Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15

AIM: Ursolic acid (UA) and oleanolic acid (OA) are triperpene acids having a similar chemical structure and are distributed wildly in plants all over the world. In recent years, it was found that they had marked anti-tumor effects. There is little literature currently available regarding their effects on colon carcinoma cells. The present study was designed to investigate their inhibitory effects on human colon carcinoma cell line HCT15. METHODS: HCT15 cells were cultured with different drugs. The treated cells were stained with hematoxylin-eosin and their morphologic changes observed under a light microscope. The cytotoxicity of these drugs was evaluated by tetrazolium dye assay. Cell cycle analysis was performed by flow cytometry (FCM). Data were expressed as means +/-SEM and Analysis of variance and Student' t-test for individual comparisons. RESULTS: Twenty-four to 72 h after UA or OA 60 micromol/L treatment, the numbers of dead cells and cell fragments were increased and most cells were dead at the 72nd hour. The cytotoxicity of UA was stronger than that of OA. Seventy-eight hours after 30 micromol/L of UA or OA treatment, a number of cells were degenerated, but cell fragments were rarely seen. The IC(50) values for UA and OA were 30 and 60 micromol/L, respectively. Proliferation assay showed that proliferation of UA and OA-treated cells was slightly increased at 24h and significantly decreased at 48 h and 60 h, whereas untreated control cells maintained an exponential growth curve. Cell cycle analysis by FCM showed HCT15 cells treated with UA 30 and OA 60 for 36 h and 72 h gradually accumulated in G(0)/G(1) phase (both drugs P【0.05 for 72 h), with a concomitant decrease of cell populations in S phase (both drugs P【0.01 for 72 h) and no detectable apoptotic fraction. CONCLUSION: UA and OA have significant anti-tumor activity. The effect of UA is stronger than that of OA. The possible mechanism of action is that both drugs have an inhibitory effect on tumor cell proliferation through cell-cycle arrest.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

Expression of vascular endothelial growth factor C and chemokine receptor CCR7 in gastric carcinoma and their values in predicting lymph node metastasis

AIM:To study the expression of vascular endothelial growth factor C (VEGF-C) and chemokine receptor CCR7 in gastric carcinoma and to investigate their associations with lymph node metastasis of gastric carcinoma and their values in predicting lymph node metastasis.METHODS:The expression of VEGF-C and CCR7 in gastric carcinoma tissues obtained from 118 patients who underwent curative gastrectomy was examined by immunohistochemisty.Among these patients,39 patients underwent multi-slice spiral CT (MSCT) examination.RESULTS:VEGF-C and CCR7 were positively expressed in 52.5 and 53.4% of patients. VEGF-C expression was more frequently found in tumors with lymph node metastasis than those without it (P<0.001).VEGF-C expression was also closely related to lymphatic invasion (P<0.001), vascular invasion (P<0.01),and TNM stage (P<0.001). However,there was no significant correlation between VEGF-C expression and age at surgery, gender, tumor size, tumor location,Lauren classification,and depth of invasion. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P<0.001) and was also associated with tumorsize (P<0.01), depth of invasion (P<0.001), lymphatic invasion (P<0.001),and TNM stage (P<0.001).However,the presence of CCR7 had no correlation to age at surgery,gender, tumor location, Lauren classification,and vascular invasion. Among the 39 patients who underwent MSCT examination,only CCR7 expression was related to lymph node metastasis determined by MSCT (P<0.05).In the current retrospective study,the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of VEGF-C and CCR7 expression in the diagnosis of lymph node metastasis for patients with gastric carcinoma were 73.8%, 70.2%,72.6%,71.4% and 72.0%,and 82.0%,77.2%,79.4%,80.0% and 79.7%,respectively.After subdivision according to the combination of VEGF-C and CCR7 expression, receiver operating characteristic (ROC) analysis showed that the accuracy of the combined examination of VEGF-C and CCR7 expression in predicting lymph node metastasis was relatively high (area under ROC curve [Az]=0.83).CONCLUSION:The expression of VEGF-C and CCR7 is related to lymph node metastasis of gastric carcinoma and both of them may become new targets for the treatment of gastric carcinoma.Furthermore,the combined examination of VEGF-C and CCR7 expression in endoscopic biopsy specimens may be useful in predicting lymph node metastasis of gastric carcinoma and deciding the extent of surgical lymph node resection.

Inhibitory effect of oxymatrine on serum hepatitis B virus DNA in HBV transgenic mice

AIM:To study the inhibitory effect of oxymatrine on serum hepatitis B virus (HBV) DNA in HBV transgenic mice. METHODS:HBV transgenic mice model was established by microinjection,and identified by HBV DNA integration and replication.Transgenic mice with replicating HBV were divided into 3 groups,and injected with normal saline (group A,n=9),50 mglkg (group B,n=8) and 100 mg/kg (group C,n=9) oxymatrine intraperitoneally once a day for 30d,respectively.Quantitation of serum HBV DNA in HBV transgenic mice was performed by competitive polymerase chain reaction (PCR) in combination with DNA hybridization quantitative detection technique before and after treatment. RESULTS:Compared with pre-treatment,the serum HBV DNA in group A (F=1.04,P=0.9612) and group B (F=1.13, P=0.8739) had no changes after treatment.However,in group C serum HBV DNA was significantly decreased (F=13.97, P=0.0012).The serum HBV DNA after treatment was lower in group C than in groups B and A (F=8.65,P=0.0068; F=12.35,P=0.0018;respectively).The serum HBV DNA after treatment was lower in group B than in group A,but there was no statistical significance (F=1.43,P=0.652). CONCLUSION:Oxymatrine has inhibitory effects on serum HBV DNA in HBV transgenic mice.

Anti-cancer effects of COX-2 inhibitors and their correlation wit angiogenesis and invasion in gastric cancer

AIM- To observe the anti-cancer effects of COX-2 inhibitors and investigate the relationship between COX-2 inhibitors and angiogenesis, infiltration or metastasis in SGC7901 cancer xenografts.METHODS: Thirty athymic mice xenograft models with human stomach cancer cell SGC7901 were established and divided randomly into 3 groups of 10 each. Sulindac, one non-specific COX inhibitor belonging to non-steroidal antiinflammatory drugs (a series of COX inhibitors known as NSAIDs) and celecoxib, one selective COX-2 inhibitor (known as SCIs) were orally administered to mice of treatment groups. Immunohistochemistry was used to examine the expression of PCNA, CD44v6 and microvessel density (MVD).Apoptosis was detected by using TUNEL assay.RESULTS: Tumors in sulindac and celecoxib groups were significantly smaller than those in control group from the second week after drug administration (P<0.01). In treatment group, the cell proliferation index was lower (P<0.05) and apoptosis index was higher (P<0.05) than those in control groups. Compared with the controls,microvessel density was reduced (P<0.01) and expression of CD44v6 on tumor cells was weakened (P<0.05) in treatment groups.CONCLUSION: COX-2 inhibitors have anticancer effects on gastric cancer. They play important roles in angiogenesis and infiltration or metastasis of stomach carcinoma. The anticancer effects of COX-2 inhibitors may include inducing apoptosis, suppressing proliferation, reducing angiogenesis and weakening invasiveness.

Clinical characteristics and prognostic factors of severe acute pancreatitis

AIM: To investigate the clinical characteristics and prognostic factors of a consecutive series of patients with severe acute pancreatitis (SAP). METHODS: Clinical data of SAP patients admitted to our hospital from January 2003 to January 2004 were retrospectively reviewed. Collected data included the age, gender, etiology, length of hospitalization, APACHE Ⅱ score at admission, local and organ/systemic complications of the patients. RESULTS: Of the 268 acute pancreatitis patients, 94 developed SAP. The mean age of SAP patients was 52 years, the commonest etiology was cholelithiasis (45.7%), the mean length of hospitalization was 70 d, the mean score of APACHE Ⅱ was 7.7. Fifty-four percent of the patients developed necrosis, 25% abscess, 58% organ/systemic failure. A total of 23.4% (22/94) of the SAP patients died. Respiratory failure was the most common organ dysfunction (90.9%) in deceased SAP patients, followed by cardiovascular failure (86.4%), renal failure (50.0%). In the SAP patients, 90.9% (20/22) developed multiple organ/systemic failures. There were significant differences in age, length of hospitalization, APACHE Ⅱ score and incidences of respiratory failure, renal failure, cardiovascular failure and hematological failure between deceased SAP patients and survived SAP patients. By multivariate logistic regression analysis, independent prognostic factors for mortality were respiratory failure, cardiovascular failure and renal failure. CONCLUSION: SAP patients are characterized by advanced age, high APACHE Ⅱ score, organ failure and their death is mainly due to multiple organ/systemic failures. In patients with SAP, respiratory, cardiovascular and renal failures can predict the fatal outcome and more attention should be paid to their clinical evaluation.

Gallbladder motor function, plasma cholecystokinin and cholecystokinin receptor of gallbladder in cholesterol stone patients

AIM: To study the interactive relationship of gallbladder motor function, plasma cholecystokinin (CCK) and cholecystokinin A receptor (CCK-R) of gallbladder in patients with cholesterol stone disease.METHODS: Gallbladder motility was studied by ultrasonography in 33 patients with gallbladder stone and 10 health subjects as controls. Plasma CCK concentration was measured by radioimmunoassay in fasting status (CCK-f) and in 30 min after lipid test meal (CCK-30).Radioligand method was employed to analyze the amount and activity of CCK-R from 33 gallstone patients having cholecystectomy and 8 persons without gallstone died of severe trauma as controls.RESULTS: The percentage of cholesterol in the gallstone composition was more than 70%. The cholesterol stone type was indicated for the patients with gallbladder stone in this study. Based on the criterion of gallbladder residual fraction of the control group, 33 gallstone patients were divided into two subgroups, contractor group (14 cases)and non-contractor group (19 cases), The concentration of CCK-30 was significantly higher in non-contractor group than that in both contractor group and control group (55.86±3.86 pmol/l vs 37.85±0.88 pmol/l and 37.95±0.74 pmol/L, P＜0.01), but there was no difference between contractor group and control group. Meanwhile no significant difference of the concentration of CCK-f could be observed among three groups. The amount of CCK-R was lower in non-contractor group than those in both control group and contractor group (10.27±0.94 fmol/mg vs24.59±2.39 fmol/mg and 22.66±0.55 fmol/mg, P＜0.01).The activity of CCK-R shown as KD in non-contractor group decreased compared to that in control group and contractor group. Only was the activity of CCK-R lower in contractor group than that in control group. The ejection fraction correlated closely with the amount of CCK-R (r = 0.9683,P＜0.01), and the concentration of CCK-30 correlated negatively with the amount of CCK-R closely (r = -0.9627,P＜0.01).CONCLUSION: The distinctive interactive relationship of gallbladder emptying, plasma CCK and CCK-R in gallbladder from this study suggested that the defect of CCK-R may be a key point leading to the impairment of gallbladder motor function and the pathogenesis of cholesterol gallstoneformation may differ in two subgroups of gallstone patient,gallbladder non-contractor group or contractor group.

Functional brain imaging in irritable bowel syndrome with rectal balloon-distention by using fMRI

AIM: Irritable bowel syndrome (IBS) is characterized by abdominal pain and changes in stool habits. Visceral hypersensitivity is a key factor in the pathophysiology of IBS. The aim of this study was to examine the effect of rectal balloon-distention stimulus by blood oxygenation leveldependent functional magnetic resonance imaging (BOLDfMRI) in visceral pain center and to compare the distribution,extent, and intensity of activated areas between IBS patients and normal controls. METHODS: Twenty-six patients with IBS and eleven normal controls were tested for rectal sensation, and the subjective pain intensity at 90 ml and 120 ml rectal balloon-distention was reported by using Visual Analogue Scale. Then, BOLDfMRI was performed at 30 ml, 60 ml, 90 ml, and 120 ml rectal balloon-distention in all subjects. RESULTS: Rectal distention stimulation increased the activity of anterior cingulate cortex (35/37), insular cortex (37/37),prefrontal cortex (37/37), and thalamus (35/37) in most cases.At 120 ml of rectal balloon-distention, the activation area and percentage change in MR signal intensity of the regions of interest (ROI) at IC, PFC, and THAL were significantly greater in patients with IBS than that in controls. Score of pain sensation at 90 ml and 120 ml rectal balloon-distention was significantly higher in patients with IBS than that in controls. CONCLUSION: Using fMRI, some patients with IBS can be detected having visceral hypersensitivity in response to painful rectal balloon-distention. fMRI is an objective brain imaging technique to measure the change in regional cerebral activation more precisely. In this study, IC and PFC of the IBS patients were the major loci of the CNS processing of visceral perception.

Correlation of thymidylate synthase,thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2′-deoxyuridine

AIM:To determine the expression levels of three metabolic enzymes of fluoropyrimidines:thymidylate synthase (TS),thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines,and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2′-deoxyuridine (FdUrd).METHODS:TS,TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR,TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50),representing the sensitivity to drugs,were determined by MTT assay.RESULTS:IC50 values ranged from 1.28 to 12.26μM for 5-FU,and from 5.02 to 24.21μM for FdUrd,respectively.Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P<0.05), but neither TS nor TP correlated with 5-FU IC50 (P>0.05).Only TS mRNA level was sharply related with FdUrd sensitivity (P<0.05),but TP and DPD were not (P>0.05).A correlation was found between mRNA and protein levels of DPD (P<0.05),but not TP (P<0.05).CONCLUSION:DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd,respectively.

Effects of bile reflux on gastric mucosal lesions in patients with dyspepsia or chronic gastritis

AIM: To investigate the influences of bile reflux on profiles of gastric mucosal lesions in patients with dyspepsia or chronic gastritis.METHODS: A total of 49 patients diagnosed with dyspepsia and chronic gastritis underwent 24-h ambulatory andsimultaneous monitoring of intragastric bilirubin absorbance and pH values, and then they were divided into bile refluxpositive group and bile reflux negative group. Severity of pathological changes in gastric mucosa including activeinflammation, chronic inflammation, intestinal metaplasia, atrophy and dysplasia as well as Helicobacter pylori (H pylori) infection at the corpus, incisura and antrum were determined respectively according to update Sydney system criteria. The profiles of gastric mucosal lesions in the two groups were compared, and correlations between time-percentage of gastric bilirubin absorbance ＞0.14 and severity of gastric mucosal lesions as well as time-percentage of gastric pH ＞4 were analyzed respectively. RESULTS: Thirty-eight patients (21 men and 17 women, mean age 44.2 years, range 25-61 years) were found existing with bile reflux (gastric bilirubin absorbance ＞0.14) and 11 patients (7 men and 4 women, mean age 46.2 years,range 29-54 years) were bile reflux negative. In dyspepsia patients with bile reflux, the mucosal lesions such as active inflammation, chronic inflammation, intestinal metaplasia, atrophy or H pylori infection in the whole stomach, especially in the corpus and incisura, were significantly more severe than those in dyspepsia patients without bile reflux. Moreover, the bile reflux time was well correlated with the severity of pathological changes of gastric mucosa as well as H pylori colonization in the near-end stomach, especially in the corpus region. No relevance was found between the time of bile reflux and pH ＞4 in gastric cavity. CONCLUSION: Bile reflux contributes a lot to mucosal lesions in the whole stomach, may facilitate H pylori colonization in the corpus region, and has no influence on acid-exposing status of gastric mucosa in patients with dyspepsia or chronic gastritis.

Clinical and experimental study of oxaliplatin in treating human gastric carcinoma

AIM: To evaluate the therapeutic effectiveness of oxaliplatinon human gastric carcinoma and to explore its mechanisms.METHODS: Twenty-two cases of stage IV gastric carcinomareceived 4-6 (mean 4.6) cycles of first line combinedchemotherapy with oxaliplatin (oxaliplatin 85 mg/m^2, iv, gtt,1 h, d 1; leukovorin 200 mg/m^2, iv, gtt, 1 h, d 1 and d 2; 5-FU 300 mg/m^2,iv, d I and d 2, 5-FU, continuous iv, gtt, 48 h;1 cycle/2 wk). Response rate, progression-free survival(PFS), total survival time, toxic side effects were evaluated.The inhibitory effect of oxaliplatin on human gastric cell lineSGC-7901 was detected and ICs0 was calculated by MTT.Transmission electron microscopy, flow cytometry andTUNEL were performed to evaluate the apoptosis of cellline induced by the drug. The expression of Caspase-3m-RNA was detected by RT-PCR. AC-DEVD-CHO, aCaspase-3 specific inhibitor, was used to elucidate the roleof activated Caspase-3 in the process of apoptosis inducedby oxaliplatin.RESULTS: Total response (complete and partial) occurredin 9 (40.9%) patients. Mean PFS was 4.2 mo and meantotal survival time was 7.2 mo. Cumulative neurotoxicity(all grade I-II), vomiting and diarrhea, myelosuppressionappeared in 93.5%, 20%, 32.9% patients, respectively.ICs0 was calculated to be 0.71 mg/L by Ml-r assay. A maximalinhibitory rate reached 85.3%. Apoptosis index was elevatedafter incubated with 1 mmol/L oxaliplatin for 30 rain, butwithout statistic significance (P>0.05). However it couldbe detected at a much higher degree both by fiowcytometryand by TUNEL with a statistical significance (68.47+7.92%and 8.23+2.67%, respectively, P<0.05) at^er incubated withi mmol/L oxaliplatin for 2 d. By means of RT-PCR, we detectedan enhancement of Caspase-3 m-RNA expression inducedby oxaliplatin which was also in positive correlation withthe apoptotic level. AC-DEVD-CHO, a Caspase-3 specificinhibitor, could significantly inhibit and delay apoptosisinduced by oxaliplatin.CONCLUSION: Oxaliplatin is effective and well-toleratedin patients with advanced gastric carcinoma. Oxaliplatincould significantly inhibit the growth of human gastric cell lineSGC-7901. The induction of Caspase-3 m-RNA expression,activation of Caspase-3 and promotion of apoptosis maybe some of the therapeutic mechanisms of oxaliplatin ongastric carcinoma. Annexin-V-fluorescein labeling flowcytometry is much more sensitive than TUNEL in detectingearly stage apoptosis.

Polymorphisms at cholesterol 7α-hydroxylase,apolipoproteins B and E and low density lipoprotein receptor genes in patients with gallbladder stone disease

AIM: To investigate the relationship between gallbladder stone disease (GSD) and single nucleotide polymorphisms of cholesterol 7α-hydroxylase (CYP7A) gene promoter,apolipoprotein (APO) B gene exon 26, APOEgene exon 4 or microsatellite polymorphism of low density lipoprotein receptor (LDLR) gene exon 18.METHODS: Genotypes of CYP7A, APOB, APOE and LDLR genes were determined in 105 patients with GSD diagnosed by B-mode ultrasonography and 274 control subjects.Serum lipids were analyzed with HITACHI 7060 automaic biochemical analyzer.RESULTS: Body mass index (BMI) was significantly higher in patients with GSD (24.47±3.09) than in controls (23.50±2.16).Plasma total cholesterol was lower in patients with GSD (4.66±0.92 mmol/L) than in controls (4.91±0.96 mmol/L),P<0.01 after adjusted for age, sex and BMI. The significantly higher frequency of A allele of CYP7A gene polymorphism and X+ allele of APOBgene polymorphism was seen in GSD patients. Percentages of A allele in patients and controls were 62.86% and 54.38% (P <0.05) and those of X+ allele 8.57% and 4.01% (P<0.01). Subjects with A allele had significantly lower plasma total cholesterol and LDL cholesterol than subjects with CC homozygote. In a multiple variable logistic regression model, the BMI (OR=1.13, 95% CI: 1.05-1.22), A allele (OR=1.48, 95% CI: 1.05-2.09) and X+ allele (OR=2.28, 95% CI: 1.14-4.59) were positively associated with GSD (P <0.05). Plasma total cholesterol (OR=0.69, 95% CI: 0.64-0.74) was negatively related to GSD (P<0.05).CONCLUSION: With an association analysis, it was determined that A allele of CYP7A gene and X+ allele of APOB gene might be considered as risk genes for GSD. These alleles are related with differences of serum lipids among subjects.Multiple-variable logistic regression model analysis showed that besides BMI, GSD was affected by polygenetic factors.But the mechanism for these two alleles responsible for GSD requires further investigations.

Helicobacter pylori infection, glandular atrophy and intestinal metaplasia in superficial gastritis, gastric erosion, erosive gastritis, gastric ulcer and early gastric cancer

AIM: To evaluate the histological features of gastric mucosa, including Helicobacter pylori infection in patients with early gastric cancer and endoscopically found superficial gastritis, gastric erosion, erosive gastritis, gastric ulcer. METHODS: The biopsy specimens were taken from the antrum, corpus and upper angulus of all the patients. Giemsa staining, improved toluidine-blue staining, and Hpylori-specific antibody immune staining were performed as appropriate for the histological diagnosis of H pylori infection. Hematoxylin-eosin staining was used for the histological diagnosis of gastric mucosa inflammation, gastric glandular atrophy and intestinal metaplasia and scored into four grades according to the Updated Sydney System. RESULTS: The overall prevalence of H pylori infection in superficial gastritis was 28.7%, in erosive gastritis 57.7%, in gastric erosion 63.3%, in gastric ulcer 80.8%, in early gastric cancer 52.4%. There was significant difference (P<0.05), except for the difference between early gastric cancer and erosive gastritis. H pylori infection rate in antrum, corpus, angulus of patients with superficial gastritis was 25.9%, 26.2%, 25.2%, respectively; in patients with erosive gastritis 46.9%, 53.5%, 49.0%, respectively; in patients with gastric erosion 52.4%, 61.5%, 52.4%, respectively; in patients with gastric ulcer 52.4%, 61.5%, 52.4%, respectively; in patients with early gastric cancer 35.0%, 50.7%, 34.6%, respectively. No significant difference was found among the different site biopsies in superficial gastritis, but in the other diseases the detected rates were higher in corpus biopsy (P<0.05). The grades of mononuclear cell infiltration and polymorphonuclear cell infiltration, in early gastric cancer patients, were significantly higher than that in superficial gastritis patients, lower than that in gastric erosion and gastric ulcer patients (P<0.01); however, there was no significant difference compared with erosive gastritis. The grades of mucosa glandular atrophy and intestinal metaplasia were significantly highest in early gastric cancer, lower in gastric ulcer, the next were erosive gastritis, gastric erosion, the lowest in superficial gastritis (P<0.01). Furthermore, 53.3% and 51.4% showed glandular atrophy and intestinal metaplasia in angular biopsy specimens, respectively; but only 40.3% and 39.9% were identified in antral biopsy, and 14.1% and 13.6% in corpus biopsy; therefore, the angulus was more reliable for the diagnosis of glandular atrophy and intestinal metaplasia compared with antrum and corpus (P<0.01). The positivity rate of glandular atrophy and intestinal metaplasia of superficial gastritis with H pyloripositivity was 50.7%, 34.1%; of erosive gastritis 76.1%, 63.0%; of gastric erosion 84.8%, 87.8%; of gastric ulcer 80.6%, 90.9%; and of early gastric cancer 85.5%, 85.3%, respectively. The positivity rate of glandular atrophy and intestinal metaplasia of superficial gastritis with H pylorinegativity was 9.9%, 6.9%; of erosive gastritis 42.5%, 42.1%; of gastric erosion 51.1%, 61.9%; of gastric ulcer 29.8%, 25.5%; and of early gastric cancer 84.0%, 86.0%, respectively. The positivity rate of glandular atrophy and intestinal metaplasia of superficial gastritis, erosive gastritis, gastric erosion, and gastric ulcer patients with H pylon positivity was significantly higher than those with H pylori negativity (P<0.01); however, there was no significant difference in patients with early gastric cancer with or without H pylori infection. CONCLUSION: The progression of the gastric pre-cancerous lesions, glandular atrophy and intestinal metaplasia in superficial gastritis, gastric erosion, erosive gastritis and gastric ulcer was strongly related to H pylori infection. In depth studies are needed to evaluate whether eradication of H pylori infection will really diminish the risk of gastric cancer.

TK gene combined with mIL-2 and mGM-CSF genes in treatment of gastric cancer

AIM: Cancer gene therapy has received more and moreattentions in the recent decade. Various systems of genetherapy for cancer have been developed. One of the mostpromising choices is the suicide gene. The product ofthymidine kinase (TK) gene can convert ganciclovir (GCV)to phosphorylated GCV, which inhibits the synthesis of cellDNA, and then induces the cells to death. Cytolines play animportant role in anti-tumor immunity. This experiment wasdesigned to combine theTK gene and mIL-2/mGM-CSFgenes to treat gastric cancer, and was expected to producea marked anti-tumor effect.METHODS: TK gene was constructed into the retroviralvector pLxSN, and the mIL-2 and mGM-CSF genes wereinserted into the eukaryotic expressing vector pIRES. Thegastric cancer cells were transfected by retroviral serum thatwas harvested from the package cells. In vitro study, thetransfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect andbystander effect. In vivo experiment, retroviral serum andcytokines plasmid were transfected into tumor-bearing mice,to observe the changes of tumor volumes and survival ofthe mice.RESULTS: In vitro experiment, 20 % TK gene transducedcells could cause 70-80 % of total cells to death. In vivoresults showed that there was no treatment effect in controlgroup and TK/GCV could inhibit the tumor growth. Thestrongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis ofthe cancer in the treated groups.CONCLUSION: TK/GCV can kill tumor cells and inhibit thetumor growth in vivo IL-2 and GM-CSF strongly enhancethe anti-tumor effect. Through the retrovirus and liposomemethods, the suicide gene and cytokine genes are allexpressed in the tissues.

Adenoviral transfer of human intedeukin-10 gene in lethal pancreatitis

AIM: To evaluate the therapeutic effect of adenoviral-vector-delivered human interleukin-10 (hIL-10) gene on severe acute pancreatitis (SAP) rats. METHODS: Healthy Sprague-Dawley (SD) rats were intraperitoneally injected with adenoviral IL-10 gene (AdvhIL-10), empty vector (Adv0) or PBS solution. Blood, liver, pancreas and lung were harvested on the second day to examine hIL-10 level by ELISA and serum amylase by enzymatic assay. A SAP model was induced by retrograde injection of sodium taurocholate through pancreatic duct. SAP rats were then administered with AdvhIL-10, Adv0and PBS solution by a single intraperitoneal injection 20 min after SAP induction. In addition to serum amylase assay, levels of hIL-10 and tumor necrosis factor-α (TNF-α) were detected by RT-PCR, ELISA and histological study. The mortality rate was studied and analyzed by Kaplan-Meier and log rank analysis.RESULTS: The levels of hIL-10 in the pancreas, liver and lung of healthy rats increased significantly after AdvhIL-10 injection (1.42 ng/g in liver, 0.91 ng/g in pancreas); while there was no significant change of hIL-10 in the other two control groups. The concentration of hIL-10 was increased significantly in the SAP rats after AdvhIL-10 injection (1.68 ng/g in liver, 1.12 ng/g in pancreas) compared to the other two SAP groups with blank vector or PBS treatment (P<0.05). The serum amylase levels remained normal in the AdvhIL-10 transfected healthy rats. However, the serum amylase level was significantly elevated in the other two control SAP rats. In contrast, serum amylase was down-regulated in the AdvhIL-10 treated SAP groups.The TNF-α expression in the AdvhIL-10 treated SAP rats was significantly lower compared to the other two control SAP groups. The pathohistological changes in the AdvhIL-10 treated group were better than those in the other two control groups. Furthermore, the mortality of the AdvhIL-10 treated group was significantly reduced compared to the other two control groups (P<0.05). CONCLUSION: Adenoviral hIL-10 gene can significantly attenuate the severity of SAP rats, and can be used in the treatment of acute inflammation process.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Antitumor effects of vaccine consisting of dendritic cells pulsed with tumor RNA from gastric cancer

AIM:To investigate the immunotherapeutic potential of vaccine consisting of dendritic cells (DCs) pulsed with total RNA from MFC gastric cancer cells.METHODS:DCs were prepared from the spleens of strain 615 mice by magnetic cell sorting (MACS). Alter culture for 24h, DCs were pulsed with total RNA from MFC gastric cancer cells. Mice of one group were immunized with tumor RNA pulsed DC (RNA/DC) at the dosage of 1×10^6 on d 14 and 7 by s c inoculation before tumor implantation. Mice of another group were immunized with unpulsed DC (UDC) at the same dosage on days as the RNA/DC group. The third group of control mice was untreated. On d 0, all the mice were challenged with s c injections of 5×10^5 MFC gastric cancer cells.Alter inoculation, the mice were monitored closely with respect to tumor growth. Activities of NK cells in PBL and splenocytes and CTL were tested.RESULTS:On d 21 alter tumor cell inoculation, the mice of control group manifested the largest tumors with volume at a mean of 2.6323±1.1435cm^3, followed by the UDC and RNA/DC groups with mean volumes at 0.7536±0.3659cm^3 and 0.3688±0.6571cm^3, respectively. The activities of NK cells in PBL and splenocytes in RNA/DC group were 66.2% and 65.4%, respectively, higher than that in the control group. The tumor specific CTL activity in RNA/DC group was 49.5%, higher than that in the control group.CONCLUSION:The tumor vaccine with DCs pulsed with total RNA from gastric cancer cells possesses the ability to stimulate tumor specific CTL activity and to establish antitumor immunity when administered in vivo.

Capsule endoscopy in diagnosis of small bowel Crohn's disease

AIM: To evaluate the effectiveness of wireless capsule endoscopy in patients with suspected Crohn's disease (CD) of the small bowel undetected by conventional modalities,and to determine the diagnostic yield of M2A Given Capsule.METHODS: From May 2002 to April 2003, we prospectively examined 20 patients with suspected CD by capsule endoscopy. The patients had the following features:abdominal pain, weight loss, positive fecal occult blood test, iron deficiency anaemia, diarrhoea and fever. All the patients had normal results in small bowel series (SBS) and in upper and lower gastrointestinal endoscopy beforethey were examined. Mean duration of symptoms before diagnosis was 6.5 years.RESULTS: Of the 20 patients, 13 (65%) were diagnosed as CD of the small bowel according to the findings of M2A Given Capsule. The findings detected by the capsule were mucosal erosions (2 patients), aphthas (5 patients),nodularity (1 patient), large ulcers (2 patients), and ulcerated stenosis (3 patients). The distribution of the lesions was mainly in the distal part of the small bowel,and the mild degree of lesions was 54%.CONCLUSION: Wireless capsule endoscopy is effective in diagnosing patients with suspected CD undetected by conventional diagnostic methods. It can be used to detect early lesions in the small bowel of patients with CD.

Effect of tetramethylpyrazine on P-selectin and hepatic/renal ischemia and reperfusion injury in rats

AIM: To investigate the effect of tetramethylpyrazine on hepatic/renal ischemia and reperfusion injury in rats. METHODS: Hepatic/renal funclJon, histopathotogical changes, and hepatic/renal P-selectin expression were studied with biochemical measurement and immunohistochemistry in hepatic/renal ischemia and reperfusion injury in rat models. RESULTS: Hepatic/renal insufficiency and histopathological damage were much less in the tetramethylpyrazine-treated group than those in the saline-treated groups. Hepatic/renal P-selectin expression was down regulated in the tetramethylpyrazine-treated group. CONCLUSION: P-selectin might mediate neutrophil infiltration and contribute to hepatic/renal ischemia and reperfusion injury. Tetramethylpyrazine might prevent hepatic/renal damage induced by ischemia and reperfusion iniury throuclh inhibition of P-selectin.