Biomedical subjects

Ya-Li Zhang

Publications and source records attributed to Ya-Li Zhang.

At least 19 recordsLinked to original sources

PubMed2006

The effects of different sample labelling methods on signal intensities of a 60-mer diagnostic microarray.

The effects of four different labelling methods on signal intensities of a 60-mer diagnostic microarray were studied. Eighty of virus-specific oligonucleotide probes for human influenza virus were prepared in an array of 15x16 spots. RNA samples from cultured human influenza virus strains were labelled with four different methods, including direct cDNA labelling (DL), universal primer labelling (UPL), direct cDNA labelling with restriction display (DL-RD), and Cy-dUTP incorporated cDNA labelling with restriction display (IL-RD) in a signal color format. The background-subtracted signal intensities from five replicate hybridization experiments of each labelling method were analyzed using one-way analysis of variance (one-way ANOVA) and linear regression techniques. The effect of sample labelling method on background-subtracted signal intensities was significant (p<0.001) and multiple comparisons showed the differences existed mainly between DL and the other three labelling methods. The sample labelling method explained about 4.3% of signal intensity. The results demonstrated that UPL and the RD-based methods are more efficient than the conventional DL method for sample labelling, an important variation factor affecting the signal intensities in diagnostic microarrays.

Analysis of Variance↗

PubMed2005

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.

Adhesins, Bacterial↗

PubMed2005

Preventive and therapeutic effects of NF-kappaB inhibitor curcumin in rats colitis induced by trinitrobenzene sulfonic acid.

AIM: To ascertain the molecule mechanism of nuclear factor-kappaB (NF-kappaB) 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-kappaB activity in colonic mucosa was evaluated by using electrophoretic mobility shift assay. Cytoplasmic IkappaB 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-kappaB activation in colonic mucosa was suppressed in the curcumin-treated groups. Degradations of cytoplasmic IkappaB 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-kappaB inhibitor curcumin could prevent and improve experimental colitis in murine model with inflammatory bowel disease (IBD). The findings suggest that NF-kappaB inhibitor curcumin could be a potential target for the patients with IBD.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Adhesins, Bacterial↗

PubMed2004

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.

Adhesins, Bacterial↗

PubMed2004

Relationship between plasma D-dimer levels and clinicopathologic parameters in resectable colorectal cancer patients.

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 14(th) 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.

Adult↗

PubMed2004

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.

Colorectal Neoplasms↗

PubMed2004

Effect of environmental exposure to cadmium on pregnancy outcome and fetal growth: a study on healthy pregnant women in China.

The objective of the present study was to evaluate the potential effect of environmental exposure to cadmium on pregnancy outcome and fetal growth. Normal pregnant women were selected from Da-ye city of Hubei province, a cadmium-polluted area, from November 2002 through January 2003. Whole blood of pregnant women, cord blood, and placenta were collected and cadmium levels were determined by inductively coupled plasma emission mass spectroscopy. Incidence rate of preterm labor (gestational age < or = 37 weeks) and neonatal asphyxia, neonatal birth height, and birth weight were compared between lower and higher cadmium exposure level groups. Whole blood cadmium of 44 mothers ranged from 0.80 to 25.20 microg/L. Cadmium concentration in maternal blood was significantly higher than that in cord blood (t = 11.44, P < 0.01). Placenta cadmium ranged from 0.084 to 3.97 microg/g dry weight. After adjustment for maternal age, history of gestation, abortion and lactation, Logistic regression analysis showed that there was no significant association between cadmium exposure levels and pregnancy outcome (premature labor or neonatal asphyxia). Multiple linear regression analysis showed that, cord blood cadmium level, but not maternal blood cadmium and placenta cadmium, was significantly negatively associated with neonatal birth height (t= -2.33, P < 0.05). Compared with lower cord blood cadmium level (< or = 0.40 microg/L), higher level of cord blood cadmium (>0.40 microg/L) was associated with 2.24cm decrease in neonatal birth height. There was no significant association between cadmium exposure and birth weight. It was concluded that environmental exposure to cadmium significantly lower neonatal birth height.

Adult↗

PubMed2004

Acute diarrhea during army field exercise in southern China.

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.

Acute Disease↗

PubMed2004

[Association of TGFalpha and cyclin E expression in gastric carcinoma and precancerous lesions].

BACKGROUND & OBJECTIVE: Previous studies have indicated that increased expression of TGFalpha and cyclin E are correlated with the oncogenesis and progress of cancer; however, their expression patterns in gastric precancerous lesions have been not clear yet. In addition, the association between expression of TGFalpha and cyclin E has not been reported. This study was designed to investigate the expression of TGFalpha and cyclin E in chronic superficial gastritis (CSG), gastric precancerous lesions, and gastric carcinoma (GCA), and analyze the association of TGFalpha and cyclin E expression, and the relationship between their expression and different development stages of oncogenesis of GCA. METHODS: The expression of TGFalpha and cyclin E in CSG, intestinal metaphases (IM), atypical hyperplasia (AH), and GCA were examined using immunohistochemical staining. The association of TGFalpha and cyclin E expression was also statistically analyzed. RESULTS: The expression rates of TGFalpha and cyclin E were 15.1%, 53.6%, 51.7%, 61.7%, and 7.5%, 28.6%, 37.9%, 42.6% in tissue samples of CSG, IM, AH and GCA, respectively. The positive expression rates of TGFalpha and cyclin E were higher in IM, AH, GCA than in CSG; the difference was significant (each P< 0.05). In addition, the positive expression rates of TGFalpha and cyclin E were higher in low differential adenocarcinoma (TGFalpha 81.0%, cyclin E 57.1%) than in mediate to high differential one (TGFalpha 41.7%, cyclin E 16.7%) (P< 0.05,for both TGFalpha and cyclin E). It is also revealed that the expression of TGFalpha and cyclin E were closely associated (P< 0.001 for the tissue of gastric chronic inflammation, and P=0.005 for gastric carcinoma, respectively). CONCLUSION: In the tissues of CSG, IM, AH, and GCA, the expression rates of TGFalpha and cyclin E are increased gradually with the severity of the pathological lesions and the malignancy of GCA. In addition, the expression of TGFalpha and cyclin E were obviously associated.

Adolescent↗

PubMed2003

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.

Adult↗

PubMed2003

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.

Animals↗

PubMed2003

Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase.

AIM: To study the detail mechanism of interaction between PKC and GRK(2) and the effect of GRK(2) on activity of PKC. METHODS: The cDNA of pleckstrin homology (PH) domain located in GRK(2) residue 548 to 660 was amplified by PCR with the mRNA of human GRK(2) (beta1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK(2) PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK(2) PH domain fusion protein, BTK (Bruton's tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK(2) 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 GRK(2) PH domain, GST-GRK(2) 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 GRK(2) to PKC was also detected by western blot and Co-immunoprecipitation. RESULTS: The binding of GRK(2) 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, GRK(2) PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK(2) PH domain. CONCLUSION: GRK(2) can bind with PKC and down-regulated PKC activity.

Amino Acid Sequence↗

PubMed2003

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.

Base Sequence↗

PubMed2003

Contribution of eIF-4E inhibition to the expression and activity of heparanase in human colon adenocarcinoma cell line: LS-174T.

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.

Adenocarcinoma↗