7.288，P=0.000）. Liver indicator of mice were significantly higher in inflammation group than in control group（t=8.452，P=0.000）. Increased lipid accumulation demonstrated by Oil red O staining was observed in inflammation group. Conclusion：Inflammatory stress probably promotes cholesterol synthesis in fatty liver induced by CCl4 via SREBP-1-HMGCR pathway，which may contribute to the abnormal lipid accumulation in the liver.

Abstract:Objective：To clone the full length gene of Chromate reductase T（ChrT） from serratia sp. CQMUS2，to construct the prokary－otic expression vector and to transform it into E.coli to express protein，and to analyze the activity of expression products. Methods：The ChrT gene was cloned from DNA template of serratia sp. CQMUS2，a chromium-resistant bacteria by PCR and was connected with prokaryotic vector pET-28a（+）. Then the recombinant vector was transformed into the E.coli BL21（DE3） to express protein. The activity and the reduction ability of hexava－lent chromium of recombinant ChrT enzyme were determined by the enzymatic reaction. The optimal pH and temperature of recombi－nant ChrT enzyme were also studied. Results：The ChrT gene was an ORF of 567 bp that encoded a 188-aa protein with a relative molecular mass of 20 kD At. 10 h after the induction with IPTG，the target protein was successfully expressed in E.coli and the base sequences and relative molecular mass were consistent with ChrT enzyme. After the purification，ChrT enzyme reduced Cr（Ⅵ） in the enzymatic system and the Cr（Ⅵ） concentration was significantly lower in experiment group than in control group（P=0.000）. The ac－tivity of ChrT enzyme was up to 997 U/L，the optimum pH of ChrT enzyme was 6.5-7.5 and the optimum temperature was 37 ℃. Conclusion：ChrT enzyme can reduct Cr（Ⅵ），which provide solid foundation for further study on the high-level expression of Cr（Ⅵ）-removal engineering bacteria.

Abstract:Objective：To investigate the changes of interleukin- 17（IL-17）and interleukin-4（IL-4） in nasal lavage fluid and inter－leukin-23（IL-23） mRNA in nasal associated lymphoid tissue of animal model of allergic rhinitis（AR）. Methods：Guinea pig model of AR was established. Nasal lavage fluid was collected，then IL-17 and IL-4 levels in nasal lavage fluid were measured by ELISA. Nasal associated lymphoid tissue was separated，then IL-23 mRNA expression level was detected by RT-PCR. Differences of cytokines level were compared between model group and control group. Results：Level of IL-17 in nasal cavity lavage fluid was （（4.98±1.54） pg/ml） in model group and （（12.68 ± 3.32） pg/ml） in the control group. Level of IL-17 decreased in model group than in control group with statistically significant differences（t=10.35，P=0.006）. mRNA level of IL-23 in nasal-associated lymphoid tissue was the same with IL-17. But the level of IL-4 in the nasal cavity lavage fluid（（31.20±4.25） pg/ml） was significantly higher in model group than in control group（（6.60±1.36） pg/ml），with statistically significant differences（t=17.26，P=0.001）. Conclusion：Levels of IL-17 and IL-23 decrease in AR model. But level and levels of IL-4 are significant higher in model group than in control group. Thereby we infer that there is the imbalance of Th2/Th17 cytokine network in AR，which may play an important role in allergic inflammation.

Abstract:Objective：To investigate the risk factors for hepatocellular carcinoma（HCC） combined with portal vein tumor thrombus（PVTT）. Methods：Retrospective analysis was conducted for 126 HCC patients chosen at random in the department of hepatobiliary surgery in the First Affiliated Hospital of Chongqing Medical Uni－versity from April 2009 to April 2011. Alpha-fetoprotein（AFP） level（400 ng/ml is the dividing line），with or without the PVTT，tumor size，cell differentiation grade（Edmondson-Steiner grade），tumor number，gender and vascular invasion were observed. Chi-square test and non-conditional logistic regression analysis were adopted to do statistical analysis. Results：Totally 126 patients were diagnosed as HCC including 56 HCC patients combined with PVTT（44.4%）. Vascular invasion，tumor size，high level of AFP，cell differentiation grade，tumor number were the risk factors for PVTT formation（χ2=20.97，13.67，4.73，5.04，all P<0.05）. Vascular invasion，tumor size，cell differentiation grade were the risk factors for AFP（χ2=8.121，4.038，all P＜0.05）. AFP and vascular invasion were the independent risk factors for PVTT. Conclusion：High leveled AFP could become one risk factor for HCC patients combined with PVTT，which can help to find or treat PVTT at early stage.

Abstract:Objective：To evaluate the application characteristics of glucocorticoid after liver transplantation in the treatment of autoim－mune hepatitis（AIH）. Methods：All data was collected from the First Affiliated Hospital of Chongqing Medical University and West China Hospital of Sichuan University，including 99 adult patients with liver transplantation（from January 2005 to January 2012）. All patients were assigned to two groups：36 cases with AIH（observation group） and 63 cases without AIH（control group）. Differences in perioperative parameters and glucocorticoid usages between the two groups were observed. Results：In order to combat rejection，liver and various organ functions remained stable. Glucocorticoid usage was significantly larger in observation group than in control group（P=0.000）. Dosage of glucocorticoid was significantly higher in observation group than in control group（（17.8±5.3） mg/d vs. （9.4±3.9） mg/d）. Duration of using glucocorticoid was significantly longer in observation group than in control group. （385.2±21.7） d vs. （47.1±11.3） d）（P=0.000）. Conclusion：Long-term application of glucocorticoid during immune rejection period can improve trans－plant outcome during immune rejection period for AIH.

Abstract:Objective：To investigate the role of Th17/Treg and some cytokines imbalance in the pathogenesis of childhood Henoch-Schonlein purpura（HSP） and to further explore the clinical effectiveness and immunomodulatory effects of compound glycyrrhizin（GL） in HSP. Methods：Twenty-seven HSP patients were chose as GL-treated group，another 26 patients were used as conventional therapy group，and 25 age-and sex-matched healthy children were used as healthy controls. Intravenous injection of 0.5-2.0 ml/kg GL for 7 days was provided in GL-treated group on the basis of treatments in conventional therapy group. Blood samples of patients were ob－tained at the acute stage and after 7 days’ treatment.Postive cell rates of peripheral Th17 and Treg cells were detected by flow cytom－etry using intracellular staining. Serum levels of interleukin-17，interferon-γ，TNF-α，interferon inducible protein-10 were detected by ELISA. Results：①Clinical effectiveness：renal complications in GL-treated group were relieved and recurrence of HSP within 1 month was lower. ②Positive cell rate of Treg and Th17 cell：compared with that of healthy controls，the positive cell rate of Treg in GL-treated group was significantly down-regulated during acute phase（P=0.010） and was up-regulated after the treatment（P=0.059）. In conventional therapy group，positive cell rate of Treg was lower after the treatment compared with that before treatment and that of healthy control group（P=0.006，P=0.013）. The positive cell rate of Th17 in GL-treated group was significantly higher before the treat－ment compared with that after the treatment and that of conven－tional therapy group（P=0.024，P=0.013）. After the treatment，there was no difference in the positive cell rate of Th17 between GL-treated group and conventional therapy group（P=0.337）. The positive cell rate of Th17 in conventional therapy group was significantly higher before the treatment compared with that after the treatment（P=0.014）. ③Cytokines：Serum levels of IL-17 and IFN-γ were higher in GL-treated group and conventional therapy group than in healthy control group before the treatment（P=0.013，P=0.010）. There was no difference in serum levels of IL-17 and IFN-γ between GL-treated group after the treatment and healty control group（P=0.052，P=0.154）. There was no difference in serum levels of IL-17 and IFN-γ in conventional therapy group before and after the treatment（P=0.218，P=0.970）. Serum levels of TNF-α and IP-10 in GL-treated group were down-regulated after the treatment（P=0.011，P=0.037）. But no difference was detected in conventional therapy group before and after the treatment（P=0.247，P=0.709）. Conclusion：①GL could relieve the renal complications and decrease recurrence of HSP in a short period of time. ②Treg and Th17 might in part contribute to the process of HSP. ③GL could help to up-regulate Treg and down-regulate Th17，IL-17，IFN-γ，TNF-α，IP-10 level，thus regulating the Th17/Treg deviation and cytokine im－balance and alleviating cellular immunity in HSP.

Abstract:Objective：To summarize the clinical characteristics and to do etiological diagnosis for one confirmed case of plastic bronchitis. Methods：One child of more than 5 years old was enrolled. Clinical manifestations were fever，cough，gasp，anhelation and progressive difficulty of breath. Thoracic CT showed that the left lung had large parts of consolidation，possibly being pulmonary atelectasis. Bronchoscopy and lavage were conducted and the ingrown foreign body in the air passage being taken out. The child was diagnosed as plastic bronchitis. Bronchoalveolar lavage fluid of the sick child was taken out for detecting 16 kinds of respiratory viruses. Results：Detecting results of 16 kinds of respiratory viruses indicated that the sick child was influenza A virus positive，and the nucleoprotein sequencing of the further amplified virus was clarified as H3N2. At the same time，human bocavirus（HBoV） were found out and the number of virus copy was 3×105 copies/ml. The child was discharged from the hospital after the endogenous foreign matter being taken out. Conclusion：As a pediatric intensive case，plastic bronchitis can be clearly diagnosed by bronchoscopy and removing endogenous foreign body through lavage，which is essential for the effective treatment. The patient’s bronchoalveolar lavage etiological diagnosis remind us of mixed infection of human influenza viruses and HBoV，which is worthy of attention.

Abstract:Objective：To study the effect of respiratory frequency on spectral components of rabbit heart rate variability（HRV） and to discuss how to exclude it. Methods：Totally 12 healthy male rabbits aged 4-5 months and weighting 2.5-3.0 kg were selected. Rabbit respiratory control system was established. Electrocardiograph，instant blood pressure and respiratory waveform under different respira－tory frequencies（RF）（40，50，60 times/min） were recorded synchronously via SKY-A4（a three channel electrophysiolograph）. HRV ＆ BRS2.0-a HRV analysis system was used to observe the shift of high frequency peak（HFP） and to analyze power spectra density（PSD）. Two different analysis methods were adopted to analyze PSD：traditional heart rate variability（tHRV） analysis method and enhanced heart rate variability（eHRV） analysis method. Comparison between the results of the two methods was made. Results：①the shift of HFP：HFP was located at the conjunction of low frequency（LF） segment and high frequency（HF） segment when RF 50 times/min；HFP shifted into the LF segment when RF 40 times/min；total HFP shifted to HF segment when RF 60 times/min. ②tHRV results：RF exerted no effect on total power while significantly affected LF，LFnorm，HF，HFnorm. Compared with those when RF 50 times/min，HF and HFnorm decreased（P=0.179，P=0.011）while LF，LFnorm，LF/HF increased（P=0.04，P=0.001，P=0.028） when RF 60 times/min. Compared with those when RF 50 times/min，HF and HFnorm decreased while LF，LFnorm，LF/HF increased when RF 40 times/min.③eHRV results：there was no significant difference in LF，LFnorm，HF，HFnorm，LF/HF when RF 60 times/min and RF 40 times/min compared with those when RF 50 times/min. Conclusion：RF slowing down would shift HFP to relatively low frequency，sometimes to LF segment，which would increase LF and decrease HF. Enhanced HRV analysis could exclude this influence.

Abstract:Objective：To verify the antigen-antibody（P24 polyclonal and monoclonal antibody） binding in detecting Borna disease virus（BDV）-P24 protein in paraffin tissue sections and to explore the work conditions. Methods：Forty Sprague-Dawley rats born within 24 h were intracranially（i.c.） inoculated in the right cerebral hemisphere with either BDV solution or phosphate-buffered saline（con－trol：sham inoculated）. Forty Sprague-Dawley rats and 40 C57 rats without injection were chosen as negative controls. The brains of rats on 60 d after birth were perfused and were subsequently embedded into paraffin. Then the immunohistochemical staining was per－formed following Envision two-step protocol. The primary antibody was chosen as P24 polyclonal and monoclonal antibody and the di－lution ranged from 1∶50 to 1∶5 000. The primary antibody was replaced by PBS as blank control. The brownish yellow or brown parti－cles distributing in the cells were considered as positive and total score was got by multiplying positive cell proportion score and posi－tive intensity score. Results：There were significant differences in staining scores between experimental group and control group（polyclonal P24 group Z=-3.108，P=0.000，monoclonal P24 group Z=-4.605，P=0.000. It showed a high sensitivity with slightly stained background in dilution 1∶200，1∶400 using polyclonal P24 staining while a high sensitivity without stained background in dilution 1∶100，1∶200，1∶400 using monoclonal P24 staining. Conclusion：BDV P24 polyclonal and monoclonal antibody can bind the antigen distributed in paraffin sections of BDV infectious rat. The recommended dilution of polyclonal P24 antibody is 1∶200 and 1∶400. The recommended dilution of monoclonal P24 antibody is 1∶100，1∶200 and 1∶400.

Abstract:Objective：To optimize urinary protein preparation method，to obtain a two-dimensional gel electrophoresis（2-DE） map with more urinary protein spots and higher resolution，and to provide technical supports for urinary proteomic researches. Methods：Firstly，urinary protein was extracted by 6 different preparation methods and then the total protein content between them were compared；secondly，protein spots as well as three-time repeated matching rate of 2-DE maps of these six methods were ana－lyzed，and P value for statistical analysis was set；finally，for the best method，2 different polyacrylamide gel electrophoresis（PAGE） concentrations and 2 different immobilized pH gradient（IPG） strips were utilized to compare protein spots and resolution of 2-DE maps. Results：Method named ultrafiltration got the most proteins（（791±17） μg，P=0.000） as well as maximum protein spots（724±29，P=0.000）. Utilization of 12.5% PAGE gel combined with IPG pH 3-10 strip was beneficial for the whole analysis of urinary proteins，es－pecially for the acid and basic proteins，however，when combined with pH 4-7 strip，a 2-DE map of higher resolution within the iso－electric point range of 4-7（P<0.05） can be obtained. Conclusion：This research establishes a 2-DE compatible urinary protein prepa－ration method and obtains more comprehensive urinary protein spots information，which lays a solid foundation for urinary proteomics research of clinical physiology and diseases.

Abstract:Objective：To study the high performance liquid chromatography（HPLC） fingerprint chromatogram and chemical pattern recognition method of Euphorbia chrysocoma Levl.etVant. Methods：Fifteen batches of Euphorbia chrysocoma Levl.etVant. samples were harvested at different time periods in different producing areas of Guizhou and were carried out to gain fingerprints by HPLC. Cluster analysis was conducted to do chemical pattern recognition of fingerprints of Euphorbia chrysocoma Levl.etVant.. Results：Fin－gerprints of 15 batches of samples demonstrated 13 characteristic peaks. Chromatographic profiles of 15 batches of samples were simi－lar；major differences resulted from intensity of chromatographic peaks. Samples were grouped into 2 types based on the harvest time. Conclusion：Components of Euphorbia chrysocoma Levl.etVant. can be well separated on HPLC chromatogram. HPLC fingerprint chromatogram has good repeatability and stability and can be used for quality control of Euphorbia chrysocoma Levl.etVant．

Abstract:Objective：To establish a high performance liquid chromatography-diode array detection（HPLC-DAD） method for the si－multaneous determination of four alkaloids in corydalis yanhusuo，and to compare the contents of four alkaloids at different production places. Methods：Angilent Eclipse XDB-C18（5 μm，4.6 mm×250 mm） was used as the analytical column. Acetonitrile-0.05% H3PO4（pH 6.0）was used as the mobile phase with gradient elution and the flow rate was 1.0 ml/min. The detection wavelengths were 270 nm for palmatine and corydaline，280 nm for tetrahydropalmatine and tetrahydroberineper respectively. Results：Palmatine，coryda－line，tetrahydropalmatine and tetrahydroberineper have a good linear relation within ranges of 1-10 μg/ml，10-100 μg/ml，5-30 μg/ml，1-10 μg/ml，respectively. Spiked recovery of high，middle and low concentration ranged from 98.2% to 101.3%. Conclusion：The method is simple，accurate and reproducible and can be used to determine the above four alkaloids．

Abstract:Objective：To establish reversed phase-high performance liquid chromatography（RP-HPLC） for the determination of sily－bin in rabbit plasma and to provide a method for studying pharmacokinetics and relative bioavailability of bioadhesive microspheres of silybin. Methods：Separation was achieved using an Agilent Zorbax C18 column（250 mm×4.6 mm）.The mobile phase were：A，20 mmol/L KH2PO4 buffer，pH=3.0；B，methanol（A∶B=55∶45，V/V）. A randomized cross-over design was performed on 12 healthy rabbits. Results：Main pharmacokinetic parameters of bioadhesive microspheres and suspensions：Cmax（905.94±230.16） ng/ml and （321.32±86.65） ng/ml；T（peak）（4.70±1.02） h and （1.66±1.00） h；t1/2（4.07±0.12） h and （1.64±0.09） h；AUC0-24（11 841.35±579.58）（ng·h）/ml and （1 537.34±156.43） （ng·h）/ml，respectively. Relative bioavailability of the bioadhesive microspheres to suspensions was （770.2±370.5）%. Conclusion：Relative bioavailability of bioadhesive microspheres is 770% to suspensions of silybin and bioadhesive micro－spheres promote the absorption of silybin.

Abstract:Objective：To evaluate the clinical efficacy of telbivudine（LdT） in the treatment of patients with hepatitis B virus-related cirrhosis. Methods：A total of 43 patients with HBV DNA level more than 5 log10 copies/ml were enrolled in this study：23 patients received LdT 600 mg orally daily（LdT group） and 20 patients received no antiviral treatment（control group）. Liver function and serum HBV DNA level were assessed every 3 months. Results：The parameters of liver function（alanine aminotransferase（ALT），as－partate aminotransferase（AST），total bilirubin（TBIL），Child-pugh score）and serum HBV DNA level were significantly improved in LdT group compared with those in control group with the extension of the treatment（HBV DNA level was reduced to 4.83±0.70 at the 4th week；AST，TBIL and Child-pugh score were reduced to （55.19±22.48） U/L，（36.81±22.11） μmol/L，6.65±1.94 respec－tively at the 12th week；ALT was reduced to （63.67±19.52） U/L at the 24th week）（P<0.05）. The serum ALB level showed no remarkable change in LdT group than in control group. Two patients in LdT group experienced virological breakthrough after 24 and 36 weeks（rtM204 mutations） and were salvaged treat－ment with adefovir dipivoxil. Conclusion：LdT suppresses viral replication and improves liver function in patients with chronic hepatitis B cirrhosis，but there is still a risk of emergence of LdT resistance.

Abstract:Objective：To investigate the correlation of plasma platelet activating factor（PAF） level of patients with sepsis at admission to ICU with their illness severity and prognosis. Methods：Totally 102 patients with sepsis were divided into three subgroups：sepsis group（n=30），severe sepsis group（n=26） and septic shock group（n=46）. All patients were divided into survival group and non-sur－vival group according to their prognosis during the hospital. Forty volunteers were selected as normal controls. The plasma PAF level，serum concentration of inflammatory biomarker procalcitonin（PCT），acute physiology and chronic health evaluationⅡ（APACHEⅡ），simplified acute physiology scoreⅡ（SAPSⅡ） and sequential organ failure assessment（SOFA） scores of patients at admission to ICU were compared. Correlation of plasma PAF level with serum concentration of PCT，scores of APACHEⅡ，SAPSⅡ and SOFA were an－alyzed. Results：Plasma PAF levels were significantly higher in sepsis group，severe sepsis group and septic shock group than in control group（z=-5.424，-5.512，-7.662，P=0.000），and the PAF level was significantly higher in septic shock group than in sepsis and severe sepsis groups（z=-4.559，-3.910，P=0.000） while there was no statistically difference in PAF level between sepsis group and severe sepsis group（z=-0.427，P=0.669）. Plasma PAF level of patients with sepsis was positively correlated with the serum concentration of PCT，scores of SAPSⅡ and SOFA（rs=0.376，0.326，0.483，P=0.000，0.001，0.000），while it was not correlated with APACHEⅡ score（rs=0.143，P=0.152）. Plasma PAF level of patients in non-survival group was significantly higher than that in sur－vival group（z=-3.152，P=0.002）. Receiver operating character－istics（ROC） curve showed that both PAF and APACHEⅡ score could be used as predictors of mortality during the hospitalization（area under the curve=0.708，0.787）. Conclusion：PAF level of patients with sepsis at admission to ICU is essentially and positively correlated with illness severity and prognosis，which could be used to guide the assessment of illness severity and prognosis of patients with sepsis.

Abstract:Objective：To study and compare the different curative effects on Barrett’s esophagus（BE） by using esomeprazole magnesium，omeprazole and lansoprazole as the basic therapeutic regimen respectively. Methods：Totally 87 patients were randomly selected and divided into three groups. Esomeprazole magnesium，omeprazole and lansoprazole were given to the three groups respectively. Changes of BE before and after the treatment（1-5 weeks） and during the long-term maintenance treatment（12-24 months） were carefully observed. Results：Data indicated that there were significantly statistical differences among the three groups；esomeprazole magnesium group displayed more positive results than omeprazole group and lansoprazole group in the control of helicobacter pylori（Hp） infection and alleviation of gastroesophageal reflux disease（GERD） and BE symptoms（P=0.019，P=0.007）. Concerning the direct treatment effect on BE，evaluation indicators including metaplasia length reduction rate（P=0.028 2），metaplasia area reduction rate（P=0.022 6），squamous epithelium detection rate（P=0.001） and mixed epithelium detection rate（P=0.003） were better in esomeprazole magnesium group than in omeprazole group and lansoprazole group. Concerning the prevention of BE，there was no statistical difference among esomeprazole magnesium group，omeprazole group and lansoprazole group in number of cases with metaplasia aggravation after the treatment（P=0.483）. Adverse reaction detection showed that esomeprazole magnesium group had lower incidence rate than omeprazole group and lansoprazole group（P=0.013），indicating that the former treatment was much safer than the latter ones. Conclusion：Therapy with esomeprazole magnesium is much safer and more dependable with lower adverse effects. It is able to control the Hp infection more effectively，inhibit the secretion of gastric acid，alleviate or eliminate the erosion of GERD on the mucous membrane epithelium of esophagus，promote the esophageal epithelial cell proliferation，which，to some extent，contributes to the remedy of the pathological tissues and restrain BE from developing into esophageal adenocarcinoma.

Abstract:Objective：To investigate the protective effect of antibiotic prophylaxis against postoperative infection in perioperative peri－od of intravascular interventional therapy. Methods：Clinical data of inpatients with liver cancer undergoing transarterial chemoem－bolization（TACE） from January 2007 to March 2013 or inpatients with cirrhosis undergoing transjugularintrahepatic portosystemic shunts（TIPS） from January 2011 to March 2013 were collected and retrospectively analyzed. There were 538 patients undergoing TACE，including 501 males and 37 females，with an average age of （52.87±12.33）-year-old. There were 61 patients undergoing TIPS including 47 males and 14 females，with an average age of （47.52±9.55）-year-old. According to antibiotic prophylaxis in periopera－tive period of TACE and TIPS，inpatients were classified into TACE antibiotic prophylaxis treatment group，TACE non antibiotic pro－phylaxis treatment group，TIPS antibiotic prophylaxis treatment group and TIPS non antibiotic prophylaxis treatment group. Postopera－tive infection rate and risk factors of infection were analyzed and compared. Results：The postoperative infection rate of TACE antibi－otic prophylaxis treatment group and TACE non antibiotic prophylaxis treatment group were 16.71%（60/359） and 20.11%（36/179） respectively（P=0.33）. Univariate and multivariate analyses indicated that Child-Pugh score and operation time over 2 h were indepen－dent risk factors. The postoperative infection rate of TIPS antibiotic prophylaxis treatment group was 33.33（5/15） and that of TIPS non antibiotic prophylaxis treatment group was 34.78（16/46）（P=0.92）. Multivariate analysis showed that age and Child-Pugh score were independent risk factors. Conclusion：Antibiotic prophylaxis in patients with Child-Pugh A undergoing TACE or TIPS therapy is not routinely necessary. However，it is more reasonable to use appropriate antibiotics for the high-risk group who have poor liver func－tion，long operation time and advanced age.

Abstract:Objective：To assess the value of indocyanine green clearance test in selective surgical treatment of portal hypertension. Methods：Clinical data of 47 portal hypertension patients who received surgical treatment in the First Affiliated Hospital of Chongqing Medical University from April 2011 to May 2013 were collected. According to indocyanine green clearance at 15 min（ICGR15），these patients were divided into group A（ICGR15<10%），group B（10%≤ICGR15<30%） and group C（ICGR15≥30%）. According to postoperative liver dysfunction，they were divided into team M with mild liver dysfunction and team S with sever liver dysfunction. Clinical data were compared among these groups. Results：Among groups A，B，C，Child-Pugh score，blooding amount and operation time were similar（P >0.05）；the lowest the postoperative serum albumin（P=0.002），the highest the postoperative serum bilirubin（P=0.012）；the postoperative recovery time of liver function（P=0.002） and rate of postoperative severe liver dysfunction（P=0.023） were significantly different among groups. Between team M and team S，preoperative ICGR15 was the only variant parameter（P=0.027）. Conclusion：For selective surgical treatment of portal hypertension，postoperative liver function mainly depends on functional liver re－serve，not surgical procedure. Indocyanine green clearance test is useful for accurate assessment of liver function and it could be a predic－tor of severe liver dysfunction. Recovery of functional liver reserve should be one of the goals preoperative treatment.

Abstract:Objective：To study the clinical treatment of severe dental ankylosis by orthognathic-orthodontic treatment. Methods：A total of 2 patients suffering from severe dental ankylosis together with maxillofacial discrepancy were operated by a special orthognathic-or－thodontic treatment based on their individual characteristics. Results：All the surgeries were performed as planned with stable long term effects. Dento-maxillofacial deformities were corrected and the masticatory function was improved. All patients were satisfied with the results. Conclusion：For patients with severe dental ankylosis，the individualized treatment should be made based on actual situation of each patient. In this study，a variety of improved orthognathic-orthodontic treatment approaches including unconventional mandibu－lar subapical osteotomy and ‘sandwich’ onlay bone grafting were used with a satisfactory clinical effect and minimal complications，making this surgery valuable for clinical application.