Effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl4/ethanol / 中华肝脏病杂志

To study the effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl(4)/ethanol. The wild-type mice (Smad4 +/+) and the Smad4 knockout mice (Smad4 +/-) were injected subcutaneously with carbon tetrachloride(CCl(4))/ethanol twice a week for twenty weeks. The expression of Smad4, TGFbeta1, Smad2, Smad3, Smad6, TIMP1, MMP2 and MMP9 was detected by RT-PCR. In the cirrhotic liver, the expression of Smad4 mRNA was significantly higher than that in the normal liver. Comparing with wild-type mice (Smad4 +/+), the TGFbeta1-Smad4 signaling was markedly attenuated in the Smad4 knockout mice (Smad4 +/-). After induction by CCl(4)/ethanol, the hepatic fibrosis in the Smad4 knockout mice (Smad4 +/-) was obviously alleviated compared with the wild-type mice (Smad4 +/+), and the incidence rate of hepatocarcinogenesis of the former was also lower than that of the latter(32.0% vs 41.9%). These results indicate that knocking out Smad4 can delay the progression of liver fibrosis and liver cancer.

Effects of galectin-3 on proliferation and apoptosis of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the effects of galectin-3 on proliferation and apoptosis of hepatic stellate cells.</p><p><b>METHODS</b>RT-PCR and Western blot were used to detect the expression of galectin-3 in hepatic stellate cells. Short hairpin DNA targeting galectin-3 of rat was was ligated into the recombinant vector pGCsilencer U6/Neo/GFP/shRNA plasmid. Then the plasmid was transfected into rat hepatic stellate cells. RT-PCR and Western blot were used to detect the interfering efficiency. Cell proliferation level was observed by CCK8 method at 24, 48 and 72 hours after transfection. Cell apoptosis was measured by Annexin V/PI-labeled flow cytometric analysis.</p><p><b>RESULTS</b>Expression of galectin-3 in HSC was verified by both RT-PCR and Western blot. The recombinant vector was successfully constructed and verified, and was transfected into rat hepatic stellate cells. Western Blot and RT-PCR results demonstrated that the expression level of Galectin-3 was significantly down-regulated in galectin-3 shRNA transfected cells compared to control vector transferred cells. CCK8 assay indicated that proliferation of Galectin-3 knockdown cells was lower than that of control cells 48 and 72 hours post-transfection. Apoptotic cells in shRNA-interfering group were higher than those in control group both in early stage and advanced stage.</p><p><b>CONCLUSION</b>Hepatic stellate cells can express galectin-3. Inhibition of galectin-3 using RNAi technique can suppress proliferation and induce apoptosis in HSC.</p>

NY-ESO-1 and cancer immunotherapy / 中国医学科学院学报

NY-ESO-1 is an important member of cancer-testis antigen family and is widely distributed among many cancer types. As a tumor-specific antigen with the strongest immunogenicity so far identified, it can induce spontaneous antibody and T-cell responses in patients with NY-ESO-1-positive tumors. Therefore, it has been a good vaccine candidate in the immunotherapy against many malignancies. This article reviews the recent research advances in NY-ESO-1 and its relevant vaccines.

Identification of a naturally presented MAGE-A3 epitope on the surface of HLE cell line by mass spectrometry / 中华外科杂志

<p><b>OBJECTIVE</b>To identify a naturally presented HLA-A2-restricted epitope of MAGE-A3 antigen, FLWGPRALV (MAGE-A3(271 - 279)), on the surface of a human hepatocellular carcinoma (HCC) cell line HLE.</p><p><b>METHODS</b>Synthetic peptide FLWGPRALV, served as positive control target, was analyzed by HPLC and HPLC-ESI-TOF-MSMS, in order to determine its HPLC elution time, mass-spectrometric characteristics and the lowest detection limitation by the two approaches. 3 x 10(9) HLE cells were collected, peptides naturally presented by major histocompatibility complex (MHC) molecules on the cell surface were isolated by mild acid elution, and concentrated by lyophilization, then the mixtures of peptides were fractioned by HPLC. The ingredient ranged from 2 min before the elution time determined by the synthetic peptide to 2 min after that was collected, concentrated by lyophilization, and analyzed by HPLC-ESI-TOF-MSMS, to identify the existence of the MAGE-A3(271 - 279) peptide.</p><p><b>RESULTS</b>The HPLC-ESI-TOF-MSMS detection provided an evidence for the existence of a doubly charged ion of (m/z)(2) 529.9, which was further analyzed by collision induced dissociation. The doubly charged ion was ultimately identified as the MAGE-A3(271 - 279) peptide, its amino sequence was FLWGPRALV and its molecular weight was 1058.4 Da.</p><p><b>CONCLUSIONS</b>MAGE-A3(271 - 279) epitope could be naturally presented by HLA-A2 molecules to the surface of HCC cell line and MAGE-A3(271 - 279) peptide may have potential immunotherapeutic value in HCC patients.</p>

Downregulation of transforming growth factor-beta1 and platelet-derived growth factor gene expression by interleukin-10 in murine hepatic stellate cells in response to experimental liver injury / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of interleukin-10 (IL-10) on the expression of transforming growth factor-beta(1) (TGFbeta(1)) and platelet-derived growth factor (PDGF) in hepatic stellate cells (HSC) during liver injury.</p><p><b>METHODS</b>The adenovirus vector (the titer was 1 x 10(7) efu/ml) encoded IL-10 gene was used to transfect the rat via the vein of caudal. At the same time, CCl(4) was injected into rat by a hypodermic injection. These processes went on twice a week. After eight weeks, the liver were perfused with collagenase IV and purified by density gradient centrifugation with Nycodenz for separate HSC. The level of IL-10 was measured by ELISA method; The expression of PDGF and TGFbeta(1) in HSC was detected by semi-quantitative RT-PCR and Western-blot methods.</p><p><b>RESULTS</b>The level of IL-10 in therapy group (adenovirus vector encoding IL-10 gene group) was higher than that in non-therapy group (adenovirus vector without IL-10 gene and PBS group); The expression of TGFbeta(1) mRNA, TGFbeta(1) protein and PDGF mRNA, PDGF protein in therapy group were significantly lower than that in non-therapy group (P < 0.05).</p><p><b>CONCLUSION</b>Downregulating the TGFbeta(1) and PDGF expression could be the passageway by which IL-10 alleviate the degree of proliferation and activation in hepatic stellate cells.</p>

Establishment of dendritomas by fusion of human dendritic cells with human hepatocellular carcinoma cell line HLE cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct dendritomas by fusion of human dendritic cells with HLE cells, a human hepatocellular carcinoma cell line.</p><p><b>METHODS</b>HLE cells were cultured in RPMI 1640 with 15% FCS. Human dendritic cells (DCs) were obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4 for 7 days, matured with TNF-alpha and PGE(2) for 2 days. The DCs and HLE cells were labeled with green fluorescence dye PKH67-GL and red fluorescence dye PKH26-GL, respectively, and fused in 50% polyethylene glycol (PEG) + 10% dimethyl sulfoxide (DMSO) to generate dendritomas for rapid fluorescence-activated cell sorting (FACS).</p><p><b>RESULTS</b>Dendritomas with dual red-green fluorescence were constructed successfully, and FACS analysis showed the effective fusion rate was 16.8%.</p><p><b>CONCLUSION</b>With fluorescence dyes PKH67-GL and PKH26-GL as fusion markers, dendritomas for rapid fluorescence-activated cell sorting are constructed, which may throw new light on immunotherapy of hepatocellular carcinoma.</p>

Screening and analysis of genes encoding hepatocellular carcinoma associated tumor antigens / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To screen and clone the genes encoding hepatocellular carcinoma associated tumor antigens.</p><p><b>METHODS</b>A hepatocellular carcinoma cDNA express library was constructed with ZAP vector and analyzed by serological analysis of recombinant cDNA expression library (SEREX) with sera from autologous and allogenous patients. Monoclonalized positive phage clones were converted into pBK-CMV phagemid forms by in vivo excision. The cDNA inserts were determined by restriction endonuclease digestion with EcoR I and Xho I. The cDNA inserts were sequenced and analyzed with bioinformatics. LIMS1 insert was cut from the clone HCL5-70 and constructed into pQE 31 express vector. The recombinant LIMS1 was expressed in M15 and analyzed with SDS-PAGE and Western blot.</p><p><b>RESULTS</b>Fourteen genes were cloned from autologous screening and eleven genes were obtained with allogeneous analysis. One gene, kinectin, was identified in both autologous and allogeneous screening. Eight of the total twenty-four genes were unknown for their functions; the other sixteen genes can be classified into eight groups according to their established or putative function. Recombinant LIMS1 was expressed in M15.</p><p><b>CONCLUSION</b>The identification of hepatocellular carcinoma associated tumor antigens provides potential targets for immunotherapy of hepatocellular carcinoma patients and will help in the understanding of the carcinogenesis of hepatocellular carcinoma.</p>

An in vivo study on interlukin-10 inhibiting the expression and secretion of collagen I and IV in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effect of interlukin-10 (IL-10) on expression and secretion of collagen I, IV in rat's hepatic stellate cells (HSC) of livers injured by CCl4.</p><p><b>METHOD</b>The adenovirus vector encoded IL-10 gene was used to transfect rats with liver injury via the caudal veins. HSC were isolated and purified from the rat livers by collagenase IV perfusion and density gradient centrifugation with Nycodenz. The expression of collagen I, IV mRNA in HSC was detected by semi-quantitative RT-PCR method and the secretion of collagen I, IV in culture serum of HSC by ELISA method. The quantity of collagen was measured in the van Gieson stained histological liver preparations.</p><p><b>RESULTS</b>The expression and secretion of collagen I, IV in the adenovirus vector encoding IL-10 gene group were significantly lower than those in the adenovirus vector without IL-10 gene group and the control group (P < 0.05). The quantity of collagen in the treatment group was lower than that in the control group.</p><p><b>CONCLUSION</b>IL-10 can inhibit collagen I, IV expression and secretion in rat HSC.</p>

Regulation of hepatic stellate cell activation by interleukin-10/platelet derived growth factor/mitogen-activated protein kinase pathway / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the regulatory effect of interleukin-10 (IL10) on the activation of hepatic stellate cells (HSC) through platelet derived growth factor (PDGF) and mitogen-activated protein kinase (MAPK) pathways.</p><p><b>METHODS</b>HSC were divided randomly into 4 groups. Group 1 served as a control. HSC were incubated with 1 ng/ml, 5 ng/ml, and 25 ng/ml IL-10 in groups 2, 3 and 4. RT-PCR and western blot were used to detect the expression of PDGF and MAPK protein ERK and p38 and alpha-SMA.</p><p><b>RESULTS</b>Compared with the control group, expressions of ERK, p38 and alpha-SMA of groups 2, 3 and 4 were significantly lower (F values were 240.47, 21.39, 28.86 respectively. IL-10 inhibited PDGF and MAPK protein ERK and p38 and alpha-SMA expression in a dose-dependent way.</p><p><b>CONCLUSION</b>IL-10 inhibits activation of HSC through the PDGF/MAPK pathway.</p>

The diagnosis and treatment of hepatic artery complications in 107 cases of orthotopic liver transplantation / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the diagnosis and managements of hepatic artery complications in orthotopic liver transplantation.</p><p><b>METHODS</b>The clinical data of 107 consecutive orthotopic liver transplantation patients was reviewed retrospectively to assess the risk factors and the diagnosis and treatment of the vascular complications.</p><p><b>RESULTS</b>The incidence of the artery related complications in orthotopic liver transplantation was associated with the quality of the donor organ artery and the reconstruction way of donor-recipient artery intimately. The main hepatic artery related complications were hepatic artery thrombosis and stenosis. The incidence of the vascular complications was 6.54%, and the mortality rate was 85.7%.</p><p><b>CONCLUSIONS</b>The main influence factors of vascular complications were the quality of the donor organ artery and the reconstruction way of donor-recipient artery. The key steps of organ salvaging and the patients' life saving were early diagnosis and treatment of those complications.</p>

Expression of MAGE-B genes in hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of MAGE-B genes in hepatocellular carcinoma (HCC) in order to find new targets for immunotherapy.</p><p><b>METHODS</b>The expression of MAGE-B1, B2, A1 and A3 mRNA was detected using RT-PCR in HCC tissues and the corresponding adjacent non-HCC tissues from 47 HCC patients, 30 samples of cirrhosis and normal liver tissues. Four samples selected randomly from MAGE-B1 or B2 with positive RT-PCR results were sequenced to confirm the results of RT-PCR. The relationship between the expression of MAGE-B and some clinicopathological parameters was analyzed.</p><p><b>RESULTS</b>MAGE-B1 mRNA and MAGE-B2 mRNA were detected in 44.7% (21/47) and 61.7% (29/47) of HCC samples, respectively, while neither MAGE-B1 nor MAGE-B2 could be detected in the corresponding adjacent non-HCC liver tissues. In addition, none of 30 samples of cirrhosis and normal liver tissues was shown to express both MAGE-B genes. The DNA sequence confirmed that the RT-PCR products were truly target cDNA. The frequency of the expression of MAGE-A1 and A3 was 74.5% (35/47) and 44.7% (21/47), respectively. There was significant correlation between the expression of MAGE-B and MAGE-A (P < 0.05). However, the positive expression of MAGE-B was observed in 5 out of 12 HCC tissues without expression of MAGE-A1 and/or A3. When all four MAGE genes were examined, the positive rate of expression of one, two, three and four genes was 83.0% (39/47), 55.3% (26/47), 48.9% (23/47), and 38.3% (18/47) of 47 HCC tissues, respectively. No correlation was found between the expression of MAGE-B and clinical parameters such as age, sex, tumor size, degree of tumor differentiation, serum alpha-fetoprotein level and hepatitis B virus or hepatitis C virus infection (P > 0.05).</p><p><b>CONCLUSION</b>MAGE-B genes are expressed with relatively high frequency and specificity in HCC. Most HCC patients with positive expression of at least one member of MAGE-B or MAGE-A gene family are adequate candidates to receive specific immunotherapy. Frequent co-expression of multiple members of MAGE-B and MAGE-A subfamilies provides the possibility of using polyvalent vaccines to achieve more effective immunotherapeutic results.</p>

Inhibitory effect of retroviral vector containing anti-sense Smad4 gene on Ito cell line, LI90 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Transforming growth factor-beta1 (TGF-beta1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90.</p><p><b>METHODS</b>The recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed.</p><p><b>RESULTS</b>mRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus.</p><p><b>CONCLUSIONS</b>The antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-beta1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy.</p>

The effect of somatostatin analogue on expression of connective tissue growth factor gene of rat hepatic stellate cells / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of somatostatin analogue-octreotide (OCT) on expression of connective tissue growth factor (CTGF) gene of murine hepatic stellate cells (HSCs) in vitro.</p><p><b>METHODS</b>HSCs separated from Sprague Dawley rats by in situ perfusion and Nycodenz gradient were divided into 5 groups. HSCs in 4 out of 5 groups were co-cultured with octreotide at different dosages, and the remaining group served as control. The expression of CTGF and TGF-beta mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>OCT down-regulates the expression of CTGF and TGF-beta mRNA in HSCs. The effect is increased with a dose dependent manner.</p><p><b>CONCLUSIONS</b>OCT could exert the inhibitory effect on HSCs by down-regulating the expression of CTGF and TGF-beta. This provides a potential for the prevention and management of hepatic fibrosis.</p>

Expression of 5-hydroxytamine receptors in hepatic stellate cell and action of 5-hydroxytamine on biological characteristics of hepatic stellate cell / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of 5-hydroxytamine receptors in hepatic stellate cells HSCs and action of 5-hydroxytamine on biological characteristics of HSC.</p><p><b>METHODS</b>Liver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate hepatic stellate cell. RT-PCR was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. HSCs were cultured on silicone membrane. The effect of 5-hydroxytamine, ketanserin and ondanosetron on cell contraction were studied.</p><p><b>RESULTS</b>HSC expressed 5-hydroxytamine receptors subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P < 0.05). This was antagonized by ketanserin, not by ondanosetron. 5-hydroxytamine induced cell contraction in a dose-dependant manner. Ketanserin antagonized this action, but ondanosetron did not.</p><p><b>CONCLUSIONS</b>HSCs express 5-hydroxytamine receptors. 5-hydroxytamine could affect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.</p>

Expression and clinical significance of hepatocellular cancer antigen genes in human hepatocellular carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of four hepatocellular cancer antigen (HCA) gene mRNA in hepatocellular carcinoma.</p><p><b>METHODS</b>The expression of HCA90, HCA519, HCA520, HCA587 mRNA was detected using RT-PCR in HCC tissues and the corresponding adjacent non-HCC tissues from 46 HCC patients, cirrhosis tissues from 10 samples and normal liver tissues from 10 samples. The relationship between positive expression rate of HCA gene and clinical and lab data was evaluated.</p><p><b>RESULTS</b>Of 46 HCC tissues, HCA90, HCA519, HCA520 and HCA587 mRNA were detectable in 65.2%, 76.1%, 45.7% and 32.6%, respectively. At least one HCA gene mRNA was positive in 82.6% of HCC tissues. Only weak expression of HCA519 could be detectable in 6.5% of the corresponding adjacent non-HCC tissues. None of 10 samples of cirrhosis and normal liver tissues expressed any HCA gene mRNA. No correlation was found between the expression of HCA and clinical date such as age, sex, tumor size, tumor differentiation, serum alpha-fetoprotein level and hepatitis B virus infection or hepatitis C virus infection (P > 0.05). However, in some patients with normal serum alpha-fetoprotein (< 25 ng/L), specific expression of HCA genes was observed.</p><p><b>CONCLUSION</b>HCA gene mRNA is expressed with a high percentage and specificity in hepatocellular carcinomas and their products are new potential promising targets for immunotherapy of HCC.</p>