Aquaporin-9 downregulation prevents steatosis in oleic acid-induced non-alcoholic fatty liver disease cell models.

Aquaporin-9 (AQP9) is an aquaglyceroporin that acts as the adipose glycerol channel. However, the role of AQP9 in steatosis in non-alcoholic fatty liver disease (NAFLD) has not yet been fully elucidated. In the present study, the coding sequence of the AQP9 gene was obtained from LO2 cells by RT-PCR, and cloned into the pEGFP-N1 vector. Short hairpin RNA (shRNA) targeting the AQP9 gene was inserted into the pGenesil-1 vector. Recombinant plasmids were confirmed by enzyme digestion and sequence analysis, and transfected into cell models (derived from LO2 cells) of oleic acid-induced NAFLD. Our results demonstrated that AQP9 recombinant plasmids can be effectively expressed in cell models of NAFLD. Furthermore, in comparison with the control group, AQP9 overexpression significantly increased intracellular lipid content, triglyceride (TG), free fatty acid (FFA) and glycerol levels; however, the silencing of AQP9 exerted the opposite effects. Taken together, recombinant plasmids used to induce AQP9 overexpression and to silence AQP9 expression were successfully constructed. AQP9 overexpression aggravated the degree of steatosis; however, the silencing of AQP9 alleviated these effects. Based on these data, we suggest that AQP9 may serve as a novel molecular target for therapeutic intervention in NAFLD.

[Construction of short hairpin RNA targeting aquaglyceroporin 9 and screening its effect on molecular mechanisms of nonalcoholic fatty liver disease using a cell model system].

OBJECTIVE: To construct a short hairpin (sh)RNA targeting aquaglyceroporin 9 (AQP9) that effectively silences gene expression in liver cells in order to investigate of the role of AQP9 in nonalcoholic fatty liver disease (NAFLD) pathogenesis using an in vitro cell model system. METHODS: Small interfering (si)RNAs were designed against the human gene sequences encoding AQP9 (NCBI GenBank Accession No. AB008775) and unrelated control sequences, synthesized, annealed to form double-strands, and inserted into the pGenesil- 1 shRNA-expression plasmid. The silencing effects of the four pshRNA-AQP9 constructs (a-d) and the pshRNA-negative control construct were investigated by transfecting into the L02 human normal liver cell line and detecting expression of AQP9 mRNA and protein (relative to beta-actin) by reverse transcription-PCR and western blotting. The NAFLD cell model was established by treating L02 cells with oleic acid to induce fatty degeneration. After transfecting the NAFLD cell model with various constructs, the effects on NAFLD-related features were investigated by staining with Oil Red O (to detect lipid droplets) and performing enzymatic assays (to quantitate triglyceride (TG), free fatty acid (FFA) and glycerol content). The significance of intergroup differences was assessed by analysis of variance test. RESULTS: Of the four pshRNA-AQP9 constructs, pshRNA-AQP9a produced the most robust silencing effect on AQP9 mRNA (25.1 - 1.2% vs. untransfected: 39.3 +/- 1.7% and pshRNA-negative control: 39.4 +/- 1.5%, P < 0.01) and protein (25.4 - 2.0% vs. untransfected: 35.1 +/- 1.9% and psh-RNA-negative control: 35.6 +/- 2.3%, P < 0.01). Oleic acid-induced L02 cells showed enhanced AQP9 mRNA and protein expression, and increased intracellular content of lipid, TG, FFA, and glycerol, which were significantly reduced by pshRNA-AQP9a transfection (all P <0.05). CONCLUSION: The new pshRNA-AQP9a construct can efficiently reduce AQP9 expression in cultured human liver cells and relieve steatosis-related features in an NAFLD cell model, pshRNA-AQP9a represents a novel tool for studying the role ofAQP9 in NAFLD pathogenesis and its potential as a gene therapy strategy.

Oral immunization with recombinant Mycobacterium smegmatis expressing the outer membrane protein 26-kilodalton antigen confers prophylactic protection against Helicobacter pylori infection.

Helicobacter pylori infection is prevalent worldwide and results in chronic gastritis, which may lead to gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. We have previously reported that oral immunization with recombinant Mycobacterium smegmatis expressing the H. pylori outer membrane protein 26-kilodalton (Omp26) antigen affords therapeutic protection against H. pylori infection in mice. In the present study, we investigated the prophylactic effects of this vaccine candidate on H. pylori challenge in mice. We found that oral immunization with recombinant Mycobacterium Omp26 significantly reduced H. pylori colonization in the stomach compared to inoculation with wild-type M. smegmatis in control mice. Six of the recombinant Mycobacterium-immunized mice (60%) were completely protected from H. pylori infection. The severity of H. pylori-associated chronic gastritis assessed histologically was significantly milder in mice vaccinated with recombinant Mycobacterium than in control animals. Mice immunized with recombinant Mycobacterium showed enhanced antigen-specific lymphocyte proliferation and antibody responses. Moreover, immunization with recombinant Mycobacterium resulted in an increased expression of interleukin-2 and gamma interferon in the stomach and spleen, as determined by reverse transcription-PCR analysis. Our results collectively suggest that vaccination with recombinant Mycobacterium Omp26 confers prophylactic protection against H. pylori infection. The inhibition of H. pylori colonization is associated with the induction of antigen-specific humoral and cell-mediated immune responses.

Attenuated Salmonella typhimurium carrying TRAIL and VP3 genes inhibits the growth of gastric cancer cells in vitro and in vivo.

BACKGROUND: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and apoptin (VP3) of chicken anemia virus can selectively induce apoptosis in human tumor cell lines by two different pathways. Salmonella not only delivers functional genes to mammalian cells but also possesses antitumor activity and therefore could be adopted as a novel vector for anticancer therapy. MATERIALS AND METHODS: TRAIL and VP3 genes were cloned into a pBudCE4.1 vector and delivered by attenuated Salmonella typhimurium into gastric cancer cells, and their expression and antitumor effects in nude mice were monitored by Western blot, fluorescence microscopy, MTT assay, TUNEL staining, and immunohistochemistry. RESULTS: pBud-VP3 and pBud-TRAIL-VP3 plasmids were constructed to express TRAIL and apoptin in gastric cancer cells, leading to inhibition of cancer cell proliferation after 48 hours (P < 0.05). TRAIL and VP3 genes in pBudCE4.1 vector were also successfully delivered by attenuated S. typhimurium into gastric cancer cells in vivo, in which both TRAIL and apoptin were expressed. In vivo data indicated that S. typhimurium carring pBud-TRAIL-VP3 induced significant cell growth inhibition and tumor regression (P < 0.05). Moreover, expression of TRAIL and apoptin increased the expression of caspase-3 and caspase-9, resulting in enhanced apoptosis. CONCLUSION: Delivery of TRAIL and VP3 genes by attenuated S. typhimurium can significantly inhibit the growth of gastric cancer cells in vitro and in vivo.

[Overexpression of adiponectin prevents hepatocyte steatosis].

OBJECTIVE: To investigate the effect of adiponectin on hepatocyte steatosis. METHODS: L02 cells were transfected with pEGFP-N1-AdipoQ, a plasmid encoding pEGFP-adiponectin fusion protein, or pEGFP-N1. Lipid droplets in the hepatocytes were observed by oil red staining at 72 h. The contents of TG, FFA and glycerol in hepatocytes were measured. RESULTS: Compared to cells transfected with pEGFP-N1-AdipoQ plasmid, much more lipid droplets were observed in cells transfected with pEGFP-N1 plasmid. TG, FFA and glycerol contents in L02 cells and L02/pEGFP-N1 cells were significantly higher than those in L02/pEGFP-N1-AdipoQ cells. CONCLUSIONS: Overexpression of adiponectin prevent hepatocyte steatosis.

[Anti-tumor effect of eukaryotic expressing plasmid containing soluble tumor necrotic factor-related apoptosis inducing ligand combined with human angiostatin Kringle (1 - 3) genes on human gastric cancer xenografts in nude mice].

OBJECTIVE: To investigate the anti-tumor effect of eukaryotic expressing plasmid containing human angiostatin Kringle (1 - 3) [hAG (K1-3)] combined with soluble tumor necrotic factor-related apoptosis inducing ligand (sTRAIL) genes on human gastric cancer xenografts in nude mice. METHODS: Recombinant plasmids of pBud-hAG and pBud-hAG-TRAIL were constructed by subcloning technique. Twenty nude BALB/c mice were inoculated with human gastric cancer cells of the line BGC-823 subcutaneously into the back. One week later after the appearance of implanted tumors the mice were randomly divided into 4 groups with pBud-hAG, pBud-hAG-TRAIL, pBud blank plasmid, and normal saline (NS) injected into the tumors respectively once the other day for 7 times. The size of tumor was observed. 7 days later the mice were killed with their tumors taken out. RT-PCR was used to detect the expression of hAG and sTRAIL. The microvessel density (MVD) of tumor was observed and recorded by detecting the protein of CD34 with immunohistochemistry on the microscopy. RESULTS: The MVD of tumor in the pBud-hAG-TRAIL and pBud-hAG groups were (4.8 +/- 0.9)/HP and (4.6 +/- 1.2)/HP respectively, significantly lower than those of the pBud and NS groups [(17.4 +/- 2.4)/HP and (18.2 +/- 2.7)/HP respectively, all P < 0.05], but there was no significant difference between the pBud-hAG-TRAIL and pBud-hAG groups. mRNA expression and protein expression of sTRAIL and hAG were positive in the pBud-hAG-TRAIL and pBud-hAG groups. The tumor volumes of tumors of the pBud-hAG-TRAIL and pBud-hAG groups were (1.325 +/- 0.012) cm(3) and (1.862 +/- 0.017) cm(3) respectively, both significantly lower than those of the pBud and NS groups [(3.637 +/- 0.032) cm(3) and (3.521 +/- 0.028) cm(3) respectively, all P < 0.05]. CONCLUSION: Angiostatin inhibits tumor angiogenesis through inhibiting the growth of vascular endothelial cells, and TRAIL induces tumor cell apoptosis. hAG (K1-3) combined with TRAIL can inhibit tumor growth more efficiently.

Recombinant Mycobacterium smegmatis mc(2)155 vaccine expressing outer membrane protein 26 kDa antigen affords therapeutic protection against Helicobacter pylori infection.

Orally administered recombinant Mycobacterium smegmatis (rM. smegmatis) vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, in the present study, we evaluated the capacity of the outer membrane protein 26kDa antigen (Omp26) of Helicobacter pylori (H. pylori) to induce therapeutic protection against H. pylori infection in mice. Omp26 was cloned and expressed in M. smegmatis mc(2)155 as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of the up-regulated M. fortuitum beta-lactamase promoter, pBlaF. The rM. smegmatis strain was shown to be relatively stable in vitro in terms of plasmid stability and bacterial persistence. We found that oral immunization of H. pylori-infected mice with rM. smegmatis-Omp26 induced protection, i.e., significant reduction in bacterial colonization in the stomach. The protection was strongly related to serum specific antibodies with a Th(1) and Th(2) profile as well as to local cytokines in the stomach and spleen. These findings suggest that Omp26 is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori. The rM. smegmatis expressing Omp26 antigen could constitute an effective, low-cost combined vaccine against H. pylori.

RNA interference-mediated silencing of the Hsp70 gene inhibits human gastric cancer cell growth and induces apoptosis in vitro and in vivo.

AIMS AND BACKGROUND: The role of heat shock protein (HSP) 70 in gastric cancer has been extensively examined in many studies for the past decade. It has been demonstrated that over-expression of Hsp70 might play important role in malignant transformation and maintenance of malignant phenotypes. Therefore, silencing the Hsp70 gene could be applicable in molecular therapies of human gastric cancer. Herein, we designed a small interfering RNA targeting Hsp70 to knock down its expression and investigated its effect on cell proliferation and apoptosis in a gastric cancer cell line. METHODS: Two plasmids (phsp1-siRNA, phsp2-siRNA), along with a negative control (phsp3-siRNA), were created using a genic recombination technique. BGC823 cell lines were used to perform experiments. Western blotting and RT-PCR were used to detect Hsp70 expression in vitro and in vivo. Cell morphology was observed under light microscope. Cell cycle and apoptosis were analyzed by flow cytometry and acridine orange/ethidium bromide double stain, and cell proliferative activity was measured by alamarblue assay. In all experiments, a negative control served as a baseline measure. RESULTS: We successfully constructed phsps-siRNA plasmids and transfected them into BGC823 cells. RT-PCR and western blotting revealed that the expression of Hsp70 was down-regulated in transfection groups compared with the control group. Flow cytometric analysis indicated that less S-phase fraction accumulated in small interfering RNA transfected cells than in parental cells and the cells transfected with empty vector. CONCLUSIONS: Our results demonstrated that RNAi against Hsp70 could effectively knock down gene expression, inhibit growth of cancer cells, induce cell cycle arrest and increase cell apoptosis in vitro and in vivo. Hsp70 might serve as a therapeutic target for human gastric cancer.

Comparison of esomeprazole enteric-coated capsules vs esomeprazole magnesium in the treatment of active duodenal ulcer: a randomized, double-blind, controlled study.

AIM: To evaluate the efficacy and tolerability of two different preparations of esomeprazole in healing duodenal ulcers. METHODS: A total of 60 patients with active duodenal ulcers were enrolled and randomized to receive esomeprazole enteric-coated capsules (40 mg) or esomeprazole magnesium (40 mg), once daily, for 4 consecutive wk, with ulcer healing being monitored by endoscopy. Safety and tolerability were also assessed. RESULTS: Fifty seven patients completed the whole trial. The ulcer healing rates at the end of wk 2 were 86.7% and 85.2% in the esomeprazole enteric-coated capsules and esomeprazole magnesium groups, respectively (P = 0.8410), and reached 100% at the end of wk 4 in both groups. Symptom relief at the end of wk 2 was 90.8% in the esomeprazole enteric-coated capsules group and 86.7% in the esomeprazole magnesium group (P = 0.5406); at the end of wk 4 symptom relief was 95.2% and 93.2%, respectively (P = 0.5786). Adverse events occurred in 16.7% of the esomeprazole enteric-coated capsules group and 14.8% of the esomeprazole magnesium group (P = 1.0000). CONCLUSION: The efficacies of esomeprazole enteric-coated capsules and esomeprazole magnesium in healing duodenal ulcer lesions and relieving gastrointestinal symptoms are equivalent. The tolerability and safety of both drugs were comparable.

[Effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer: experiments in vitro and in vivo].

OBJECTIVE: To investigate the effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer. METHODS: Recombinant eukaryotic expression plasmid pshRNA-DNMT1 containing the sequence of the gene of DMNT1 that methylates the specific pyrimidine residue in the DNA promoter region was constructed. Human gastric cells of the line AGS were cultured and transfected with pshRNA-DNMT1. Western blotting was used to detect the protein expression of DNMT1 of the AGS cells, and RT-PCR was used to detect the mRNA expression of DNMT1 of the AGS cells. MTT method was used to dynamically monitor the surviving cells and the cell apoptotic was observed by electron microscopy and TUNEL method. Forty nude the mice were inoculated with suspension of AGS cells. When the tumor reached the size of 5 - 6 mm in diameter the mice were randomly divided into 5 equal groups to be injected intravenously with PBS, liposome, pTZU6 + 1, pshRNA-DNMT1 of medium dose, and pshRNA-DNBMT1 of large dose for 4 times with an interval of 3 days. The tumor size was measured every day. Three days after the last injection the mice were killed and the tumors were taken out to undergo light and electron microscopy and TUNEL method to detect the cell apoptosis. Immunohistochemistry was used to detect the proliferating cell nuclear antigen (PCNA) of the cells. RESULTS: The protein and mRNA expression levels of DNMT1 in the cultured AGS cells 24, 48, and 72 hours after transfection of the pshRNA-DNMT1 group were all lower than those of the control group. The numbers of surviving AGS cells of the pshRNA-DNMT1 group became significantly gradually lower than those of the liposome and pTZU6 + 1 groups since 24 hours after transfection (all P < 0.05). The apoptotic rate of AGS cells in the pshRNA-DNMT1 group was 34.78% +/- 0.52%, significantly higher than those of the liposome and pTZU6 + 1 groups (4.86% +/- 0.17% and 5.12% +/- 0.76% respectively, both P < 0.05). The subcutaneous tumors of the mice of the PGS, liposome, and pTZU6 + 1 groups augmented along with time without significant differences among these 3 groups (all P > 0.05). The tumor of the 2 pshDNMT1 groups began to augment since the 5(th) day and began to be reduced in size since the 10(th) day in comparison with the other 3 groups (all P < 0.05), and the tumor size of the pshRNA-DNMT1 (large dose) group was significantly smaller than that of the pshRNA-DNMT1 (medium dose) group 15 days after the injection (P < 0.05). The rates of cell apoptosis of the pshRNA-DNMT1 (large dose) and pshRNA-DNMT1 (medium dose) groups were both significantly higher than those of the other 3 groups (all P < 0.05) and with a sufficient difference between these 2 pshRNA-DNMT1 groups (P < 0.05). PCNA analysis showed that the proliferation activity of the cells in the pshRNA-DNMT1 groups was significantly suppressed. CONCLUSION: The recombinant plasmid pshRNA-NMT1 effectively and specifically inhibits the expression of the gene DNMT1, thus inhibiting the proliferation and inducing the apoptosis of gastric cancer cells.

[Effects of dnmt1 gene silencing on cell cycle, proliferation, and apoptosis of gastric cancer cell line AGS].

BACKGROUND & OBJECTIVE: Dnmt1, a major DNA methyltransferase gene, is highly expressed in many cancers and lowly expressed in normal adult cells, therefore, its overexpression is closely related to tumorigenesis. This study was to assess effects of dnmt1 gene silencing on cell cycle, proliferation, and apoptosis of gastric cancer cell line AGS. METHODS: The eukaryotic expression plasmid pshRNA-dnmt1, containing the sequence of short hairpin RNA (shRNA) targeting dnmt1, was constructed and transfected into AGS cells. PBS-treated cells and pTZU6+1-transfected cells were set as control. The expression levels of dnmt1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell cycle was analyzed by flow cytometry; cell survival was analyzed by MTT assay; cell apoptosis was evaluated by AO/EB double staining, electron microscopy, and TUNEL. RESULTS: pshRNA-dnmt1 targeting dnmt1 was successfully constructed, and confirmed by sequencing. Relative to control, 24, 48, and 72 h after transfection of pshRNA-dnmt1, the inhibitory rates of dnmt1 protein levels in AGS cells were 28.24%, 68.54%, and 81.47%, respectively, and those of dnmt1 mRNA levels were 21.63%, 52.97%, and 72.06%, respectively. The growth of AGS cells was suppressed 72 h after transfection: S phase cells were reduced from (36.58+/-1.76)% to (18.54+/-6.59)% (P<0.05), and G(2)/M phase cells were increased from (6.18+/-0.32)% to (18.53+/-1.42)% (P<0.05). Cell survival rates were 79.49%, 51.63%, and 39.16%, respectively, 24, 48, and 72 h after transfection of pshRNA-dnmt1. A lot of apoptotic and necrosis cells were observed after transfection. CONCLUSIONS: The recombinant plasmid pshRNA-dnmt1 can efficiently and specifically inhibit the expression of dnmt1 gene and the proliferation of AGS cells, and induce cell apoptosis. It provides evidence for gene therapy of human cancers.

[Establishment of cellular hypoxia model and its effects on biological behavior of gastric cancer cell line SGC-7901].

BACKGROUND & OBJECTIVE: Hypoxia is an elementary characteristic of tumor microenvironment. Establishing hypoxia microenvironment in vitro will be of help in studying the effects of hypoxia on tumor cells. Previous methods of establishing hypoxic cell culture model are fussy, and researches on the effects of hypoxia on gastric cancer cells are rare. This study was to establish hypoxia microenvironment with GasPaK method in vitro, and observe the effects of hypoxia on biological features of gastric cancer cell line SGC-7901. METHODS: The hypoxia microenvironment was established by GasPaK method; the changes of PO2, PCO2, and pH in RPMI-1640 were monitored by blood gas analysis. The effect of hypoxia on cell cycle of SGC-7901 cells was analyzed by flow cytometry (FCM). The morphology of SGC-7901 cells was observed under light microscope and transmission electron microscope. Live cells were counted with trypan blue staining. The adhesiveness of SGC-7901 cells was detected by MTT assay; migration ability of SGC-7901 cells was assessed by movement experiment. RESULTS: GasPaK formed hypoxia microenvironment in 0.5-48 h with stable PO2, PCO2, and pH. When cultured in hypoxia for 16 h, no significant change in cell cycle of SGC-7901 cells was observed; morphology of some SGC-7901 cells was changed; the number of live cells didn't significantly decreased; the adhesiveness and migration ability of SGC-7901 cells were significantly enhanced. CONCLUSIONS: GasPaK method can stably establish hypoxia microenvironment with good repetition. Hypoxia has slight effect on SGC-7901 cells, and may induce morphologic change of SGC-7901 cells.

Diagnosis of Helicobacter pylori infection and diseases associated with Helicobacter pylori by Helicobacter pylori outer membrane proteins.

AIM: To examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylori (H pylori) infection to two H pylori outer membrane proteins (OMPs) (Mr18,000 and Mr 26,000) acquired by gene recombinant technique, and to determine the diagnostic significance of serological tests derived from these OMPs. METHODS: Recombinant vectors encoding the two H pylori OMPs were used to transform and express in BL21 (DE3) E.coli. After purification with Ni2+-NTA agarose resin, colloid gold kits were prepared with purified recombinant proteins to detect H pylori infection and H pylori-associated diseases by the immunity-marker technology. We selected 150 patients with H pylori infection and digestive symptoms without previous treatment, including chronic gastritis (n = 60), duodenal ulcer (n = 30), gastric ulcer (n = 30), and gastric cancer (n = 30). As controls, 33 H pylori-negative healthy volunteers were also recruited. Serum samples were collected from all subjects, and the antibodies to specific proteins of H pylori were tested with the colloid gold test kits. The sensitivity, specificity and accuracy of the colloid gold tests were evaluated, by using the combination of standard diagnostic methods (13C urea breath test and bacteria culture) and classic enzyme-linked immunosorbent assay (ELISA) as reference. RESULTS: After purification with Ni2+-NTA agarose resin, the purity of recombinant fusion proteins was about 95%. The recombinant fusion proteins were recognized by the specific monoclonal antibodies against the two H pylori OMPs, as demonstrated by the ELISA. Of the 150 serum samples from patients infected with H pylori 141 (94.0%) responded positively to the recombinant protein with Mr 26,000, while the seropositive rates were 95.0%, 96.7%, 96.7% and 90.0% for patients with H pylori-associated chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer respectively. The sensitivity, specificity, and accuracy of the colloid gold kit with Mr 26,000 protein were 94.0%, 97.0%, and 94.5%, respectively. Compared with the classic ELISA, bacteria culture and 13C urea breath test results in detecting H pylori-infection, there was no significant difference (P>0.05). For the colloid gold kit with Mr 18,000, the seropositive rates were 52.0%, 40.0%, 40.0%, 53.3% and 86.7%, respectively, in H pylori-infected patients, and those with H pylori-associated chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer. There was a significant difference (P<0.05) in seropositivity between patient with gastric cancer (86.7%) and those with other diseases (43.3%). CONCLUSION: The two colloid gold kits derived from the recombinant OMPs are useful tools either for detecting H pylori infection, or for, predicting H pylori-associated gastric malignancy.

Cloning and sequence analysis of gene oipA encoding an outer membrane protein of human Helicobacter pylori.

AIM: To construct a recombinant E. coli strain that would highly express the proinflammatory outer membrane protein of human Helicobacter pylori (H pylori). METHODS: The oipA DNA was amplified by PCR, inserted into pET-32a, and transformed into Top10 E. coli strain. This recombinant plasmid of Top10 was sent out for nucleotide sequence analysis. Finally this sequence AF479754 was compared with HP0638 and JHP0581. RESULTS: The sequence of the aim gene was obtained. It had 924 base pairs. The identity was 95.32% against HP0638, 95.02% against JHP0581, which was higher than the identity between HP0638 and JHP0581. CONCLUSION: Although the aim gene was obtained, but it was different from the published sequence of GenBank. It is not clear what makes this difference. Maybe it is because different strain was used or because there were some variations. So more researches are required to prove it.

[Construction, expression and antigenicity of bivalent vaccine candidate of human Helicobacter pylori].

AIM: To construct a recombinant vector containing fused gene of heat shock protein A(HspA) and outer membrane protein(OMP) with M(r) 18,000, from human Helicobacter pylori(Hp) and express the fusion protein in E.coli BL21. METHODS: The gene encoding HspA was amplified from Hp chromosome by PCR. After digestion with kpn I and BamH I, the HspA gene was inserted into the prokaryotic expression vector pET32a(+). After recombinant vectors pET32a(+)/HspA and pET32a(+)/Omp (18) were digested with Hind III and BamH I, the pET32a(+)/HspA and 18,000 OMP gene segments were recovered through agarose electrophoresis, and connected by T4 ligase. The recombinant vector pET32a(+)/HspA-Omp(18) was transformed into E.coli BL21(DE3). The antigenicity of recombinant fusion protein was analysed by Western blot. RESULTS: Enzyme digestion analysis and sequencing showed that the fused gene had 891 base pairs. As compared with gene reported in GenBank, there were 1.15% and 1.26% differences in cloned nucleotide sequence and amino acid sequence respectively. SDS-PAGE analysis showed that recombinant vector could be expressed in E.coli BL21. The relative molecule mass (M(r)) of expressed fusion protein was 51x10(3). And soluble expression product accounted for 18.96% of total bacterial protein.After purification via Ni-NTA agarose resin, the purity of recombinant fusion protein was about 95%. The Western blot analysis showed that recombinant fusion protein could be recognized by anti-Hp positive serum and anti-18,000 OMP mAb, suggesting that this protein had good antigenicity. CONCLUSION: The fused gene of HspA and OMP is cloned and expressed successfully, which lays the foundation for development of protein and DNA vaccines and a diagnostic kit of Hp infection.

[Construction, expression and antigenic study of bivalent vaccine candidate with 26,000 OMP and heat short protein A of human Helicobacter pylori].

OBJECTIVE: To construct a recombinant vector containing gene encoding heat shock protein A (HspA) and outer membrane protein (OMP) with relative molecule mass (Mr) of 13,000 and 26,000 respectively from human Helicobacter pylori (Hp) and be expressed in E. coli BL21, as well as analyse its antigenic for the exploiting vaccine of Hp. METHODS: The target gene encoding heat shock protein A was amplified from Hp chromosome by PCR. And then digested by restricted endonuclease enzyme of kpnI, BamH I simultaneously, and inserted into the prokaryotic expression vector pET32a(+) digested by corresponding restricted endonuclease enzyme. The recombinant vector was used to select and transform for sequence analysis. After pET32a(+)/HspA and pET32a(+)/Omp(26) digested by restricted endonuclease enzyme of Hind III, BamH I simultaneously, the pET32a(+)/HspA and 26,000 OMP were taken out of agarose electrophoresis, and connected by T4 ligase. The recombinant vector pET32a(+)/HspA-Omp(26) was used to select and transform, meanwhile expressed in E. coli BL21(DE3). The antigenic of recombinant fusion protein was analysed by western blotting. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes was found to be 951 base pairs, and had been inserted into recombinant vector, but as compared with gene reported by GenBank, 1.15% of the gene mutation and 1.26% of amino acid residues change in Hp happened respectively. SDS-PAGE analysis showed that recombinant vector could be expressed in E. coli BL21, its relative molecule mass of expressed product was 59 x 10(3), while Mr of protein expressed by pET32a(+) of them was about 20 x 10(3), and soluble expression product accounted for 19.96% of total bacterial protein. After purification with Ni-NTA agarose resin, the purity of recombinant fusion protein was about 95%. The western blot result showed that recombinant fusion protein could be recognized by anti-Hp positive serum and monoclonal antibody of 26,000 OMP, suggesting that this protein had good antigenic. CONCLUSION: The genes coding HspA and OMP with Mr 13,000 and 26,000 respectively are cloned and expressed successfully. The results obtained lay the foundation for research on development of Hp protein and DNA vaccine and a quickly diagnostic kit applying to detection of Hp infection.

Construction and characterization of bivalent vaccine candidate expressing HspA and M(r)18,000 OMP from Helicobacter pylori.

AIM: To construct a recombinant vector which can express outer membrane protein (OMP) with M(r)18,000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection. METHODS: The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, BamH I simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp(18), digested by restrictive endonuclease enzyme Hind III and BamH I simultaneously. pET32a(+)/HspA and Omp(18) were recovered from 1 % agarose gel by gel kit, and ligated with T(4) ligase by BamH I digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp(18) was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3 % and 1.4 % differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (M(r)) of the expressed product was M(r) 51,000, M(r) of protein expressed by pET32a (+) was about M(r) 20,000, and soluble expression product accounted for 18.96 % of total bacterial protein. After purification with Ni(+2)-NTA agarose resins, the purification of recombinant fusion protein was about 95 %. Western blot showed that recombinant fusion protein could be recognized by the patients' serum infected with H. pylori and anti-Omp(18) monoclone, suggesting that this protein had good antigenicity. CONCLUSION: The gene coding for H. pylori M(r)18,000 OMP and HspA was cloned and expressed successfully. The results obtained lay the foundation for development of H. pylori protein vaccine and a quick diagnostic kit.

Effect of compound rhodiola sachalinensis A Bor on CCl4-induced liver fibrosis in rats and its probable molecular mechanisms.

AIM: To explore the anti-fibrotic effect of a traditional Chinese medicine, compound rhodiola sachalinensis A Bor on CCl(4)-induced liver fibrosis in rats and its probable molecular mechanisms. METHODS: Ninety healthy male SD rats were randomly divided into three groups: normal group (n=10), treatment group of compound rhodiola sachalinensis A Bor (n=40) and CCl(4)-induced model group (n=40). The liver fibrosis was induced by CCl(4) subcutaneous injection. Treatment group was administered with compound rhodiola sachalinensis A Bor (0.5 g/kg) once a day at the same time. Then the activities of several serum fibrosis-associated enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), N-acetyl-beta-D-glucosaminidase (beta-NAG) and the levels of serum procollagen III (PCIII), collagen IV (CIV), hyaluronic acid (HA) were assayed. The histopathological changes were observed with HE, VG and Masson stain. The expression of TGF-beta1 mRNA, alpha1 (I) mRNA and Na(+)/Ca(2+) exchanger (NCX) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in situ. RESULTS: Compound rhodiola sachalinensis A Bor significantly reduced serum activities of ALT, AST, beta-NAG and decreased the levels of PCIII, CIV, HA, improved the liver histopathological changes, inhibited the expression of TGF-beta1 mRNA, alpha (I) mRNA and Na(+)/Ca(2+) exchanger mRNA in rats. CONCLUSION: Compound rhodiola sachalinensis A Bor can intervene in CCl(4)-induced liver fibrosis in rats, in which potential mechanisms may be decreasing the production of TGF-beta1, reducing the production of collagen, preventing the activation of hepatic stellate cell (HSC) and inhibiting the expression of TGF-beta1 mRNA, alpha1(I) mRNA and Na(+)/Ca(2+) exchanger mRNA.