Screening up-regulated genes in hepatic stellate cells treated with PDGF-BB using suppression subtractive hybridization technique / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a subtractive cDNAs library of up-regulated genes in rat hepatic stellate cells (HSCs) stimulated with platelet-derived growth factor (PDGF)-BB by suppression subtractive hybridization (SSH) technique, to clone the up-regulated genes associated with its regulation effects, and to elucidate the mechanism of the molecular biology of hepatic fibrosis involved in PDGF-BB.</p><p><b>METHODS</b>The mRNA was isolated from HSCs stimulated with PDGF-BB and controlled with identical cells untreated with PDGF-BB, and then the cDNAs were synthesized. The cDNAs were designated as tester and driver. After being digested by restriction enzyme Rsa I, small-sized cDNAs were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, individually. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.</p><p><b>RESULTS</b>The subtractive cDNAs library of up-regulated genes in HSCs stimulated with PDGF-BB was constructed successfully. The amplified library contained 102 positive clones. Colony PCR showed that 93 clones contained 200-1000 bp inserts. Sequence analysis was performed in 31 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank; altogether 13 coding sequences were obtained, which were known ones. The genes mainly included voltage-dependent anion channel (VDAC), heat shock protein 47 and RAN-member RAS oncogene family genes.</p><p><b>CONCLUSION</b>Acting as one of the most effective mitogens, PDGF-BB up-regulated some gene expressions during stimulation of the HSCs, including some cell growth associated proteins, some proteins participating in intracellular metabolism and some molecular chaperone proteins. This work brings some new clues for studying the molecular biological mechanism involved in the up-regulated genes in PDGF-BB transactivated HSCs in hepatic fibrosis.</p>

Screening proteins in hepatocytes interacting with the middle surface protein of hepatitis B virus using the yeast-two hybrid technique / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To screen proteins in hepatocytes interacting with hepatitis B virus surface antigen middle protein (MHBs) with yeast-two hybrid technique for studying the biological functions of MHBs.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) was used to amplify the gene of MHBs from the plasmid A7 containing the whole fragment of adr subtype of HBV and the PCR product was cloned into pGEM-T vector and then evaluated by sequencing. The gene of MHBs was cut by EcoRI and BamH I from pGEM-T vector and then cloned into the yeast expression plasmid pGBKT7. MHBs bait plasmid was constructed by ligating MHBs gene with yeast expression vector pGBKT7 with yeast-two hybrid system 3 and then was transformed into yeast AH109 (a type). The transformed yeast cells were mated with yeast Y187 (alpha type) containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X- alpha -gal for selecting and screening. After extracting and sequencing the plasmid from true positive blue colonies, the results were analyzed by bioinformatics.</p><p><b>RESULTS</b>A pGBKT7- MHBs yeast expressed vector was successfully constructed. Two colonies were sequenced. One colony was Homo sapiens aldolase B fructose-bisphosphate, the other was a new gene with unknown function, which was named MHBs-binding protein 1.</p><p><b>CONCLUSION</b>MHBs gene was successfully cloned. Two genes of MHBs interacting proteins in hepatocytes were obtained by yeast-two hybrid system 3. Our results brought some new clues for studying the biological functions of MHBs and the mechanisms of HBV carcinogenesis.</p>

Effect of tumor antigen specific CTL induced by dendritic cells on a model of human hepatocellular carcinoma in nude mice (LCI-D20) / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the cure effect of tumor antigen specific CTL on a model of human hepatocellular carcinoma in nude mice LCI-D20.</p><p><b>METHODS</b>Dendritic cells (DCs) were induced from peripheral blood mononuclear cells of healthy people in vitro by using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and interleukin-4 (rhIL-4) and were pulsed with tumor antigen from hepatocellular carcinoma cell line MHCC97H. Then tumor antigen specific cytotoxic T lymphocytes (CTLs) were induced. By intraperitoneal injection of tumour antigen specific CTLs into the LCI-D20, the preventive and therapeutic effects of these CTLs to HCC in the LCI-D20 model were assessed. Cytokine-induced killer (CIK) cells and phosphate buffer solution were used as controls at the same time.</p><p><b>RESULTS</b>The weights of tumors in the tumor antigen specific CTL group, in the CIK cell group and in the blank group were (1.11+/-0.63), (1.12+/-0.36) and (2.68+/-0.53) grams respectively (t = 5.18, t = 6.06, P < 0.01). The amount of blood alpha fetal protein in the tumor antigen specific CTL and CIK groups were (52.1+/-9.7) microg/L and (48.6+/-5.2) microg/L, and was (82.2+/-7.2) microg/L in the blank group (t = 17.26, t = 22.07, P < 0.01 respectively). The metastasis rates in livers were 16.7%, 16.7% and 58.3% in the tumor antigen specific CTL, CIK cell and blank control groups respectively (chi2= 4.44, P < 0.01). The survival time of the mice in the tumor antigen specific CTL group was (79.0+/-5.02) days, (73.3+/-7.0) days in the CIK group, and (52.3+/-5.2) days in the blank group (t = 14.56, t = 17.54, P < 0.01).</p><p><b>CONCLUSION</b>Tumor antigen specific CTLs may prevent metastasis in the LCI-D20 model and prolong the survival time.</p>

Molecular mechanisms of the protection of SNMC in HepG2 cell apoptosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Apoptosis of the cells of liver cancer cell line HepG2 could be induced by TNF alpha and actinomycin D (Act D). In the current study, the molecular mechanism of the apoptosis protection of stronger neo-minophagen C (SNMC) to HepG2 cells was investigated.</p><p><b>METHODS</b>SNMC was added to the HepG2 cell culture medium when the cell concentration reached 0, 2, 20, 100, 200, 800 microg/ml 30 min before their apoptosis were inducted with TNF alpha and Act D. A flow cytometry assay was performed to detect the cell apoptosis rate; electromicroscopy was employed to visualize the subcellular structure after apoptosis. DNA ladder formation was checked with genomic DNA agarose electrophoresis. The expression pattern of apoptosis related protein Caspase-3, Bcl-2 and Bax was detected by Western blot.</p><p><b>RESULTS</b>After pretreatment with various concentrations of SNMC and 12 hours after treatment with TNF alpha and Act D, the HepG2 cell apoptosis rate and DNA ladder formation decreased dramatically when the SNMC concentration was higher in the media; the intracellular inactive form of Caspase-3 increased while the 17*10(3) active Caspase-3 decreased gradually. In addition, the expression of Bcl-2 increased and the expression of Bax decreased. Under the electromicroscope, the typical nucleolus condensation of HepG2 induced by TNF alpha and Act D was not seen among the 100 microg/ml SNMC treated cells.</p><p><b>CONCLUSION</b>SNMC inhibits TNF alpha and Act D induced HepG2 cell apoptosis. This protective action may be regulated by intracellular apoptosis related factors.</p>

Protective effect of SNMC on mice with fulminant liver failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of stronger neo-minophagen C (SNMC) on fulminant liver failure (FLF).</p><p><b>METHODS</b>D-Gal N and LPS were injected once into the abdominal cavity of rats to establish an experimental model of FLF. The level of plasma ALT, Alb, TBil, TNFalpha, NO, ET-1, IL-6 and liver histopathology of the rats were examined.</p><p><b>RESULTS</b>In the D-Gal N and LPS model of FLF, there was an obvious decline of plasma TNFalpha (F = 52.84), NO (F = 15.81), ET-1 (F = 15.68), IL-6 (F = 15.32) and there was less hepatic tissue damage in SNMC-treated groups using different doses (high dose, medium dose, low dose) and at different times (pre-protection, simultaneous protection, post-protection) compared with those not treated with SNMC. These results indicated that SNMC could be used to treat FLF. It was better to use a low dose of SNMC and use it at the same time as inducing the FLF. There were no differences in the results of those treated with SNMC of different dosages and treated at different times.</p><p><b>CONCLUSION</b>SNMC can decrease the mortality of FLF by preventing hepatocyte apoptosis induced by D-Gal N and LPS and inhibit liver inflammation caused by all kinds of factors.</p>