Cloning and analysizing the up-regulated expression of transthyretin-related gene （LR1） in rat liver regeneration following short interval successive partial hepatectomy

AIM: Cloning and analysing the up-regulated expression of transthyretin-related gene following short interval successive partial hepatectomy (SISPH) to elucidate the mechanism of differentiation, division, dedifferentiation and redifferentiation in rat liver regeneration (LR). METHODS: Lobus external sinister and lobus centralis sinister, lobus centralis, lobus dexter, lobus candatus were removed one by one from rat liver at four different time points 4, 36, 36 and 36 hr (total time: 4 hr, 40 hr, 76 hr, 112 hr) respectively. Suppression subtractive hybridization (SSH) was carried out by using normal rat liver tissue as driver and the tissue following short interval successive partial hepatectomy (SISPH) as tester to construct a highly efficient forward-subtractive cDNA library. After screening, an interested EST fragment was selected by SSH and primers were designed according to the sequence of the EST to clone the full-length cDNA fragment using RACE (rapid amplification of cDNA end). Homologous detection was performed between the full-lenth cDNA and Genbank. RESULTS: Forward suppression subtractive hybridization (FSSH) library between 0 h and 112 h following SISPH was constructed and an up-regulated full-length cDNA (named LR1), which was related with the transthyretin gene, was cloned by rapid amplification of cDNA end. It was suggested that the gene is involved in the cellular dedifferentiation in LR following SISPH. CONCLUSION: Some genes were up-regulated in 112 h following SISPH in rat. LR(1) is one of these up-regulated expression genes which may play an important role in rat LR.     

Gene expression differences of regenerating rat liver in a short interval successive partial hepatectomy

AIM: To identify the genes expressed differentially in the regenerating rat liver in a short interval successive partial hepatectomy (SISPH), and to analyze their expression profiles. METHODS: Five hundred and fifty-one elements selected from subtractive cDNA libraries were conformed to a cDNA microarray (cDNA chip). An extensive gene expression analysis following 0-36-72-96-144 h SISPH was performed by microarray. RESULTS: Two hundred and sixteen elements were identified either up- or down-regulated more than 2-fold at one or more time points of SISPH. By cluster analysis and generalization analysis, 8 kinds of ramose gene expression clusters were generated in the SISPH. Of the 216 elements, 111 were up-regulated and 105 down-regulated. Except 99 unreported genes, 117 reported genes were categorized into 22 groups based on their biological functions. Comparison of the gene expression in SISPH with that after partial hepatectomy (PH) disclosed that 56 genes were specially altered in SISPH, and 160 genes were simultaneously up-regulated or down-regulated in SISPH and after PH, but in various amount and at different time points. CONCLUSION: Genes expressed consistently are far less than that intermittently; the genes strikingly increased are much less than that increased only 2-5 fold; the expression trends of most genes in SISPH and in PH are similar, but the expression of 56 genes is specifically altered in SISPH. Microarray combined with suppressive subtractive hybridization can in a large scale effectively identify the genes related to liver regeneration.     

Cloning and sequencing of cagA gene fragment of Helicobacter pylori with coccoid form

AIM:To clone and sequence the cagA gene fragment ofHelicobacter pylori(H pylon)with coccoid form.METHODS:Hpyloristrain NCTC11637 were transformedto coccoid form by exposure to antibiotics in subinhibitoryconcentrations.The coccoid Hpyloriwas collected,cagAgene of the coccoid Hpyloristrain was amplified by PCR.After purified,the target fragment was cloned into plasmidpMD-18T.The recombinant plasmid pMD-18T-cagA wastransformed into E.coli JM109.Positive clones were screenedand identified by PCR and digestion with restrictionendonucleases.The sequence of inserted fragment wasthen analysed.RESULTS:cagA gene of 3 444 bp was obtained from thecoccoid Hpylori genome DNA.The recombinant plasmidpMD-18T-cagA was constructed,then it was digested byBamH I Sac I,and the product of digestion was identicalwith the predicted one.Sequence analysis showed that thehomology of coccoid and the reported original sequenceH pylori was 99.7%.CONCLUSION:The recombinant plasmid containing cagAgene from coccoid H pylorihas been constructed successfully.The coccoid H pylori contain completed cagA gene,whichmay be related to pathogenicity of them.     

Differential gene expression in human hepatocellular carcinoma Hep3B cells induced by apoptosis-related gene BNIPL-2

AIM:Bd-2/adenovirus E1B 19 ku interacting protein 2-like(BNIPL-2) is a novel protein recently identified in ourlaboratory.BNIPL-2 is homologous to human BNIP-2,apotentially proapoptotic protein,and can interact with Bcl-2 and Cdc42GAP and promote apoptosis in BEL-7402 cells.Here we report the gene-expression profile regulated byBNIPL-2 in human hepatocarcinoma Hep3B cells and theanalysis of its potential roles in cell apoptosis.METHODS:BNIPL-2 was overexpressed in Hep3B cellsusing tetracycline inducible or Tet-on system.Screened byWestern blot,the cells with low background and highinduction fold of BNIPL-2 were obtained.We performedAtlas human cDNA expression array hybridization on thesecells and analyzed the data with Quantarray~(?) software toidentify BNIPL-2-regulated genes and their expressionprofile.RT-PCR was used to confirm the altered expressionlevel of part of genes identified by the Atlas array hybridization.RESULTS:Fifteen of 588 genes spotted on the Atlasmembrane showed altered expression levels in BNIPL-2-transfected Hep3B-Tet-on cells,in which 8 genes involvedin cell apoptosis or growth inhibition were up-regulatedand 7 genes involved in cellular proliferation were down-regulated following overexpression of BNIPL-2.CONCLUSION:cDNA array is a powerful tool to exploregene expression profiles under inducible conditions.Thedata obtained using the cDNA expression microarraytechnology indicates that BNIPL-2 may play its roles inapoptosis through regulating the expression of genesassociated with cell apoptosis,growth inhibition and cellproliferation.     

Ischemic preconditioning decreases C-X-C chemokine expression and neutrophil accumulation early after liver transplantation in rats

AIM: Polymorphonuclear neutrophil (PMN) plays a major role in liver ischemia/reperfusion injury. Protective effect of ischemic preconditioning (IP) has been confirmed in liver ischemia/reperfusion injury. The purpose of this study was to investigate the effect of IP on C-X-C chemokine expression and PMNs recruitment early after liver transplantation.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT). The donor liver was stored 24 hours in University of Wisconsin (UW) solution at 4℃ pre-implantation. IP was done by clamp of the portal vein and hepatic artery of the donor liver for 10minutes followed by reperfusion for 10 minutes before harvesting. The neutrophilic infiltration in liver was quantified using a myeloperoxidase (MPO) assay. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) mRNA was investigated with in situ hybridization. The serum levels of MIP-2 and tumor necrosis factor (TNF)-α were also monitored.RESULTS: After liver transplantation without IP, the hepatic MPO increased significantly compared with sham operated group. In IP group, PMN in liver indicated by MPO was reduced significantly. In situ hybridization showed no MIP2 mRNA in sham group but dramatic expression in hepatocytes in non-IP group. In IP group, MIP-2 mRNA was significantly down-regulated. Similarly, serum MTP-2 and TNF-α levels were significantly elevated in non-TP group and both were reduced in IP group.CONCLUSION: IP might protect graft liver from preservationreperfusion injury after OLT through down-regulating C-X-C chemokine expression of hepatocytes, and alleviating PMNs recruitment after reperfusion.     

Effect of matrine on Kupffer cell activation in cold ischemia reperfusion injury of rat liver

AIM: To study the effect of matrine on activation of Kupffer cell during cold ischemia and reperfusion injury in rat orthotopic liver transplantation (OLT).METHODS: 168 syngeneic SD rats were randomly divided into four groups: untreated group, small-dose treated group, large-dose treated group and sham operation group. After 5 hours of preservation in Ringer's (LR) solution, orthotopic implantation of the donor liver was performed. At 1 h, 2 h, 4 h and 24 h after reperfusion of the portal vein, 6 rats were killed in each group to collect the serum and the liver for assay and pathology.RESULTS: Matrine markedly inhibited the activation of Kupffer cells and their release of tumor necrosis factor (TNF). TNF cytotoxicity level at 2 h decreased significantly by matrine treatment (7.94±0.42, 2.39±0.19 and 2.01±0.13 U/ml,respectively; P＜0.01), so did the other three time points. The level of hylluronic acid (HA) and alanine transaminase (ALT) decreased significantly in both treated groups, and matrine treatment markedly ameliorated focal necrosis of hepatocytes, inflammatory cells aggregating, rounding and detachment of sinusoidal endothelial cells (SEC). And no significant difference was observed between the treated groups.CONCLUSION: Matrine can inhibit the activation of Kupffer cell and prevent the donor liver from cold preservation and reperfusion injury in rat orthotopic liver transplantation.     

Effect of 2-(3-carboxy-1-oxopropyl) amino-2-deoxy-D-glucose on human esophageal cancer cell line

AIM: To determine whether 2-(3-carboxy-1-oxopropy1)amino-2-deoxy-D-glucose (COPADG), a derivative of Damino-glucose, inhibited the growth of human esophageal cancer cell line Eca-109.METHODS: Effects of COPADG on Eca-109 cells cultured in RPMI 1640 medium were examined by a tetrazoliumbased colorimetric assay (MTT assay).RESULTS: COPADG inhibited the growth of Eca-109 cells in a dose- and time-dependent manner; the maximum inhibition rate was 83.75%.CONCLUSION: COPADG can directly inhibit the proliferation of Eca-109 cells, which may serve as the experimental evidence for development of new drugs for esophageal cancer therapy.     

Distribution of constitutive nitric oxide synthase in the jejunum of adult rat

AIM: To study the distribution of the constitutive nitric oxidesynthase (NOS) in the jejunum of adult mt.METHODS: The distribution of endothelial NOS (eNOS) wasdetected by immunohistochemistry. Immunofluorescencehistochemical dual staining technique were used forstudying the distribution of neuronal NOS (nNOS) andeNOS. The dual stained slides were observed under aconfocal laser scanning microscopeRESULTS: Positive neuronal NOS (nNOS) and endothelialNOS (eNOS) cells were found to be distributed in laminapropria of villi, and the epithelial cell was not stained. eNOSwas mainly located in subrnucosal vascular endothelia,while nNOS was mainly situated in myenteric plexus. Somecells in the villi had both nNOS and eNOS. More than 80 %of the cells were positive for both nNOS and eNOS, the restcells were positive either for nNOS or for eNOS.CONCLUSION: The two constitutive nitric oxide synthasesam distributed differently in the jejunum of rat. nNOSdistributed in myenteric plexus is a neurotransmitter in thenon-adrenergic non-cholinergic (NANC) inhibitory nerves.eNOS distributed in endothelial and smooth muscle cells ofblood vessesels plays vasodilator role. eNOS and nNOS arecoexpressed in some cells of lamina propria of villi. NOgeneratedl by those NOS is very important in thephysiological and pathological process of small intestine.     

Clinical significance of vascular endothelial growth factor expression and neovascularization in colorectal carcinoma

AIM: To clarify the association of vascular endothelial growth factor (VEGF) and microvascular density (MVD)expression with the angiogenesis and prognosis of colorectal cancer.METHODS: A total of 97 cases of colorectal carcinomas were examined by immunohistochemical staining (SP method), using anti-VEGF and anti-factor CD34+ monoclonal antibodies. RESULTS: VEGF positive staining was obtained in 68 out of 97 cases (70.1%), and observed mainly in the cytoplasm of tumor cells, and also frequently in stromal cells. VEGF expression was more intense in poorly differentiated adenocarcinoma in comparison with others, but there was no significant correlation between VEGF expression and age,sex and stage. A significant correlation was found between the MVD and grades, and there was no significant relationship between the MVD and age, sex, and stage. The MVD in the VEGF positive group (68 cases) was higher than that in the negative group. Upon multivariate analysis, the significant variables were stage, tumor grade and MVD; VEGF expression was not an independent prognostic factor. CONCLUSION: The expression of VEGF has a significant correlation with MVD; MVD expression has prognostic value but VEGF has not in colon cancer.     

Relationship between the expression of iNOS, VEGF, tumor angiogenesis and gastric cancer

AIM: To investigate the relationship between theexpression of inducible nitric oxide synthase(iNOS),vascular endothelial growth factor(VEGF),themicrovascular density(MVD) and the pathologicalfeatures and clinical staging of gastric cancerMETHODS: Immunohistochemical staining was used fordetecting the expression of iNOS and VEGF in 46resected specimens of gastric carcinoma; themonoclonal antibody against CD34 was used fordisplaying vascular endothelial cells, and MVD wasdetected by counting of CD34-positive vascularendothelial cells.RESULTS: Of 46 resected specimens of gastriccarcinoma,the rates of expressions of iNOS and VEGFwere 58.70% and 76.09%, respectively,and MVDaveraged 55.59± 19.39.Judged by the standard TNMcriteria, the rate of expression of iNOS in stage ⅣV(84.46%) was higher than those in stage Ⅰ, Ⅱ, Ⅲ(Fishexact probabilities test, P=0.019,0.023 and 0.033,respectively);the rates of expression of VEGF in stageⅢ, Ⅳ (76.0%,92.31%, respectively) were higher thanthose in stage Ⅰ, Ⅱ (Fish exact probabilities test,P=0.031,0.017,0.022 and 0.019). MVDs in stage Ⅲ, Ⅳ(64.72 ± 14.96,67.09± 18.29,respectively) werehigher than those in stage Ⅰ ,Ⅱ(t=2.378,4.015,2.503and 2.450,P＜0.05,P＜0.001,P＜0.001,P＜0.05,respectively). In 37 gastric carcinoma specimens withlymph node metastasis, MVD(68.69±18.07) and therates of expression of iNOS and VEGF(70.27%,83.78%,respectively) were higher than those in the specimenswith absence of metastasis (t=2.205, x2=6.3587,x2=6.2584,P＜0.01, P＜0.05,P＜0.05, respectively). MVDand the expressions of iNOS and VEGF were notcorrelated to the location ,size or grade of tumor, nor withthe depth of invasion of tumor; MVDs in the positive iNOSand VEGF specimens(59.88±18.02,58.39±17.73,respectively) were higher than those in the negative iNOSand VEGF specimens (x2=6.3587 and 6.1574, P＜0.05,P＜0.05,respectively) ;thus the expressions of iNOS andVEGF was correlated to MVD, but the expression of iNOSwas not correlated to that of VEGF. In addition,of the46 surviving patients, the 5-year survival rate ofpatients with positive iNOS or VEGF tumors wassignificantly less than that of patients with negativeiNOS-or VEGF tumors(x2=4.3842 and 5.4073,P＜0.05,P＜ 0.05,respectively).CONCLUSION: The expressions of iNOS and VEGF areclosely related to tumor angiogenesis, and are involvedin the advancement and the lymph node metastasis;thus MVD and the expressions of iNOS and VEGF mayserve indexes for evaluating staging of gastriccarcinoma and forecasting its risk of metastasis, whichwill help establish a comprehensive therapeuticalmeasure of post-operative patients and provide a newapproach to tumor therapy.     

Loss of DPC4 expression and its correlation with clinicopathological parameters in pancreatic carcinoma

AIM: DPC4 is a tumor suppressor gene on chromosome 18q21.1 that has high mutant frequencies in pancreatic carcinogenesis. The purpose of this study was to investigate the role of DPC4 alterations in tumorigenesis and progression of pancreatic carcinomas.METHODS: We studied the immunohistochemical markers of DPC4 in 34 adenocarcinomas and 16 nonmalignant specimens from the pancreas. The 16 nonmalignant specimens from the pancreas included 8 non-neoplastic cysts and 8 normal pancreatic tissues. The relationship between DPC4 alterations and various clinicopathological parameters was evaluated by chi-square test or Fisher's exact test.Survivals were calculated using Kaplan-Meier method (by a log-rank test).RESULTS: All the 16 nonmalignant cases of the pancreas showed expression of DPC4 gene. Loss of DPC4 expression was seen in 8 of 34(23.5 %) pancreatic adenocarcinomas.The frequency of loss of DPC4 expression was higher in poorly differentiated adenocarcinoma (G3) than in well and moderately differentiated adenocarcinoma (G1 and G2)histologically (P=0.037). Loss of DPC4 expression of the patients at TNM stage Ⅳ was also higher than that of the patients at TNM stages Ⅰ, Ⅱ and Ⅲ (60.0 % at stage Ⅳ,versus14.3 % atstage Ⅰ, 18.2 % at stage Ⅱ, and 18.2 % at stage Ⅲ) (P=0.223). The mean and median survival in patients with DPC4 expression was longer than those in patients with loss of DPC4 expression. Kaplan-Meier survival analysis demonstrated patients with DPC4 expression had a higher survival rate than patients with loss of DPC4 expression, but the difference did not reach statistical significance (P =0.879).CONCLUSION: This study suggests that DPC4 is involved in the development of pancreatic carcinoma and is a late event in pancreatic carcinogenesis, DPC4 expression may be a molecular prognostic marker for pancreatic carcinoma.     

Effects of heparin on liver fibrosis in patients with chronic hepatitis B

AIM: To evaluate the effects of heparin on liver fibrosis in patients with chronic hepatitis B.METHODS: Fifty-two cases under study were divided into two groups, group A and group B. The two groups were given regular treatment and heparin/low molecular weight heparin (LMWH) treatment respectively. Hepatic functions,serum hyaluronic acid (HA) and type IV collagen levels were measured before and after the treatment, and six caseswere taken liver biopsy twice.RESULTS: After treatment, hepatic functions became significantly better in both groups. Serum HA and type IV collagen levels in group B compared with group A, decreased significantly after treatment. Collagen proliferation also decreased in group B after treatment.CONCLUSION: Heparin/LMWH can inhibit collagen proliferation in liver tissues with hepatitis B.     

Polymorphisms of interleukin-1B and interleukin-1 receptor antagonist genes in patients with chronic hepatitis B

AIM:To investigate the relationships between polymorphismsof interleukin-1B(IL-1B)promoter region -511C/T andinterleukin-1 receptor antagonist gene(IL-1RN)andsusceptibility to chronic hepatitis B in Chinese population.METHODS:Genomic DNA was extracted from the peripheralblood of 190 patients with chronic hepatitis B and 249 normalcontrols and then subjected to polymerase chain reaction(PCR)amplification.The PCR products were digested byrestriction endonuclease AvaI.The products of digestion weresubjected to 20 g/L gel electrophoresis and ethidium bromidestaining.RESULTS:The frequencies of IL-1B(-511)genotypes CC,CT and Tr in patients with chronic hepatitis B were 23.7%,49.5% and 26.8%,while 26.1%,47.4% and 26.5%respectively in controls.The results showed that there wasno significant difference in the frequencies of alleles orgenotypes in IL-1B between patients with chronic hepatitisB and controls.The distributions of IL-1B(-511)genotypeCC were significantly different between the two subgroups(HBV-DNA≤1×10~3 copies/mL as subgroup I,HBV-DNA>1×10~3 copies/mL as subgroup Ⅱ)of chronic hepatitis B(P=0.029).Only four of the five kinds of polymorphism(1/1,1/2,2/2 and 1/4)were found in this study.The frequenciesof IL-1RN genotypes 1/1,1/2,2/2 and 1/4 were 88.9%,9.0%,0.5% and 1.6% in patients with chronic hepatitis B respect,while were 81.1%,16.9%,0.4% and 1.6% respectively incontrols.The frequencies of genotypel/2 and IL-1RN allele2 in patients with chronic hepatitis B were lower than thosein controls(P=0.016 and P=0.029,respectively).CONCLUSION:There is an association between polymorphismsof the promoter region -511C/T of IL-1B and IL-1RN intron 2and chronic hepatitis B virus infection.Subjects with IL-1RN2 may be resistant to HBV infection,and IL-1B(-511)genotype CC is closely related with HBV-DNA replication,which gives some new clues to the study of pathogenesis ofchronic hepatitis B.     

Effect of arsenic trioxide on rat hepatocarcinoma and its renal cytotoxicity

AIM: To study the effect of arsenic trioxide (As2O3) on rat experimental hepatocarcinoma and its renal cytotoxicity.METHODS: The hepatocarcinoma model was established by diethaylnitrosamine perfusion in stomach of 120 Wistar rats, and the treatment began at the end of 20 weeks.Before the treatment, the rat models were randomly divided into 5 groups. In the treatment groups, three doses of As2O3 were injected into rat abdominal cavity, the total time of drug administration was 4 weeks. Cisplatin control or the blank group was injected into abdominal cavity with equal amount of cisplatin or saline at the same time,respectively. On the 7th, 14th and 28th day after the treatment, the hepatocarcinoma nodules were obtained and the morphologic changes of hepatocarcinoma cells were observed under light and electron microscopes;Immunohistochemistry (S-P methods) was employed to detect the expression of bcl-2, bax and PCNA in hepatocarcinoma tissues; flow cytometry (TUNEL assay)was used to detect the apoptosis of liver cancer cells and the change of cytokinetics. On the 28th day, the kidneys were obtained and their histologic changes were observed under light microscope, and immunohistochemistry (SP stain) was also employed to detect the expression of bcl-2and PCNA. Cisplatin and saline solution were used as the control.RESULTS: As2O3 could induce the apoptosis of rat liver cancer cells and exhibited typical morphologic changes.The incidence of apoptosis of hapatocarcinoma cells was elevated (P=0.001). The elevation was the most higher in the group of middle-dose of As2O3 (1 mg.kg-1), significantly higher than that of the other arsenic groups and the controls (P=0.001). Large dose of As2O3 (5 mg.kg-1) was able to arise the incidence of apoptosis, but also produced a large amount of necrosis and inflammatory reaction. Middle dose of As2O3 dramatically increased the cell number in G2/M phase (P=0.0001), and apoptosis happened apparently.The expression of bcl-2 and bax was related to the dose of As2O3. With the up-regulation of apoptotic incidence, the ratio of bcl-2/bak decreased. But the incidence of apoptosis was not the highest status and the ratio of bcl-2/bax was at the lowest when the highest-dose of As2O3 was used.There was significant difference among the PCNA indexes (PCNA L1) of the five groups. Of them, three arsenic groups all showed decrease of different degrees, and this downregulation was most obvious in group A. There was significant difference among the three groups (P=0.016).Under the light microscope, the rat kidney in the cisplatin group exhibited tubular epithelium swelling and degeneration, protein casts in collecting tubules; While all arsenic groups didn't show the significant changes (P=0.013).In the arsenic groups, the expression of bcl-2 in the renal tubular epithelium was increased (P=0.005), no obvious changes happened to PCNA L1. But in the group of cisplatin,the PCNA L1 increased significantly (P=0.001).CONCLUSION: AS2O3 can induce apoptosis of rat hepatocellular carcinoma cells. And there is optimum dose;too high dose will induce the cytotoxic effect, while certain dose of As2O3 is able to block the cell cycle at G2/M phase.As2O3 had the most remarkable influence on G2/M cells,and it can also induce apoptosis to cells at other phases.As2O3 can restrain the proliferation of rat hepatocellular carcinoma cells, in a dose-time dependent manner.Compared with cisplatin, As2O3 didn't show obvious renal toxicity, which was related to the increasing expression of bcl-2 in renal tubular epithelium, the inhibition of apoptosis and the anti-oxidation effects.     

The article published in WJG 2005;11（7）：931-937 partly duplicated an article previously published in Critical Reviews in Eukaryotic Gene Expression （2004;14：183-202）

From the editor On November 7, 2007, we received an E-mail from He Zhi-Gang (hezhigang@gmail.com) reporting a case of duplicate publication. After our evaluation we are able to confirm this claim. The details are noted below.     

Expression and altered subcellular localization of the cyclin-dependent kinase inhibitor p27Kip1 in hepatocellular carcinoma

AIM:To investigate p27 expression in hepatocellularcarcinoma (HCC),adjacent nontumoral and normal livertissues,and to verify whether the subcellular localizationof p27 was altered in HCC.METHODS:The level of p27 in tumoral,nontumoral,andnormal liver tissues were assessed by immunohistochemical(IHC) analysis.Parallel immunostaining was done forproliferating cell nuclear antigen (PCNA) to evaluate cellproliferation.RESULTS:The labeling index (LI) of p27 in tumoral lesionswas significantly lower than that in adjacent nontumorallesions (t=2.444,P=0.017) and normal controls (t=2.268,P=0.029).The LI of p27 significantly decreased in patientswith massive type (t=2.227,P=0.037) and infiltration(t=2.197,P=0.036).The prognosis of patients with higherp27 LI was longer than that of patients with lower p27 LI(P=0.0247,log-rank test).The LI of PCNA was significantlyhigher in HCC than that in adjacent nontumoral lesions(t=2.092,P=0.041) and normal controls (t=3.533,P=0.002).There was no significant correlation between p27expression and cell proliferation in tumor samples.Thelevel of p27 in the cytoplasmic fraction was higher in tumoraland nontumoral liver tissues,and was associated withclinical stage (t=2.520,P=0.029) and the degree of invasion(t=2.640,P=0.019).Survival analysis showed that p27 wasan independent prognosis marker for HCC patients.CONCLUSION:These results suggest that p27 underexp-ressing in patients with HCC is closely associated withinfiltration,metastasis,and prognosis.Alterations in thesubcellular localization of p27 protein may occur earlyduring hepatocarcinogenesis.     

Significance of vascular endothelial growth factor expression and its correlation with inducible nitric oxide synthase in gastric cancer

AIM:To investigate the clinical significance of theexpression of VEGF_(165)mRNA and the correlation withvascular endothelial growth factor (VEGF) protein andinducible nitric oxide synthase (iNO) in human gastriccancer.METHODS:We tested VEGF_(165)mRNA expression in 31 casesof resected gastric cancer specimens and normal pairedgastric mucosae by RT-PCR.Total RNA was extracted withTRIzol reagents,transcribed into cDNA with oligo (dT_(15))priming,inner controlled with β-actin expression andagarose gel isolated after PCR.VEGF expression wasquantitated with IS1000 imaging system.Meanwhile wealso examined expression levels of VEGF protein and iNOSin 85 cases of gastric cancer.All paraffin-embeddedsamples were immunohistochemically stained by streptavidin-peroxidase method (SP).RESULTS:The mean expression of VEGF_(165)mRNA ingastric cancer was 1.125±0.356,significantly higher thanthat of normal paired mucosae,which was 0.7604±0.278.The data indicated that the expression level ofVEGF_(165)mRNA was well related to lymph node metastasisand TNM stages of UICC.The expression levels in patientswith lymph node metastasis and without lymph nodemetastasis were 1.219±0.377 and 0.927±0.205 respectively(P<0.05).The expression in stages Ⅰ,Ⅱ,Ⅲ,Ⅳ was0.934±0.194,1.262±0.386 respectively (P<0.01).Furtheranalysis showed the lymph node metastasis rate in thegroup with over-expression of VEGF was higher than thatin the group with low expression of VEGF (83.3% vs 46.2%),and the ratio of stage Ⅲ Ⅳ in the group with over-expression of VEGF was also higher than that in the groupwith low expression with VEGF (77.8% vs 33.8%) (P<0.05).The positive rates of expression of VEGF protein and iNOSin 85 cases of gastric cancer were 75.4% and 58.8%respectively,and 50.1% of the patients showed positivestaining both for iNOS and VEGF,the correlation with thetwo factors was significant (P=0.018).But more intensive analysis showed the immunoreactive grades of VEGF werenot associated with that of iNOS.CONCLUSIONS:The expression of VEGF_(165)mRNA is wellrelated with lymph node metastasis and TNM stages of UlCCin gastric cancer,and is concerned with the invasivenessand metastasis of gastric cancer.The relationship can beobserved between the expression of VEGF and iNOS in gastriccancer.     

Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens

AIM:To comparatively evaluate PCR and other diagnosticmethods (the rapid urease test and/or culture) in order todetermine which of the three PCR methods (ureA,glmMand 26-kDa,SSA gene) was most appropriate in the diagnosisof Helicobacterpylori(Hpylori) infection and also to evaluatethe detection of a putative virulence marker of H pylori,thecage,gene,by PCR in biopsy specimens.METHODS:One hundred and eighty-nine biopsy specimenswere collected from 63 patients (three biopsies each)undergoing upper gastroduodenal endoscopy for variousdyspeptic symptoms.The PCR methods used to detectH pylori DNA directly from biopsies were the glmM,26-kDa,ureA and then cagA was used to compare the culturetechnique and CLO for urease with the culture techniquebeing used as the gold standard.RESULTS:Thirty-five percent of the biopsies were positivefor H pylori DNA using the 3 PCR methods,while 68% ofthese were positive for the cagA gene.Twenty-four percentof the biopsies were negative for H pylori DNA in all PCRmethods screened.The remaining 41% were either positivefor ureA gene only,glmM only,26-kDa only,or ureA glmM,ureA 26-kDa,glmM 26-kDa.Out of the 35% positivebiopsies,41% and 82% were positive by culture and CLOrespectively,while all negative biopsies were also negativeby culture and cagA.Cag A infection was also predominantlyfound in H pylori DNA of the biopsies irrespective of theclinical diagnosis.CONCLUSION:This method is useful for correctly identifyinginfections caused by H pylori and can be easily applied inour laboratory for diagnostic purposes.