Apoptosis-inducing effect of recombinant Caspase-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901

AIM: To investigate the apoptosis-inducing effect of Caspases-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901.METHODS: PCR was employed to amplify the sequences of both small and large subunits of Caspases-3. Its products were separately cloned into the Sma I site of pBluescript KS+ to generate both plasmids pBS/SS and pBS/LS. The small subunit fragment was excised from plasmid pBS/SS with BarrH- I and then inserted into the BarnH I site of plasmid pBS/LS preceding that of the large subunit to yield plasmid pBS/Rev-Caspase-3. Rev-Caspase-3 cDNA was excised with Kpn Ⅰ+Xba I and then subcloned into plasmid pcDNA3.1(+) to construct Rev-Caspase-3 eukaryotic expression vector pcDNA/Rev-Caspase-3, which was used to transiently transfect SGC7901 cell line. Cell count, MTT assay and electron microscopy were used to confirm the antiproliferation and apoptosis-inducing effect of Rev-Caspase-3 expression on gastric cancer cells.RESULTS: Plasmid pBS/Rev-Caspase-3 and eukaryotic expression vector pcDNA/Rev-Caspase-3 were successfully constructed. SGC7901 cells were transiently transfected by either pcDNA/Rev-Caspase-3 or pcDNA3.1 (+) for 24, 48,72, and 96 h respectively. Cell growth was measured by cell count and MTT assay. In cell count assay, the cell numbers were 1.S×106, 1.55×106, 2.0×106, and 3.1×106 in the experimental group and 2.5×106, 3.1×106, 4.0×106, and 5.7×106 in the control group at 24, 48, 72 and 96 h respectively.The growth of SGC7901 cells was suppressed by RevCaspase-3 in a time-dependent manner (P＜0.05). The results of MTT assay were similar to that of cell count (P＜0.05).The characteristics of apoptosis such as chromatin condensation, crescent formation and margination were seen and more obvious with time in the given-experimental period in the experimental group, but not easily observed in the control group.CONCLUSION: The expression of Rev-Caspase-3 by the constructed eukaryotic vector can significantly induce apoptosis of gastric cancer cell line SGC7901, which may exhibit a potential way in gastric cancer gene therapy.

cagA and vacA genotype of Helicobacter pylori associated with gastric diseases in Xi'an area

AIM: To establish stock of clinical Helicobacter pylori (H.pylon) isolates, to perform cagA and vacA typing of these isolates, to evaluate the relationship between genotypes of cagA and vacA and upper gastrointestinal diseases and to assess the association of vacA genotypes with presence of the pathogenicity marker-cagA.METHODS: Clinical H.pylori strains were isolated from the antrum of 259 patients in Clumbia agar. The isolated H.pylori strains were identified by histology, and16SrRNA PCR.CagA genotypes were detected by colony hybridization, the probe was derived from the cloned plasmid PcagA, and digested by EcoRI-HindⅢ and the isolated PcagA DNA fragment was radioactively labelled by the random priming method. vacA genes types (s,m)and subtypes (s1a, s1b,s2) were typed by PCR. Vacuolating toxin was detected with neutral red absorb test. The results were treated statistically by χ2test, ttest, and rank sum test.RESULTS: A total of 192 clinical H. pylori strains were isolated and the stock of Helicobacter pylori was established. The total positive rate of cagA was 87 % in all gastric diseases,and 95 % in gastric cancer group. There was a difference between gastric cancer group and the other groups (P＜0.05)except duodenal ulcer group. The expression of type s1 of vacA was more than type s2 (P＜0.05), and, the expression of type m1 was equal to type m2. In gastric cancer group,there was a difference between s1a and s1b (P＜0.05), and s1a was more than s1b. Vacuolating toxins were more in Xi′an area isolates.CONCLUSION: The cagA+ vacA type s1 clinical isolates are more in Xi′an area, but this can not serve as an index to predict gastric cancer.

Effect of Hewei-Decoction on chronic atrophic gastritis and eradication of Helicobacter pylori

AIM: To demonstrate the effect of Hewei-Decoction(Decoction for regulating the stomach) on chronic atrophic gastritis (CAG) and eradication of Helicobacter pylori.METHODS: Ninety patients with CAG entering the investigation were divided into six differentiation syndromes, based on their major symptoms and signs.Hewei-Decoction was taken by all the patients orally for 4or 8 wk. The efficacy was assessed by both the composite accumulation of reduced scores of major symptoms and the eradication of H pylori.χ2 test was used to compare the efficacy between Hpylori-positive and negative cases,and to disclose the relationship between efficacy and eradication of H pylori.REStULTS: In patients with six different syndrome types,the efficacy of Hewei-Decoction was 91.67% (11/12),92.86% (13/14), 97.22% (35/36), 87.50% (14/16),75.00% (6/8), 75.00% (3/4) respectively. The rate of highly efficacious was 58.33% (7/12), 50.00% (7/14),77.78% (28/36), 62.50% (10/16), 12.50% (1/8) and25.00% (1/4), respectively. The total efficacy was 91.11%(82/90), and the rate of highly efficacious was 60.00%(54/90). The eradication rate of H pyloriwas 67.86%(38/56). The therapeutic effect of Hewei-Decoction was better in H pylori positive cases than that in Hpylori-negative cases with the total effect of 96.43% vs82.35% (P＜0.05).In 56 H pylori positive cases, the therapeutic effect was better in H pylori eradicated cases than that in H pyloriexistent cases with the total effect of 97.37% vs 72.22%(P＜0.01).CONCLUSION: Hewei-Decoction is effective in most cases of all the syndrome types. The results indicate that eradication of H pylori is one of the important mechanisms for alleviation of symptoms and signs. Also, the decoction is efficacious in H pylori-negative cases.

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGbl to screen a phage-displayed random peptide library fused with coat protein plII in order to get some information on mimotopes.lV^37BODS: Through affinity enrichment and EUSA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGbl-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGbl-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0±1.6) %to (39.0±2.7) %.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope

AIM:To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori ( H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of Hpyloriflagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K^2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum Hpylori-specific IgA and IgGl increased significantly in immunized group, while IgG2a only had an insignificant change. Hpylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.

采用染色内镜加靶向活检对溃疡性结肠炎进行瘤变长期随访优于白光内镜:一项多中心随机对照试验

背景:单中心或小样本研究数据显示,染色内镜用于溃疡性结肠炎(UC)患者的瘤变监测可能优于白光内镜。我们进行了一项前瞻性随机试验,通过对UC患者的长期随访,比较白光内镜加靶向活检(WLT)、白光内镜加随机活检(WLR)与染色内镜加靶向活检(CET)的肿瘤检出率。方法:前瞻性纳入2012年3月至2013年12月间11个医学中心收治的UC患者,随机分为WLT、WLR、CET三组。三组患者均仅行高清内镜检查,每年内镜随访一次,直至2017年12月。结果:中位随访55个月,122例入组患者完成了447次内镜检查,纳入最终的完成方案分析,其中WLT组43例,WLR组40例,CET组39例。在21例患者的29次肠镜检查中,共发现34个瘤变。WLR组和CET组诊断瘤变的肠镜检查比例高于WLT组(8.1%和9.7%vs 1.9%;P=0.014,P=0.004)。WLR组活检样本数量显著多于WLT组和CET组(16.4±5.1 vs 4.4±1.4和4.3±3.5;均P<0.001)。在后半程随访中(37-69个月),CET组诊断瘤变的肠镜比例显著高于WLT组(13.3%vs 1.6%,P=0.015),较WLR组则显示出了增高的趋势(13.3%vs 4.9%,P=0.107)。结论:对于UC患者的癌变/瘤变长期监测,CET比WLT更加高效,比WLR更加简单,尤其适用于3年以上的长期随访。本研究于www.chictr.org.cn进行注册(ChiCTR1900023689)。