To construct a subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus with suppression subtractive hybridization technique, the mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) NS5A and pcDNA3 1(-) empty vector, respectively, then the cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After the tester cDNA was hybridized with the driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. The amplified library contained 121 positive clones. Colony PCR showed that 115 clones contained 200- 1 000 bp inserts. Sequence analysis was performed in 90 clones, and the full length sequences were obtained with bioinformatics method. Altogether 46 kinds of coding sequences were acquired, which consisted of 31 kinds of known and 15 kinds of unknown ones. The obtained sequences might be target genes transactivated by NS5A protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. The results indicated that the subtractive library of genes transactivated by NS5A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins

To investigate the potential role of hepatitis B virus core protein (HBcAg) in the pathogenesis of hepatitis B and seek for an effective therapeatic approach to the treatment of hepatitis B, the improved yeast two hybrid technique was employed to construct HBcAg bait plasmid, and the plasmids were transformed into yeast AH109. Then the yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2?YPDA medium, diploid yeast was plated on both synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing X ? gal to be selected and screened two times. Plasmids were extracted from sixteen positive colonies, and they were sequenced. Among them, two colonies contained metallothionein. The interaction between HBcAg and metallothionein was confirmed by in vitro reticulocyte lysate translocation and immunoprecipitation. The results suggested that the pathogenesis of hepatitis B metallothionein might play an important role in the episode process.

To investigate the biological functions of hepatitis B virus e antigen (HBeAg) on pathogenesis of HBV, and to seek for effective methods to prevent and cure hepatitis B, we performed the yeast two hybrid system 3 technique to construct HBeAg bait plasmid. The bait plasmid was transformed into yeast AH109 and expressed in it. After the expression of HBeAg was identified by SDS page and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 YPDA medium to form diploid yeast and plated on synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing x ? gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. Thirty nine positive colonies were sequenced, among them three colonies were metallothionein. To further prove the interaction between HBeAg and metallothionein, translation was performed by using reticulocyte lysate, and immunoprecipitation was showed in vitro . The results indicated that hepatitis B virus e antigen could interact with metallothionein in vivo and in vitro.

AIM: To investigate the effect of L-arginine(L-arg) on the production of collagens of human mesangial cells. METHODS: Radioimmunoassay, hydroxyproline colorimetric assay and reverse transcription polymerase chain reaction (RT-PCR) were used to determine procollagen Ⅲ, total collagen level in the supernatant and expression of collagen Ⅳ mRNA in human mesangial cells. RESULTS: L-arg significantly inhibited the production of procollagenⅢ, total collagen in the supernatants ( P

OBJECTIVE: To optimize the formula and preparation technology of gliclazide controlled-release tablets. METHODS: Gliclazide controlled-release tablets were prepared in different formulations and the release rates of the prepared tablets were determined to obtain the optimum formula and preparation technology. RESULTS: The optimized formula of gliclazide controlled-release tablets were determined as follow: with HPMC(K4M)=17g and HPMC(E50)=35g as matrix materials; Calcium hydrogen phosphate=91g as the bulking agent and magnesium stearate as lubricant. Gliclazide controlled-release tablets showed a similar release rate as the reference samples. CONCLUSION: The releasing rate of gliclazide controlled-release tablets prepared in the optimized formula is in line with the standards stated in China Pharmacopeia.

Bile acid metabolism, gut microbiota, and bile acid receptors are involved in the development and progression of hepatocellular carcinoma (HCC). There are substantial increases in the levels of some bile acids, such as glycocholic acid, taurocholic acid, and taurochenodeoxycholic acid, in the liver tissue of HCC mice and the serum and feces of HCC patients. Bile acid metabolism due to the imbalance of the abundance of bacteria producing bile salt hydrolases and Clostridium in the intestine and the change in immune microenvironment may also promote the development of HCC. Moreover, some bile acid receptors, such as farnesoid X receptor, G protein-coupled bile acid receptor 1, pregnane X receptor, constitutive androstane receptor, and sphingosine-1-phosphate receptor 2, have been shown to participate in the development and progression of HCC through various pathways. Each link of bile acid metabolism plays a different role in the progression of HCC, and a systematic elaboration of the interaction between these links may help to deepen the understanding of the pathogenesis of HCC and develop the biological targets for early diagnosis, prognosis prediction, and precise treatment.

Objective To explore the mechanism of the secondary deterioration of neurological symptoms in Wilson' s disease (WD) at early stage of treatment using D-penicillamine. Methods Forty non-treated WD patients, 32 of encephalic and 8 hepatic type respectively, were enrolled in the study. Their neural symptoms were scored using modified Young grade. Cerebrospinal fluid (CSF) copper, serum copper, urinary copper, neuron specific enolase (NSE) in CSF and the albumin ratio CSF/serum (AR) were measured at the same time. After 3 months of treatment with D-penicillamine, neural symptoms of patients were scored again. All dates were analyzed. Results After 3 months of treatment with D-penicillamine, 15 patients (46. 9%) developed a secondary deterioration in neurological symptoms. The concentration of copper and the NSE in CSF of patients whose neural symptom was increasingly deteriorated. The serum copper declined after treatment((0. 37± 0. 09) vs (0. 25 ± 0. 08) mg/L, t = 3. 17, P < 0. 05). The 24 hours urinary copper of patients whose symptoms had deteriorated was much lower than that of patients who had not. No significant change was found in AR ratio before and after the treatment (9. 53 ± 3.18vs12.24±3.17) in the worsened group (t=1.45, P>0. 05). Conclusions The degree of the injury in the neural system and the dose of penicillamine may affect the deterioration of the neural symptom.

Objective: To explore the relationship between the change of blood cholesterol and prognosis in patients with sepsis. Methods: The clinical data of 236 patients with sepsis (observation group) admitted to Nanjing Hospital Affiliated to Nanjing Medical University from February 2014 to December 2018 were analyzed retrospectively.The general clinical data, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) of the two groups were compared with 236 patients without sepsis in the same period as the control group, high density lipoprotein cholesterol (HDL-C) and other biochemical indexes; sepsis patients were divided into survival group and death group according to the prognosis, and the related factors affecting the prognosis were analyzed by multi factor Logistic regression. Results: The survival rate of sepsis patients was 60.6% (143/236), the mortality rate was 39.4% (93/236), 143 cases in survival group and 93 cases in death group.Compared with control group, the levels of serum TC ((2.51±1.20) mmo/L vs.(3.42±1.33) mmo/L, t=6.385) and HDL-C ((1.62±0.91) mmo/L vs.(2.53±0.79) mmo/L, t=5.526) in observation group were significantly lower (all P<0.05), and that of LDL-C showed no statistically significant difference((1.95±0.93) mmo/L vs.(2.11±0.84) mmo/L, t=0.958, P>0.05). In observation group, the patients in death group were older than those of the survival group ((75.4±10.3) years vs.(64.3±16.0) years, t=4.984, P<0.05), serum creatinine (SCr) was higher than that of survival group((252.3±65.2) μmol/L vs.(168.3±47.8) μmol/L, t=5.604, P<0.05), the levels of serum TC ((2.20±1.46) mmo/L vs.(2.91±1.12) mmo/L, t=6.157, P<0.05), HDL-C ((1.41±0.51) mmo/L vs.(1.95±0.65) mmo/L, t=5.090, P<0.05) and LDL-C ((1.71±0.67) mmo/L vs.(2.02±0.84) mmo/L, t=4.525, P<0.05) were significantly lower than those of survival group.Logistic regression analysis showed that age was the risk factor of death in sepsis patients (OR=1.035, 95%CI 1.012-1.049, P=0.008), and TC was the protective factor on the prognosis of sepsis patients (OR=0.748, 95%CI 0.693-0.822, P=0.015). Conclusion TC and HDL-C were significantly decreased in sepsis patients, while those who died were further decreased than those who survived.As a protective factor TC can be an effective biochemical index to evaluate the prognosis of patients with sepsis.

ObjectiveTo investigate the clinical effect and safety of transcatheter arterial chemoembolization (TACE) combined with microwave ablation (MWA) in the treatment of advanced primary liver cancer. MethodsA total of 186 patients with advanced primary liver cancer who were treated in The Fifth Medical Center of Chinese PLA General Hospital from April 2015 to June 2019 were enrolled and divided into study group and control group using a random number table, with 93 patients in each group. Both groups of patients underwent TACE, and the patients in the study group were treated with ultrasound-guided percutaneous MWA. The two groups were compared in terms of clinical outcome and complications. Quantitative real-time PCR was used to measure the serum level of microRNA-202 (miR-202), ELISA was used to measure the serum levels of fragile histidine triad (FHIT) and P16 protein, and the changes in the above three indices at 3 months after treatment were compared. The two-independent-samples t test was used for comparison of continuous data between two groups, and the paired t-test was used for comparison within one group before and after treatment; The chi-square testwas used for comparison of categorical data between groups. ResultsThe study group had a significantly higher objective response rate than the control group (47.32% vs 27.96%, χ2=7.422, P=0.006), and there was no significant difference in disease control rate between the two groups(P＞0.05). Both groups had significant increases in the serum levels of miR-202, FHIT, and P16 protein at 3 months after treatment (all P＜0.05), and compared with the control group, the study group had significantly higher serum levels of miR-202 (0.84±0.14 vs 0.58±017, t=11.385, P＜0.001), FHIT (1126.35±73.05 pg/ml vs 762.87±56.71 pg/ml, t=37.904, P＜0.001), and P16 protein (52.86±651 pg/ml vs 39.06±5.37 pg/ml, t=15.770, P＜0.001). ConclusionUltrasound-guided MWA in addition to TACE can improve the short-term response of patients with advanced primary liver cancer and increase the serum levels of miR-202, FHIT, and P16 protein, with relatively high safety.

Objective:To explore the molecular mechanism of Polygalae Radix - Acori Tatarinowii Rhizoma medicinal pair for depression and Alzheimer disease (AD) with the same treatment through network pharmacology. Methods:Effective components of Polygalae Radix - Acori Tatarinowii Rhizoma medicinal pair were retrieved from TCMSP, TCMID and ETCM databases. The disease targets of depression and AD were retrieved from GeneCards, TTD and CTD databases. Targets of action of drugs on active components were predicted through SwissTargetPrediction, and then the intersection targets of medicinal pair and the diseases were taken. Cytoscape 3.6.1 was used to construct the interaction network of Polygalae Radix - Acori Tatarinowii Rhizoma medicinal pair on "component-common target-disease". The enrichment analysis of GO function and KEGG pathway was carried out with the help of Metascape platform, and molecular docking verification was carried out. Results:Through searching the databases and literature, 78 compounds in Polygalae Radix - Acori Tatarinowii Rhizoma medicinal pair were obtained, corresponding to 41 targets of different diseases with the same treatment. The GO function was mainly concentrated in response to lipopolysaccharide and cellular response to nitrogen compound. The KEGG pathway was mainly concentrated in lipid and atherosclerosis, calcium signaling pathway, serotonergic synapse, insulin resistance and so on. The core targets were PTGS2, ESR2, etc. Molecular docking showed that most of the core components could form stable conformation with the core targets. Conclusions:Polygalae Radix - Acori Tatarinowii Rhizoma medicinal pair has the characteristics of multi-component, multi-target and multi-pathway in the same treatment of depression and AD. Through their core components of senegenin, 1-carbobutoxy-β-carboline, 6-hydroxy-1,2,3,7-tetramethoxyxanthone, kaempferol and etc., the pair can act on PTGS2 and other targets, regulate lipid and atherosclerosis, calcium signaling pathway, serotonergic synapse, insulin resistance and so on, and play a therapeutic role in depression and Alzheimer's disease with the same treatment.