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ObjectiveTo investigate the preventive effects of rosiglitazone on nonalcoholic steatohepatitis (NASH) rats and to explore the potential mechanisms in modulating peroxisome proliferator-activated receptor gamma (PPARγ),nuclear factor kappa B (NF-κB),and cyclooxygenase-2 (COX-2) expression.Methods Thirty male SD rats were assigned into the normal group ( n =10),the model group ( n =10),rosiglitazone prevention group [ n =10,simultaneously 4mg/( kg · d) gavage daily at beginning].Liver appearance,liver index,and histological changes were assessed.Serum tumor necrosis factor-o (TNF-c) and prostaglandin E2 (PGE2) were determined using enzyme-linked immunosorbent assay.The expressions of PPARγ,NF-κB,and COX-2 in liver were determined using immunohistochemical methods.The mRNA and protein expressions of COX-2 were disclosed by real-time polymerase chain reaction and Western blot analysis.ResultsCompared with the normal group,the liver index significantly increased in model group (3.92 ±0.72 vs.5.71 ± 1.05,P =0.004).HE and Masson staining showed significantly increased steatosis,inflammation,and fibrosis.The serum levels of TNF-α,PGE2 in high-fat-diet-fed rats were significantly increased ( 11.72 ± 2.47 vs.29.39 ± 5.32,P =0.002 ; 236.60 ± 24.90vs.288.24 ± 17.17,P =0.004).Immunohistochemistry showed NF-κB and COX-2 in livers were significantly elevated,but PPARγ wasdecreased in nonalcoholic steatohepatitis rats.Real-time polymerase chain reaction and Western blot found mRNA and protein expressions of COX-2 were increased in the model group (0.57 ± 0.08 vs.2.83 ± 0.24,P =0.0007 ; 0.38 ± 0.03 vs.1.00 ± 0.03,P =0.004).Compared with the model group,the expressions of PPARγsignificantly increased and the expressions of NF-κB and COX-2 significantly decreased ( mRNA:2.83 ± 0.24 vs.0.46 ± 0.11,P =0.002 ; protein: 1.00 ± 0.03 vs.0.62 ± 0.02,P =0.006 ) in the rosiglitazone prevention group.ConclusionBy inhibiting NF-κB and COX-2 expressions,rosiglitazone can reduce insulin resistance and then prevent the occurrence and deve lopment of nonalcoholic steatohepatitis.
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Objective To study the expression of PIWIL1, PIWIL3 and PIWIL4 in human colon cancer and its clinical significance. Methods We collected cancerous tissues and its adjacent tissues of 106 patients with colon cancer, two tissue microarrays were constructed, with 62 and 150 points respectively. We studied the expression of PIWIL1, PIWIL3 and PIWIL4 through immunohistochemistry. Results The expression of PIWIL1, PIWIL3 and PIWIL4 were significantly higher in cancerous tissues than those in adjacent tissues (P＜0. 01). In cancerous tissues and its adjacent tissues, postive correlation were seen among PIWIL1, PIWIL3 and PIWIL4 expression (P＜0. 01). PIWIL1 expression was significant higher in low differentiation group than that in high differentiation group (t =- 2. 840, P＜0.01 ). PIWIL3 expression was higher in high clinical stage than that in low clinical stage (F= 3. 112, P＜0.05). The expression of PIWIL3 and PIWIL4 were significantly higher in patients with colon cancer with distant metastasis than those without distant metastasis (t= -3. 349, P＜0.01 ; t = - 2. 168, P＜0. 05). PIWIL3 and PIWIL4 expression were correlated with occurring of colon cancer (P＜0. 01). Conclusions The expressions of PIWIL1,PIWIL3 and PIWIL4 in colon cancer were correlated with the differentiation, clinical stage and distant metastasis of colon cancer. PIWIL3 and PIWIL4 expression were two independent related factors of occurring of colon cancer, which would be furtherly investigated to be served as novel markers for early diagnosis and promising molecular targets for colon cancer therapy.
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Objective To evaluate the impact of rosiglitazone (Ros) on liver expression of peroxisome proliferator-activated receptor γ (PPARγ),nuclear factor (NF-κB) and cyclooxygenase-2(COX-2) in treatment of nonalcoholic steatohepatitis (NASH) rats.Methods Thirty Sprague-Dawley rats were divided into normal group,model group and Ros treated group with 10 each.Except the normal group,the other two groups were given high fat diet for 12 weeks for NASH model.The rats in Ros treated group were gavaged 4 mg/kg of Ros daily at the 12th week for 8 weeks.All rats were sacrificed at the 20th week for blood sample and liver tissue.Biochemical parameters of liver function,lipid metabolism,glycometabolism and antioxidant enzyme activities were measured.The histological change of the liver were assessed with HE and Masson staining.The level of tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2) was measured using ELISA.The expression of PPARγ,NF-κB and COX-2 was detected with immunohistochemistry.The mRNA and protein expressions of COX-2 were tested by real-time PCR and Western blotting,respectively.Results In comparison with model group,Ros treated group showed significant improvement in hepatic steatosis,inflammation and fibrosis(all P value<0.05).In model group,the serum levels of fasting blood glucose,insulin and HOMA-insulin resistance index (HOMA-IRI),total cholesterol (TC),total triglyeride (TG),lowdensity lipoprotein cholesterol (LDL-C) and free fatty acids were increased,but HDL-C level was decreased.All above parameters markedly improved after Ros treatment.The levels of ALT and AST,total anti-oxidation competence,superoxide dismutase,catalase,glutathione peroxidase and malondialdehyde in Ros treated group were significantly ameliorated when compared with those in model group.Immunohistochemistry showed that the expression of NF-κB and COX-2 was significantly elevated,but PPARγ was decreased in model group.Real-time PCR and Western blot revealed that the mRNA and protein expressions of COX-2 were higher in the model group than those in normal group (0.57±0.08 vs 0.38±0.03;2.83±0.24 vs 1.00±0.03,P=0.000 and P=0.004,respectively),but significantly lower in Ros treated group (0.55±0.06 and 1.84±0.13,P<0.01).Conclusions Ros can reduce oxidative stress and insulin resistance in NASH rats by activing PPARγ expression and inhibiting expression of NF-κB and cyclooxygenases.
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Objective The primary aim of this study was to examine the proportion and natural history of Helicobacter pylori (Hp) negative bleeding peptic ulcers. Methods The study was designed as a multiple-center, case-control study conducted in 14 endoscopy centers in China from April 2006 to March 2007. Each center was expected to recruit 30 peptic ulcer patients with bleeding ( PUB group) and 30 without (PU group). All screened patients with upper gastrointestinal bleeding received endoscopy within 24 hours of admission. Biopsy specimens were taken from the antrum to determine Hp infection by rapid urease test and pathology. Patients with negative Hp infection at first examination were asked to receive urease breathe test (UBT) one month later. Results A total of 617 patients were enrolled with 263 in PUB group and 354 in PU group. There is no significant difference in demographic characters between 2 groups ( P ＞0. 05). The rate of Hp infection in PUB group ( 161/263, 61.2% ) was significantly lower than that in PUgroup (311/354, 87. 9%, P ＜0. 001 ). The incidence of complex ulcer in Hp positive PUB patients was 7.5% ( 12/161 ), which is significantly higher than that in Hp negative PUB patients ( 1/102, 1.0% , P =0. 018). In PUB group, no significant differences were found between Hp positive and negative patients in regarding of age, sex, rates of haematemesis, duodenal ulcer and gastric ulcer, and size of ulcer ( P ＞0. 05 ). Among 102 Hp negative cases in PUB, no positive case was found in UBT one month later. Conclusion We have demonstrated a rise in the incidence of Hp negative bleeding ulcers in China. The idiopathic ulcer was not rare, and might have a higher tendency to cause bleed.
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Objective To elucidate cerebral cortical response to esophageal acid exposure in normal individuals by functional magnetic resonance imaging (fMRI) and the characteristics of activity. Methods Fifteen volunteers were received intraesophageal perfusion with either 0.9% of sodium chloride or acid (0.1 mmol/L HC1) solutions. The modified block-design model of fMRI scanning was performed simultaneously. All of 32 minutes were needed for resting (A, 8 minutes), 0.9% of sodium chloride perfusion (B,8 minutes), acid perfusion (C,8 minutes) and 0.9% of sodium chloride perfusion again (D,8 minutes). Each chunk was consisted of 160 scans and every scan contained 3 seconds. Six hundred and forty scans were collected in all. The clinical response to esophageal acid exposure was observed and the changes in the cerebral regions was statistically analyzed. Results After perfusion of 0.9% of sodium chloride or acid, 10 out of 15 volunteers had chemosensitive complaints, such as pain in pars laryngen pharyngis, heartburn and chest complaint. The initial active domains involved deutocerebrum, anterior part of callosal gyrus, left side of insula, two sides of amygdale and subiculum hippocampi, two outers of forehead cortex. The provoked regions of acid perfusion (C-A) and 0.9% of sodium chloride perfusion again (D-A) were as same as that of the activated domains by initial perfusion of 0.9% sodium chloride (B-A). The intensity and amplitude of most provoked regions increased gradually(D-A> B-A, P< 0.01). Conclusions The two different stimulations of saline and acid provoke similar cerebral regions that may act in the regulation of esophageal sensitivity. There are the evidences of the central mechanism of esophageal visceral hypersensitivity by acid perfusion.
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Objective To investigate the whole genomic expression profiles of chronic atrophic gastritis with interleukin(IL)-1β-31CC/-511TT genotype as measured by oligonucleotide microarray technique.Methods Genomic RNA was extracted from gastric biopsies of 12 patients with chronic atrophic gastritis(6 with IL-1β-31CC/-511TT and 6 with IL-1β-31TT/-511CC).The genomic profiles of IL-1β gene polymorphisms 31CC/-511TT and 31TT/-511CC were compared and tested for differential expressed genes associated with 31CC/-511TT using Agilent human whole genomic oligonucleotide microarrays.The results were further analyzed in terms of gene ontology(GO).Results There were 200 differentially expressed genes associated with IL-1β-31CC/-511TT,159 of which were up-regulated and 41 were down-regulated.These genes mainly involved in macromolecule metabolic process,post-translational protein modification,ubiquitin cycle,and protein kinase cascade.Five genes had biological activities,one of which was down-regulated gene(PCSK5)and 4 were upregulated genes(PRKCA,NPLOC4,TRIB3 and MAPKAPK3).Conclusions The chronic atrophic gastritis with IL-1β-31CC/-511TT genotype has molecular phenotypes which is associated with malignance and inflammation.These individuals are needed more intensive preemptive treatment and dynamic surveillance.
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Background Magnetic activated cell sorting(MACS)is a new immunomagnetic separation technique.It's principle is based on the specialized recognition between the antibody and antigen.When directed or indirected coupled with the 50 nm magnetic beads,the antibody can linked with the cells which express the corresponding antigen,and then leads to the magnetic cell separation in a high intensity and high-gradient magnetic field.The method has higher separative purity and recovery and been made use of enriching tumor cells and tumor stem cells.It could be also utilized to enrich the rare tumor cells in fluid specimen.Objective To evaluate the enriching efficiency of detecting free cancer cells in analogue malignant ascites using MACS and a panel of tumor-specific markers.Methods Five species of tumor cell lines correlated to the diseases resulting in malignant ascites were selected and the expressions of EpCAM,CAl25,CEA,TAG-72 and their mixture in these tumor cell lines were detected using immunofluorescence reactions and flow cytometry(FCM).The tumor cells were spiked into mononuclear cells by different ratios to analogue the main ingredients of malignant ascites.The efficiency of MACS combinded with mixture antibodies in separating tumor cells was compared with single antibody.Results The FCM revealed that the expression of the mixture antibodies in 5 tumor cancer cell lines was significantly higher than that in single antibody.The mean recovery rates of spiked tumor cells were enhanced by using a panel of monoclonal antibodies combined with MACS than single antibody,especially in two gastric cancer cells and colon cancer cells(69.18%±20.84%VS 45.23%±11.54%,78.75%±15.42%vs 59.73%±16.64%and 85.63%±12.30%VS 76.88%±8.65%,respectively),the ovarian cancer cells was the next(32.49%±3.58%vs31.79%±4.82%),and the liver cancer cells was the lowest(11.78%±0.43%VS 7.16%±0.46%).Conclusions The detection of free cancer cells from malignant ascites by MACS combined with a panel of monoelonal antibodies is more effectively than single antibody.The method has the potencial value of clinical application to the malignant ascites caused by gastroenteric tumor.
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Objective To investigate the changes of esophageal visceral sence stimulated by esophageal distention in rabbits and the protein expressions of calcitonin gene related peptide(CGRP),P substance(SP),5-hydroxytryptamine(5-HT),Fos protein in central nervous system(CNS).Methods Twenty New Zealand rabbits were randomly divided into experimental group(n=8,received esophageal distention with 0.9 cm balloon for 30 s twice a day for 14 days),control group(n=6,received esophageal stimulation without balloon for 30 s twice a day for 14 days)and blank control grouop(n=6).The esophageal visceral sense was evaluated by animal behavior scores.The expressions of SP,CGRP,5-HT and Fos protein esophagus mucosa,spine,nucleus tractus solitari (NTS),periaqueductal gray(PAG)and thalamus were measured by immunochistochemistry.Results At the same behavior scores,the tube diameter of experimental group was significant lower than those of control group(P＜0.05).The expression of SP in esophagus mucosa,spine and NTS was significant increased in experimental group compared to two control groups(P＜0.05).The expression of CGRP and Fos in esophagus mucosa.spine,NTS,PAG and thalamus was increased in experimental group compared to two control groups(P＜0.05).The expression of 5-HT in esophagus mucosa and spine was higher in experimental group than that in control and blank control groups (esophagus: 27.67±3.27 vs 11.00±1.79 or 11. 17±1.33;spine:24.00±5.22 vs 11.33±2.94 or 11. 83±2. 48, P＜0. 01). But the expression of 5-HT in PAG was lower in experimental group( 13. 17±2.04) than that in control 17.67±2.07)and blank control (16.83±2.32) groups (P＜0. 05). There was significant correlation between CGRP and Fos, SP and Fos, CGRP and SP in spine (r=0. 813,0. 779,0. 772,P=0. 025,0.034, 0. 036, respectively). Conclusions Esophageal hypersensitivity may be induced by esophageal distention. The expresstion of SP, CGRP, 5-HT was increased in the esophageal mueosa and CNS, which indicate that these neurotransmitters and CNS may play an important role in the increase of esophageal visceral sense.
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Objective To investigate the association between body mass index(BMI),waist circumference(WC),waist hip ratio(WHR) and the risk of colorectal adenoma,which was considered as a precancerous lesion.Methods Subjects aging from 25 to 88 years old who underwent colonoscopy at Tongji Hospital from December 2006 to December 2007 were selected and assigned into the adenoma group( n =250) and the control group(n=289) according to the findings of the colonoscopy.The body height,weight,waist and hip circumference of every subject were measured respectively.The logistic multi-factors regression analysis was applied to analyze the data.Results When obesity was determined by BMI or WC,the risk of adenorrm in pure obesity group and abdominal adiposity group was 2.48(95%CI = 1.19～5.20,P<0.05) and 1.75(95%CI=1.15～2.66,P<0.01 ),respectively.The corresponding value in male was 4.10(95%CI = 1.26～13.31,P<0.05) and 1.70(95%CI = 1.00 -2.88,P<0.05).The risk of advanced and non-advanced adenoma in pure obesity was 2.71(95%CI=1.01～7.29,P<0.05 ) and 2.39(95%CI=1.05～5.47,P<0.05) ; the risk of non-advanced adenonm in abdominal adiposity group was 2.03(95%CI=1.25～3.28,P<0.01),but no significant difference in risk of advanced adenoma was detected.When obesity was determined by WHR,no significant difference was found in any regarding.Conclusion Obesity and abdominal adiposity are associated with the risk of colorectal adenoma,beth advanced and non-advanced,which is more obvious in male.
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Objective To compare the efficacy, toxicity and later period complications of Cf-252 neutron intracavitary brachytherapy(IBT) combined with external-beam radiotherapy (EBRT) with those of EBRT alone in patients with esophageal carcinoma. Methods Eighty-six patients were randomized into 252Cf neutron IBT and EBRT group (intracavitary group: 43 patients) and EBRT alone group (external group:43 patients). The external group was treated with three-dimensional conformal radiotherapy(3DCRT) or conventional radiotherapy of 70 Gy in 7.0 weeks using Elekta Precise medical linear accelerator. The EBRT in intraeavitary group was as same as external group, except the total dose was decreased to 60 Gy in 6.5 weeks. For IBT, the applicator with special water bursa was settled to the esophageal lesion through the mouth. The dose calculation point was 10 mm far away from the source and 1-2 em cranial-caudally from the tumor margin. 252Cf braehytherapy was delivered 3-4 fractions at 4 Gy per fraction per week. In intracavitary group, EBRT was begun on the second day of IBT. EBRT and IBT were not given on the same day. Results After the treatment,the esophageal stricture was relieved earlier in intracavitary group than external group.Six patients in intracavitary group who had drinking obstruction symptom could eat liquid food after esophageal balloon dilation, one fraction of 252 Cf neutron IBT and 5-6 days of EBRT, and could eat semiliquid food two weeks after. In the third month, the complete response rate, partial response rate and no response rate were 33%, 67% and 0% in i ntracavitary group and 19% ,76% and 5% in external group, respectively. The overall response rates of the two groups were 100% and 95% ( χ2 = 4.32, P ＜ 0.05 ). The 1 -year local control rates were 84% and 70% (χ2 =4.57 ,P ＜0.05). The 1-year survival rates were 81% and 61% (χ2 =4.17,P ＜0.05 ). The rates of acute esophageal toxicity was 61% and 51% ( χ2 = 1.75,P ＞ 0.05 ). The acute radiation esophagitis was slightly higher in "BZ ]intracavitary group than that in external group, but the difference was insignificant. The late esophageal-cardiac stricture had no significant difference between the two groups. Conclusions 252 Cf-252 neutron IBT plus EBRT, without increasing the toxicity,are better than EBRT alone.
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Objective To prepare polyclonal antibodies against human PIWIL and to identify their property and tissue distribution of PIWIL.Methods PIWIL polypeptide was synthesized and conjugated to Keyhole limpet hemocyanin(KLH) as an immunogen.Then PIWIL-KLH conjugations were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification by affinity chromatography.PIWIL were then stained on the tissue chip to study their distribution.Results Rabbit antibodies against PIWIL were prepared after injection of PIWIL-KLH conjugation.These antibodies specially recognized PIWIL peptides.Expression of PIWIL was found in the cytoplasm of epithelia cells of varied normal tissues and tumor tissues.Conclusion The successful preparation of the polyclonal antibody against PIWIL will provide an efficient reagent for further study of its role in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.
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Objective The infiltration and micrometastasis of cancer cells via lymph vessel and blood is the important factors which influence the prognosis of patients with cancer. The present study was to establish the method of cytological examination of circulating gastric cancer cell in the peripheral blood and to explore its clinical significance. Methods The monocytes in blood samples, taken from 24 patients with advanced gastric cancer, were seperated by using Ficoll density gradient centrifugation and magnetic cell sorting system in which the magnetic microbeads were wrapped with cytokeratin 7 and 8. The samples were smeared and observed with HE stain. The expression of CD 34 , CD 45 , CEA and human telomerase reverse transcriptase (hTERT) of the cells were also examined with immunocytochemical and immunofluorescence staining methods. Results Gastric cancer cells were found in 10 out of 24 cases, and the positivity rate was 41.7%, which was significantly different from pathological differentiation of primary focus. Conclusion This method can be used to detect cancer cells from peripheral blood of patients with gastric cancer and therapeutic protocol and prediction of prognosis can be made based on it.
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Objective:To determine the expression level of bone morphogenetic proteins-4 (BMP-4) and the relationship between the expression of BMP-4 and the clinical/pathological parameters in patients with gastric cancer. Methods:Using im-munohistochemical staining technique ,expressions of BMP-4 were investigated in different tissues from 90 gastric cancer specimens and 30 specimens from normal gastric mucosa as control. ResultS:The positive rate of BMP-4 was 23. 3% (21/90) in gastric cancer and 3. 3% (1/30) in normal gastric mucosa. The expression of BMP-4 in gastric cancer was closely related with locus, serosa infiltration,cell differentiation and lymph node metastasis (P
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Objective To investigate the correlation between the expression of vascular endothelial growth factor-C(VEGF-C) and lymph node metastasis in gastric cancer. Methods The expression of VEGF-C was detected in 30 pairs of fresh primary gastric cancers and their adjacent normal tissues from the same patients by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Automated image analyzer was used to quantify VEGF-C expression, which was reflected by positive index (PI). Results VEGF-C was highly expressed in gastric cancer cells,whilst its expression was undetectable in adjacent normal tissues. VEGF-C expression was higher in lymph node metastasis group(PI=1.345?0.079) and lymphatic invasive(PI=1.315?0.037) group than that in no-metastasis group(PI=1.156?0.045) and no-invasive group (PI=1.154?0.043)(P
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AIM: To investigate the effects of octreotide (Oct) on the proliferation and extracellular matrix (ECM) synthesis in hepatic stellate cells (HSCs). METHODS: HSCs were isolated from normal male Sprague-Dawley rat liver by a combination of pronase-collagenase perfusion and density gradient centrifugation. The concentration of 2.5 ?g/L transforming growth factor ?1 (TGF?1) was used in all the experiment settings. Oct at concentrations of 0.01 mg/L ,0.1 mg/L,1 mg/L and 10 mg/L,respectively,or 0.01 mg/L Oct + TGF?1,0.1 mg/L Oct+TGF?1,1 mg/L Oct+TGF?1,10 mg/L Oct+TGF?1 were respectively added to the cultured HSCs. Effects of Oct on HSC proliferation and ECM synthesis were respectively determined by MTT method,-TdR and -proline incorporation,or radioimmunoassay. RESULTS: Oct inhibited MTT intake by cultured hepatic stellate cells and down-regulated -TdR incorporation,compared with control group. The concentrations of hyaluronic acid,laminin,collagen type IV in the culture supernatant and -proline incorporation in HSCs were decreased by Oct. TGF?1 obviously up-regulated proliferation and ECM synthesis in cultured HSCs,and Oct significantly blocked these actions. CONCLUSION: Oct inhibited proliferation and ECM synthesis in cultured HSCs,and elicited the effects of anti-hepatofibrogenesis.
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Objective:To prepare rabbit polyclonal antibody against human argonaute 3(AGO3) protein,to identify its properties and investigate the tissue distribution of AGO3 using tissue array.Methods:AGO3 peptide was synthesized using chemical method,and then conjugated to Keyhole limpet hemocyanin(KLH) as immunogen.The AGO3-KLH conjugate were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification using affinity chromatography.Then the distribution of AGO3 in tissues was examined through immuno-stainning by tissue array.Results:Rabbit polyclonal antibodies against AGO3 were raised after immunization with AGO3-KLH conjugates.The anti-serum titer after the last inoculation was up to 1∶20 000.The preparations of the antibody were confirmed to raised recognize AGO3 peptides specially by ELISA and Western blot.AGO3 protein was stained positively in the cytoplasm of tumor cells and epithelial cells in many normal tissues.Conclusion:The polyclonal antibody against AGO3 protein has been achieved successfully,and it provides an efficient tool for further studying the roles of AGO3 in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.