[The diagnostic value of double balloon endoscopy in small intestine disease].

OBJECTIVE: To study the diagnostic value of double-balloon endoscopy (DBE) for small intestinal diseases. METHODS: 155 patients clinically suspicious of small intestinal diseases were studied. 110 of them were male and 45 female. Their age ranged from 6 to 75 and with an average of 41 years. They consisted of 92 cases of small intestinal hemorrhage, 39 abdominal pain, 7 diarrhoea, 13 abdominal distention, 3 malnutrition and one diarrhoea as well as refractory hypoalbuminemia. In the procedure, the operator manipulated and advanced the endoscope and the assistant helped to advance the over tube. RESULTS: Among the 155 cases lesions were found in 125 cases, with positive results accounting for 80.6%. These lesions mainly consisted of small intestinal ulcer (including Crohn's disease), chronic inflammation, Meckel's diverticulum, interstizialoma, vascular deformity and carcinoma of small intestine. In 84 of the 92 patients suspicious of intestinal hemorrhage the lesions were confirmed with a positive rate of 91.3%. In 24 of the 39 patients with abdomen pain the etiologies were confirmed with a positive rate of 61.5%. In 16 of the 23 patients with diarrhoea, abdominal distention and malnutrition the positive rate was 69.6%. The cause of the only one case with refractory hypoalbuminemia was confirmed. Among the 155 cases, 9 had lesions located in stomach and duodenum, 115 in small intestine and one in large bowel, no lesion was found in 30 cases. Among the patients, 43 were found to have small intestinal ulcer. In the 43 patients, 12 patients were with single intestinal ulcer and 31 with multiple. For cases of Meckel's diverticulum, interstizialoma, carcinoma, vascular deformity and intestinal adhesion of small intestine in this series, diagnoses made by DBE combined with morphology were completely consistent with those found in operation. However, for ulcer lesions (mainly Crohn's disease), there was diversity in the diagnoses between the two methods, the coincidence was 57.1%. Two patients had complication, one perforation of small intestine and the other acute intestinal stasis. CONCLUSIONS: DBE is efficient and safe for the diagnosis of small intestinal diseases, especially in confirming the lesions. However, for ulcer of small intestine, this method even combined with biopsy is sometimes unable to determine its nature, so surgery may be beneficial in this condition.

[Clinical value of monitoring CD14+ monocyte human leukocyte antigen (locus) DR levels in the early stage of sepsis].

OBJECTIVE: To explore the relationship of monitoring CD14(+) monocyte human leucocyte antigen (locus) DR (HLA-DR) and the outcome in the early stage of sepsis. METHODS: Thirty-six definitely diagnosed septic patients in intensive care unit (ICU) were included. CD14(+) monocyte HLA-DR levels were detected by flow cytometer on the first day of the study, and acute physiology and chronic health evaluation II (APACHE II) scores were evaluated. Their clinical values in predicting the outcome of the disease were assessed through correlation analysis. RESULTS: Among 36 sepsis patients CD14(+) monocyte HLA-DR level<30% was found in 6 patients (16.67%). The average APACHE II score was 24.17+/-4.45 (r=0.212, P=0.687), all of them die, CD14(+) monocyte HLA-DR level <40% was 27.78% (10/36), the scores of APACHE II score was 23.50+/-4.30 (r=-0.0251, P=0.484), and the mortality rate was 80% (8/10). CONCLUSION: CD14(+) monocyte HLA-DR level <30% is an immunosuppressive index. In predicting the outcome of sepsis, it might be better than APACHE II scores. Immunosuppression is primarily found in the early stage of sepsis, suggesting that the classical compensatory anti-inflammatory response syndrome (CARS) hypothesis needs to be revised and improved.

Expression of Helicobacter pylori AlpA protein and its immunogenicity.

AIM: To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 mug/mL ampicillin overnight at 37 degrees. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of beta-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.

[Preparation oral liposome-encapsulated recombinant Helicobacter pylori heat shock protein 60 vaccine for prevention of Hp infection].

OBJECTIVE: To prepare oral liposome-encapsulated recombinant Helicobacter pylori (Hp) heat shock protein 60 (Hsp60) vaccine and investigate its effect against Hp infection in mice. METHODS: The recombinant vector PET-22(+)/Hsp60 was transformed into BL21(DE3) E.coli. The recombinant protein was purified with Ni-NTA agrose resin and the oral liposome-encapsulated vaccine was prepared with phosphatidyl choline and cholesterols using film method, with the size distribution of the folate liposomes measured by transmission electronic microscopy. BALB/c mice were divided into 5 groups and immunized by intragastric administration of PBS, liposome, rHsp60 plus choleratoxin (CT), liposome-encapsulated rHsp60, and liposome-encapsulated rHsp60 plus CT, respectively, given once a week for 4 weeks. All the mice were challenged by Hp for 3 times within two weeks following the last immunization and sacrificed 3 weeks after the last challenge. Hp detection was performed by fast urease test. Semi-quantitative assessment of the bacterial colonization density observation of the inflammation severity and gastric histopathology were carried out. RESULTS: The soluble expression product accounted for 27% of the total bacterial protein. The purity of recombinant fusion protein was about 95% after purification. The mean size of the folate liposomes was 0.7+/-0.4 mum. PBS or liposome alone showed no immune-enhancing effect, and rHsp60 plus CT, liposome-encapsulated rHsp60 and liposome-encapsulated rHsp60 plus CT had the protective rates against Hp infection of 73.3%, 66.7% and 86.7%, respectively. The latter 3 preparations effected significantly reduced Hp infection and alleviated the inflammation in the gastric mucosa of the mice challenged with Hp. CONCLUSION: The oral liposome may serve as a potential adjuvant for Hp vaccine in preventing Hp infection.

Changes in ceramide levels upon catechins-induced apoptosis in LoVo cells.

It has been demonstrated that there is difference in the induction of apoptosis in LoVo cells by (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin (EC). In this study, we explored changes in ceramide levels upon the three catechins-induced apoptosis in LoVo cells. Addition of C2- and C6-ceramide to LoVo cells mimicked EGCG or EGC in leading to apoptotic death. Further measurement of intracellular ceramide content showed that the treatment of LoVo cells with EGCG or EGC resulted in a rapidly transient increase in ceramide content, and then back gradually to base line level, whereas the action of EC was just opposite to that of EGCG or EGC. These results suggest there is difference in the generation of intracellular ceramide by the three catechins and ceramide may take part in the regulation of EGCG- or EGC-induced apoptosis in LoVo cells.

Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A.

AIM: To establish an ELISA kit using monoclonal antibodies against Clostridium difficile (C. difficile) toxin A. METHODS: An indirect sandwich ELISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 flat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader. RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif. infantis, 5 strains of V. cholera, 2 strains of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL. CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.

Simplified purification method for Clostridium difficile toxin A.

AIM: To establish the purification method for Clostridium difficile (C. difficile) toxin A. METHODS: C. difficile VPI 10463 filtrate was cultured anaerobically by the dialysis bag methods. And then the toxin A was purified by precipitation with 500 g/L (NH4)2SO4 and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl. RESULTS: Purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis (native-PAGE) and Ouchterlony double immunodiffusion. The molecular weight of toxin A was estimated to be 550,000. The purified toxin A had a protein concentration of 0.881 mg/mL. The minimum lethal dose was 1X10(6) MLD/mL (i.p.mice). The cytotoxic titer was 10(7) CU/mg. The haemagglutinate activity was at a concentration of 1.72 microg/mL. The ratio of fluid volume (mL) accumulated to the length (cm) of the loop was 2.46. CONCLUSION: The modified method for purification of toxin A of C. difficile was simple and convenient. It may be even more suitable for purification of toxin A on large scales.

Pathogenicty and immune prophylaxis of cag pathogenicity island gene knockout homogenic mutants.

AIM: To clarify the role of cag pathogenicity island (cagPAI) of Helicobacter pylori (H pylori ) in the pathogenicity and immune prophylaxis of H pylori infection. METHODS: Three pairs of H pylori including 3 strains of cagPAI positive wildtype bacteria and their cagPAI knockout homogenic mutants were utilized. H pylori binding to the gastric epithelial cells was analyzed by flow cytometry assays. Apoptosis of gastric epithelial cells induced by H pylori was determined by ELISA assay. Prophylaxis effect of the wildtype and mutant strains was compared by immunization with the sonicate of the bacteria into mice model. RESULTS: No difference was found in the apoptasis between cagPAI positive and knockout H pylori strains in respective of the ability in the binding to gastric epithelial cells as well as the induction of apoptosis. Both types of the bacteria were able to protect the mice from the infection of H pylori after immunization, with no difference between them regarding to the protection rate as well as the stimulation of the proliferation of splenocytes of the mice. CONCLUSION: The role of cagPAI in the pathogenicity and prophylaxis of H pylori infection remains to be cleared.

Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity.

AIM: To construct a non-resistant and attenuated Salmonella typhimurium (S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori (H pylori) and evaluate its immunogenicity. METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supernatant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments. RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed. Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supernatant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, the entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0 x 10(10) cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response. CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of H pylori infection.

Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene.

AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori (H pylori) and to study the immunogenicity of BabA. METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore, BabA immunogenicity was studied by animal test. RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank. The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA. CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of H pylori infection.

Early diagnosis for colorectal cancer in China.

AIM: To review the present studies on early diagnosis of colorectal cancer. METHODS: The detective rate for early cancer is 1.7%-26.1% based on various statistical data, with much higher detective rate in endoscopy. Since early cancer means invasion involved in the mucosa or submucosa, the diagnosis can only be made when the invasive depth is identified. Pathological tissue materials from both surgical operation or endoscopic resection are suitable for early cancer evaluation. RESULTS: Incidence of polyp malignancy is 1.4%-20.4%. The various constitutive proportion of polyps may explain the different rates. Malignant incidence is higher in adenomatous polyps, that for villous polyps can reach 21.3%-58.3%. Type II early stage of colorectal carcinoma is rarely reported in China. It is shown that majority of them were not malignant, most of type IIa being adenoma or hyperplasia, and IIb being inflammatory and IIc might be the isolated ulcers. The occurrence of malignancy of type II is far lower than that of polypoid lesion. In China, the qualitative diagnosis and classification of neoplasm generally adopted the WHO standard, including surgical excision or biopsies. There is impersonal evaluation between colorectal pre-malignancy and cancer. The former emphasizes the dysplasia of nuclei and gland, while the latter is marked with cancer invasion. Diagnosis of early stage colorectal cancer in endoscopy is made with too much caution which made the detective rate much lower. Mass screening for asymptomatic subjects and follow-up for high risk population are mainly used to find the early stage colorectal cancer in China. Fecal occult blood test is also widely made as primary screening test, galactose oxygenase test of rectal mucus (T antigen), fecal occult albumin test are also used. The detective rate of colorectal cancer is 24-36.5 per 105 mass population. CONCLUSION: Although carcinoma associated antigen in blood or stool, microsatellite DNA instability for high risk familial history, molecular biology technology for stool oncogene or antioncogene, telomerase activity and exfoliative cytological examination for tumor marker, are utilized, none of them is used in mass screening by now.

Recombinant Helicobacter pylori catalase.

AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori (H. pylori) and assay the activity of H. pylori catalase. METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E.coli strain which expressed catalase recombinant protein. The activity of H. pylori catalase was assayed by the Beers and Sizers. RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 degrees and the activity of H. pylori catalase was high in the BL21 (DE3) E.coli strain. CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

Expression of Helicobacter pylori Hsp60 protein and its immunogenicity.

AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity. METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryote expression vector pET-22b (+), which was transformed into BL21 (DE3) E.coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments. RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2% of the total bacterial protein, and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself. CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

A new three-layer-funnel-shaped esophagogastric anastomosis for surgical treatment of esophageal carcinoma.

AIM: To reduce the incidence of postoperative anastomotic leak, stenosis, gastroesophageal reflux (GER) for patients with esophageal carcinoma, and to evaluate the conventional method of esophagectomy and esophagogastroplasty modified by a new three-layer-funnel-shaped (TLF) esophagogastric anastomotic suturing technique. METHODS: From January 1997 to October 1999, patients with clinical stage I and II (IIa and IIb) esophageal carcinoma, which met the enrollment criteria, were surgically treated by the new method (Group A) and by conventional operation (Group B). All the patients were followed at least for 6 months. Postoperative outcomes and complications were recorded and compared with the conventional method in the same hospitals and with that reported previously by McLarty et al in 1997 (Group C). RESULTS: 58 cases with stage I and II (IIa and IIb) esophageal carcinoma, including 38 males and 20 females aged from 34 to 78 (mean age: 57), were surgically treated by the TLF anastomosis and 64 by conventional method in our hospitals from January 1997 to October 1999. The quality of swallowing was improved significantly (Wilcoxon W=2 142, P=0.0 001) 2 to 3 months after the new operation in Group A. Only one patient had a blind anastomatic fistula diagnosed by barium swallow test 2 months but healed up 3 weeks later. Postoperative complications occurred in 25 (43 %) patients, anastomotic stenosis in 8 (14 %), and GER in 13 (22 %). The incidences of postoperative anastomotic leak, stenosis and GER were significantly decreased by the TLF anastomosis method compared with that of conventional methods (chi(2)=6.566, P=0.038; chi(2)=10.214, P=0.006; chi(2)=21.265, P=0.000). CONCLUSION: The new three-layer-funnel-shaped esophagogastric anastomosis (TLFEGA) has more advantages to reduce postoperative complications of anastomotic leak, stricture and GER.

A novel method for preparation of tissue microarray.

AIM: To improve the technique of tissue microarray (tissue chip). METHODS: A new tissue microarraying method was invented with a common microscope installed with a special holing needle, a sampling needle, and a special box fixing paraffin blocks on the microscope slide carrier. With the movement of microscope tube and objective stage on vertical and cross dimensions respectively, the holing procedure on the recipient paraffin blocks and sampling procedure of core tissue biopsies taken from the donor blocks were performed with the refitted microscope on the same platform. The precise observation and localization of representative regions in the donor blocks were also performed with the microscope equipped with a stereoscope. RESULTS: Highly-qualified tissue chips of colorectal tumors were produced by a new method, which simplified the conventional microarraying procedure, and was more convenient and accurate than that employing the existing tissue microarraying instruments. CONCLUSION: Using the refitted common microscope to produce tissue microarray is a simple, reliable, cost-effective and well-applicable technique.

[Cloning and immunogenicity of conservative region of adhesin gene of Helicobacter pylori].

OBJECTIVE: To construct a candidate strain of Helicobacter pylori (Hp) that expresses the proteins of the conservative region of 4 adhesins (BabA2, AlpA, AlpB, and HopZ) and study its immunogenicity. METHODS: The DNA of Hp was extracted. Primers were designed according to the C-terminal structural gene sequence (called CB) of AlpA. The CB gene was amplified by PCR and inserted into the prokaryotic expression vector pET-22b (+) and expressed in BL21 (DE3) strain of Escherichia coli. The product of expression, CB, was purified by affinity chromatography and identified by Western blot analysis. ELISA assay was used to measure the CB-specific antibody in the specimens of serum of 55 Hp infected patients. Rapid urease test (RUT) was used on biopsy specimens collected by gastroscopy as parallel control. RESULTS: A recombinant plasmid pET-22b (+)/CB was constructed with the conservative region of the 4 adhesins. DNA sequencing showed one open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The recombinant CB (rCB) protein, with a molecular weight of 22.5KD, amounted to 29% of the total bacterial protein. The purity of purified rCB was 96%. Western blot analysis showed that the rCB protein could be specifically recognized by the serum from Hp infected patients. The kappa coefficient was 0.76 for evaluation by ELISA and RUT results. CONCLUSION: CB has the potential to be used as a vaccine against Hp infection.

[Pit pattern and endoscopic mucosal resection in diagnosis and treatment of colorectal tumors].

OBJECTIVE: To explore the effective methods to diagnose and treat colorectal cancer in its early stage. METHODS: 1 205 patients were examined by colonoscopy and mucosa staining with indigocarmine. The pit patterns were observed with magnifying endoscope and stereomicroscope according to the Kudo classification. The pathological diagnoses of the lesions were compared with their pit patterns. Electrocoagulation resection was performed on the prominent lesions and endoscopic mucosal resection (EMR) or endoscopic partial mucosal resection (EPMR) was performed on the flat lesions. RESULTS: In the 282 patients 478 prominent and flat lesions were found. There were 16 cases of laterally spreading tumor (LST), including I case of pit pattern II, 6 of pit pattern IIIL, 8 of pit pattern IV, and 1 of pit pattern Va, with 2 cases of intramembrane cancer and 1 case of IIa + IIc lesions invading the superficial myometrium among them. EMR and EPMR were performed on 14 LST lesions, 22 IIa lesions, and 14 IIb lesions. CONCLUSION: The examination of pit pattern is very important in early diagnosis of colorectal cancer and differentiation tumorous from non-tumorous lesions. V type pit pattern is an indicator of colorectal cancer. EMR and EPMR are safe and effective for flat lesions.

[Comparison of curative effect for colon flat lesion between mucosa resection and fulguration with high frequency current after mucosa staining under magnifying endoscope].

OBJECTIVE: To observe the relationship between the pit patterns and pathology and compare the curative effect for colon flat lesion between endoscopic mucosa resection (EMR) and fulguration with high frequency current (FHFC). METHODS: They were divided to two groups. There were 37 cases for FHFC in group A, and 34 cases for EMR in Group B. The two groups were comparabal. Examining patients suffering with colon flat lesions with magnifying endoscope and observing the pit patterns of mucosa after staining with indicarmine. RESULTS: The pit patterns of inflammatory or hyperplastic lesions were mainly pit II, adenoma pit III and pit IV, and carcinomatous lesions pit IV and pit V. The worse the differentiation degree of lesions was, the higher the pit patterns were. There were no difference (P > 0.05) between group A and B in complication and quantity, classification and distribution of lesions. No canceration was detected in 21 cases with adenoma in group A, while 4 cases of canceration (all were adenocacinoma) was found in 20 cases whith adenoma in group B. There was significance in canceration between two groups (P < 0.05). CONCLUSIONS: The worse the differentiation degree of lesions was, the higher pit patterns were. EMR and FHFC share the same validity and security when treating the flat lesions, but by EMR, doctors could judge whether the lesions were resected completely, once the remained lesions were found, they could be resected immediately again lesion in case of omission of canceration.

[Up-regulation of c-myc expression in MCF-7/Adr human breast cancer cells and its association with resistance against doxorubicin].

OBJECTIVE: To investigate the expression of c-myc in drug-resistant MCF-7/Adr human breast cancer cells and the counteractive effect of c-myc antisense oligonucleotide on their drug-resistance. METHODS: Flow cytometry was performed to examine c-myc expression in multi-drug resistant MCF-7/Adr cell line and its parental cell line MCF-7. The IC50 value of doxorubicin was evaluated by MTT assay. RESULTS: MCF-7/Adr cell line was shown to have a significantly higher expression rate of c-myc than its parent cell line MCF-7 (70.48% vs 46.02%). The IC50 value of doxorubicin was (22.00+/-1.92) micomol/L in MCF-7/Adr cells, which was significantly decreased to (9.60+/-1.04) micromol/L (P<0.05) after coincubation with 4 micromol/L c-myc antisense oligonecleotide. CONCLUSION: c-myc expression is up-regulated in MCF-7/Adr cells as compared with their parent cell line MCF-7. Inhibition of c-myc expression may partially reverse the resistance of MCF-7/Adr against doxorubicin, suggesting that c-myc may be involved in the mechanism of drug-resistance of tumor cells.

[Construction of the non-resistant attenuated Salmonella typhimurium strain expressing Helicobacter pylori catalase].

OBJECTIVE: To construct a non-resistant attenuated Salmonella typhimurium (S.typhimurium) strain capable of expressing Helicobacter pylori (Hp) catalase. METHODS: After PCR amplification, the gene fragment encoding Hp catalase was inserted into the expression vector pYA248 containing asd gene, and the recombinant vector was then introduced into the host S.typhimurium strain X4072 depleted of genes encoding adenylate cyclase (delta cya), cyclic adenosine monophosphate receptor protein (delta crp) and aspartate-beta-semialdehyde dehydrogenase (delta asd). Bridged enzyme-linked immunosorbent assay (ELISA) was employed to measure the antigenicity of the catalase expressed in the sonicate and culture supernatant. According to Meacock's method and with the assistance of the growth curve, the stability of the recombinant strain was evaluated. A half lethal oral dose test was conducted to evaluate the safety of recombinant strain. RESULTS: S.typhimurium X4072 (pYA248-CAT) with expected capacity was successfully constructed, and bridged ELISA demonstrated higher catalase levels in the culture supernatant than in the sonicate of the recombinant strain X4072 (pYA248-CAT). After the strain was passaged for 100 generations without selection pressure, all the randomly selected colony of the recombinant strain grew well with positive catalase antigenicity as identified by ELISA. The growth curve of the recombinant strain showed comparable growth status of the 2 strains X4072 (pYA248) and X4072 (pYA248-CAT). The survival rate of C57BL/6 mice was 100% 30 d after oral administration of 1.0x10(10) cfu X4072 (pYA248-CAT). CONCLUSION: Non-resistant S. typhimurium vaccine X4072 (pYA248- CAT) is constructed successfully, which is stable in vitro and safe as confirmed by animal experiment. This vaccine provides a new candidate for viable oral vaccine against Hp infection.