Helicobacter pylori; gastric mucosa/pathology; biopsy; gastroscopy; gastritis, atrophic/pathology; metaplasia
Atrophic gastritis, is the main consequence of long-standing Helicobacter pylori infection, and is linked to the development of gastric cancer. The severity of atrophic gastritis is related to the lifetime risk of gastric cancer development, especially in terms of its degree and extent of mucosal damage. Therefore, it is important for clinicians to assess the severity of atrophic gastritis, interfere with the disease progress, and reverse gastric mucosal atrophy. In the article, we demonstrated some methods (conventional endoscopy, modern endoscopic technology and noninvasive methods) that may help assess the severity of atrophic gastritis and select the reasonable treatment protocols.
Atrophic gastritis; Endoscopy; Pepsinogen
There are limitations to the use of single biomarker levels, for example phosphate and tensin homology (PTEN) or vascular endothelial growth factor (VEGF), in the diagnosis of esophageal squamous cell carcinoma (ESCC). The present study therefore aimed to evaluate the clinical implications of combined detection of multiple biomarkers. The associations between PTEN and VEGF expression status, microvessel density (MVD), and the pathological characteristics of 50 patients with ESCC were determined using χ2, analysis of variance, and t-tests. The results indicated that the PTEN-positive rate was negatively correlated with ESCC histological grade (P<0.01), depth of ESCC invasion (P<0.01) and lymph node metastasis status. Furthermore, the VEGF-positive rate was correlated with lymph node metastasis status, while MVD was correlated with the depth of ESCC invasion (P<0.01) and lymph node metastasis status (P<0.05). The PTEN-positive rate was negatively correlated with the VEGF-positive rate. A higher MVD was identified in ESCC samples than that of the normal esophageal mucosa, particularly in VEGF-positive ESCC specimens compared with those of VEGF-negative specimens, and PTEN-negative ESCC specimens compared with that of the PTEN-positive ESCC specimens. These results suggested that combined detection of PTEN and VEGF levels, as well as evaluation of MVD in patients with ESCC may provide essential information for improvements in the diagnosis and prognosis of ESCC.
esophageal squamous cell carcinoma; phosphate and tensin homolgy; vascular endothelial growth factor; microvessel density; cluster of differentiation 31
AIM: To ascertain the molecule mechanism of nuclear factor-κB (NF-κB) inhibitor curcumin preventive and therapeutic effects in rats’ colitis induced by trinitrobenzene sulfonic acid (TNBS).
METHODS: Sixty rats with TNBS-induced colitis were treated with 2.0% curcumin in the diet. Thirty positive control rats were treated with 0.5% sulfasalazine (SASP). Thirty negative control rats and thirty model rats were treated with general diet. Changes of body weight together with histological scores were evaluated. Survival rates were also evaluated. Cell nuclear NF-κB activity in colonic mucosa was evaluated by using electrophoretic mobility shift assay. Cytoplasmic IκB protein in colonic mucosa was detected by using Western Blot analysis. Cytokine messenger expression in colonic tissue was assessed by using semiquantitative reverse-transcription polymerase chain reaction.
RESULTS: Treatment with curcumin could prevent and treat both wasting and histopathologic signs of rats with TNBS-induced intestinal inflammation. In accordance with these findings, NF-κB activation in colonic mucosa was suppressed in the curcumin-treated groups. Degradations of cytoplasmic IκB protein in colonic mucosa were blocked by curcumin treatment. Proinflammatory cytokine messenger RNA expression in colonic mucosa was also suppressed.
CONCLUSION: This study shows that NF-κB inhibitor curcumin could prevent and improve experimental colitis in murine model with inflammatory bowel disease (IBD). The findings suggest that NF-κB inhibitor curcumin could be a potential target for the patients with IBD.
IBD; Curcumin; TNBS; NF-κB
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 prokaryotie 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.
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 = 2142, P = 0.0001) 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 (χ2 = 6.566, P = 0.038; χ2 = 10.214, P = 0.006; χ2 = 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.
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 shownd 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.
AIM: To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells.
METHODS: The mRNA expression of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor-gamma 2 (PPAR-γ2), adiponectin, leptin, resistin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and the long isoform of leptin receptor (OB-Rb) and protein levels of adiponectin, leptin, and resistin were determined using fluorescent real-time PCR and enzyme linked immunosorbent assay, respectively, on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes exposed to GW4064. Moreover, mRNA expression of AdipoR2 and OB-Rb was also examined using fluorescent real-time PCR at 0, 12, 24, and 48 h in HepG2 cells treated with GW4064.
RESULTS: The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, resistin, AdipoR1, AdipoR2, and OB-Rb and protein levels of adiponectin, leptin, and resistin increased along with differentiation of 3T3-L1 preadipocytes (P < 0.05 for all). The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, and AdipoR2 in 3T3-L1 preadipocytes, and AdipoR2 and OB-Rb in HepG2 cells was significantly increased after treatment with GW4064, when compared with the control group (P < 0.05 for all). A similar trend was observed for protein levels of adipokines (including adiponectin, leptin and resistin). However, the expression of resistin, AdipoR1, and OB-Rb in 3T3-L1 cells did not change after treatment with GW4064.
CONCLUSION: The FXR agonist through regulating, at least partially, the expression of adipokines and their receptors could offer an innovative way for counteracting the progress of metabolic diseases such as nonalcoholic fatty liver disease.
Farnesoid X receptor; Adipokines; Adipokine receptors; 3T3-L1 cells; HepG2 cells; Nonalcoholic fatty liver disease
AIM: To evaluate the safety, efficacy and management of double balloon enteroscopy (DBE) carried out in those aged individuals with suspicious small intestine diseases.
METHODS: DBE is a wonderful invention of the past decade and is widely used as an examination tool for the gastrointestinal tract. From January 2003 to July 2011, data from patients who were ≥ 65 years old and underwent DBE examination in the Nanfang Hospital were included in a retrospective analysis.
RESULTS: Fifty-nine individuals were found and subsequently analyzed. The mean age was 69.63 ± 3.89 years (range 65-84), 34 were males. Indications for DBE were melena/hematochezia (36 cases), abdominal pain (15 cases), diarrhea (3 cases), stool change (1 case), weight loss (1 case), vomiting (2 cases), and debilitation (1 case). The average duration of symptoms was 33.34 ± 64.24 mo. Twenty-seven patients suffered from age-related diseases. Severe complications were not found during and after DBE. Comparison between systolic and diastolic blood pressure before and after DBE was statistically significant (mean ± SD, P < 0.01, P < 0.05, respectively). Small bowel pathologies were found by DBE in 35 patients, definite diagnoses were made in 31 cases, and detection rate and diagnostic yield for DBE were 68.6% and 60.8%, respectively.
CONCLUSION: DBE is a safe and effective method for gastrointestinal examination in the aged population. Aging alone is not a risk factor for elderly patients with suspicious gastrointestinal diseases and thorough preparation prior to the DBE procedure should be made for individuals with multiple diseases especially cardiopulmonary disorders.
Double balloon enteroscopy; Capsule endoscopy; Small bowel diseases; Multiple systematic diseases
AIM: To evaluate the anti-Helicobacter pylori (H. pylori) activity of 50 traditional Chinese herbal medicines in order to provide the primary evidence for their use in clinical practice.
METHODS: A susceptibility test of water extract from 50 selected traditional Chinese herbal medicines for in vitro H. pylori Sydney strain 1 was performed with broth dilution method. Anti-H. pylori activity of the selected Chinese herbal medicines was evaluated according to their minimum inhibitory concentration (MIC).
RESULTS: The water extract from Rhizoma Coptidis, Radix Scutellariae and Radix isatidis could significantly inhibit the H. pylori activity with their MIC less than 7.8 mg/mL, suggesting that traditional Chinese herbal medicines have anti-inflammatory and antibacterial effects and can thus be used in treatment of H. pylori infection.
CONCLUSION: Rhizoma Coptidis, Radix Scutellariae and Radix isatidis are the potential sources for the synthesis of new drugs against H. pylori.
Chinese herbal medicines; Helicobacter pylori; Minimum inhibitory concentration; Gastric; Oral
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 μg/mL ampicillin overnight at 37 °C. 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 β-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.
H pylori; Immunogenicity
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.
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.
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 pH5.5, 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 550000. The purified toxin A had a protein concentration of 0.881 mg/mL. The minimum lethal dose was 1 × 106 MLD/mL (i.p.mice). The cytotoxic titer was 107 CU/mg. The haemagglutinate activity was at a concentration of 1.72 μg/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.
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 × 1010 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.
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.
AIM: To assess the clinical significance of the D-dimer levels and the relationship between plasma D-dimer levels and clinicopathologic parameters in operable colorectal cancer patients.
METHODS: The plasma levels of D-dimer were measured pre- and postoperatively in 35 patients with colorectal cancer, and 30 healthy subjects served as controls by the method of quantitative enzyme-linked immunosorbent assay (ELISA).
RESULTS: The mean preoperative plasma levels of D-dimer in the patients with colorectal cancer (1.06 ± 0.24 mg/L) were significantly higher than those of controls (0.33 ± 0.12 mg/L, P < 0.01). The D-dimer levels were remarkably elevated on the 1st day after operation (1.22 ± 0.55 mg/L, P < 0.01). On the 3rd day the level of D-dimer began to stepwise descend and on the 14th day nearly returned to control level. The preoperative levels of D-dimer were significantly correlated with the lymph node metastasis and Dukes stage but had no association with tumor location and the degree of differentiation. A stepwise increase in the mean D-dimer levels was found with increase of the tumor stage.
CONCLUSION: Hypercoagulation and higher fibrinolytic activities occur in patients with colorectal cancer. The operative trauma could enhance the fibrinolysis in the patients with colorectal cancer. The measurement of preoperative D-dimer levels is considered to be useful for predicting lymph node metastasis and stage of colorectal cancer.
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.
AIM: During emergency period, infectious diseases can be a major threat to military forces. During field training in southern China, diarrhea is the main cause of nonbattle injury. To evaluate the causes of and risk factors for diarrhea in emergency period, we collected clinical and epidemiological data from the People’s Liberation Army (PLA) during field training in southern China.
METHODS: From September 25 to October 2 1997, 2636 military personnel were investigated. Fecal sample cultures for lapactic pathogens were obtained from 103 military personnel with diarrhea. In addition, a questionnaire was administered to 103 cases and 206 controls to evaluate the association between illness and potential risk factors. At the same time, another questionnaire of 1:4 case-case control was administered to 22 severe cases (each severe case paired 4 mild cases).
RESULTS: The training troop’s diarrhea incidence rate was significantly higher than that of garrison. The diarrhea incidence rate of officers was significantly lower than that of soldiers. A lapactic pathogen was identified in 63.1% (65/103) of the troops with diarrhea. Enterotoxigenic Escherichia coli (35.0%) and plesiomona shigelloides (16.5%) were the most common bacterial pathogens. All bacterial isolates were sensitive to norfloxacin and ceftazidine. However, almost all of them were resistant to sulfamethoxazole, trimethoprim-sulfamethoxazole, oxytetracycline, doxycycline, furazolidone, ampicillin and cloromycetin to a different degree. Risk factors associated with diarrhea included drinking raw water, eating outside, contacting diarrhea patients, lacking sanitation, depression, lacking sleep, which were established by multiple-factor logistic regression analysis. In addition, the unit incidence rate was associated with the density of flies and the average daily boiled water available by regression and discriminate analysis.
CONCLUSION: A series of risk factors are associated with the incidence rate of diarrhea. Our results may provide a useful basis for prevention and cure of diarrhea in emergency period of PLA.
AIM: Heparanase degrades heparan sulfate proteoglycans (HSPGs) and is a critical mediator of tumor metastasis and angiogenesis. Recently, it has been cloned as a single gene family and found to be a potential target for antimetastasis drugs. However, the molecular basis for the regulation of heparanase expression is still not quite clear. The aim of this study was to determine whether the expression of eukaryotic initiation factor 4E (eIF-4E) correlated with the heparanase level in tumor cells and to explore the correlation between heparanase expression and metastatic potential of LS-174T cells.
METHODS: A 20-mer antisense s-oligodeoxynucleotide (asODN) targeted against the translation start site of eIF-4E mRNA was introduced into LS-174T cells by lipid-mediated DNA-transfection. eIF-4E protein and mRNA levels were detected by Western blot analysis and RT-PCR, respectively. Heparanase activity was defined as the ability to degrade high molecular weight (40-100 kDa) radiolabeled HS (heparan sulfate) substrate into low molecular weight (5-15 kDa) HS fragments that could be differentiated by gel filtration chromatography. The invasive potential of tumor cell in vitro was observed by using a Matrigel invasion assay system.
RESULTS: The 20-mer asODN against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels. As a result, the expression and activity of heparanase were effectively retarded and the decreased activity of heparanase resulted in the decreased invasive potential of LS-174T.
CONCLUSION: eIF-4E is involved in the regulation of heparanase production in colon adenocarcinoma cell line LS-174T, and its critical function makes it a particularly interesting target for heparanase regulation. This targeting strategy in antisense chemistry may have practical applications in experimental or clinical anti-metastatic gene therapy of human colorectal carcinoma.
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&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 °C 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.
AIM: To study the detail mechanism of interaction between PKC and GRK2 and the effect of GRK2 on activity of PKC.
METHODS: The cDNA of pleckstrin homology (PH) domain located in GRK2 residue 548 to 660 was amplified by PCR with the mRNA of human GRK2 (β1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK2 PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK2 PH domain fusion protein, BTK (Bruton’s tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK2 in culture mammalian cells (6 cell lines: PC-3, MDCK, SGC7901, Jurkat cell etc.) was determined by SDS-PAGE and Co-immunoprecipitation. The binding of GRK2 PH domain, GST-GRK2 PH domain fusion protein and BTK PH domain to PKC in vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK2 to PKC was also detected by western blot and Co-immunoprecipitation.
RESULTS: The binding of GRK2 PH domain to PKC in vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK2 PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK2 PH domain.
CONCLUSION: GRK2 can bind with PKC and down-regulated PKC activity.
AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer.
METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain κ and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected.
RESULTS: The amplified fragments of Fd and κ gained by RT-PCR were about 650 bp. Fd and κ PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 × 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage anti bodies which had binding activities with antigens of colorectal cancer.
CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.
colorectal neoplasms; immunology; bacterio phages; antibody library
colorectal neoplasms; microsatellite instability; gene expression; familial predisposition; proliferation kinetics; immunohistochemistry; polymerase chain reaction; flow cytometry
AIM: To observe the tumor inhibitory effects by transfecting I L-6 cDNA into colon cancer cell line HT-29 with retroviral vector pZIP cDNA.
METHODS: Human IL-6 gene was reconstructed in retrovirus vector and transfected into incasing cells PA317 by lipofectamine mediated method, the clones of the cells transferred with hIL-6 were selected by G418, and targeted HT-29 cells were infected with the virus granules secreted from PA317 and also selected by G418. Test gene transcription and expression level by hybridization , ELISA and MTT assay, etc. Analyze tumor inhibitory effects according to the cell growth curve, plating forming rate and tumorigenicity in nude mice.
RESULT: Successfully constructed and transfected recombinant exp ressing vectors pZIPIL-6 cDNA and got positive transfected cell lines. The colon cancer cell line (HT-29 IL-6) transfected with the hIL-6 gene by retroviral vector was established. The log proliferation period and the doubling time of t his cell line was between 4 to 7 days and 2.5 days according to the direct cell count, the cell proliferation was obviously inhibited with MTT assay, the plating inhibitory rate was 50% by plating efficiency test. When HT-29 IL-6 cells w ere inoculated into the nude mice subcutaneously, carcinogenic activity of the solid tumor was found superior to the control group and the size of tumor was not significantly enlarged. Injection of combination virus fluid containing IL-6 gene into transplantation tumors could inhibit the growth and development of the tumor.
CONCLUSION: IL-6 could inhibit the growth and proliferation of colon cancer cells by retroviral vector-mediated transduction.
interleukin-6 gene; gene transfection; gene therapy; colonic neoplasms