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The Biological Activity of H. Pylori SlyD in Vitro
Author(s) -
Dan Kang,
Yuehua Gong,
yanmei Zhu,
aodi Li,
nannan Dong,
ying Piao,
Yuan Yuan
Publication year - 2013
Publication title -
helicobacter
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.206
H-Index - 79
eISSN - 1523-5378
pISSN - 1083-4389
DOI - 10.1111/hel.12057
Subject(s) - microbiology and biotechnology , western blot , in vitro , proliferating cell nuclear antigen , biology , apoptosis , cell growth , chemistry , biochemistry , gene
Aim To investigate the biological activity of the H. pylori SlyD in vitro. Methods Helicobacter pylori ( H .pylori) sly D prokaryotic expression vector was carried out in Escherichia coli ( E .coli), and recombination S ly D ( rS ly D ) was purified by immobilized metal affinity chromatography. The proliferation, apoptosis, invasion, transformation effects of rS lyD on AGS cells was detected by CCK ‐8, cell cycle, caspase‐3 activity, matrigel invasion assay, and double‐deck soft agar colony forming efficiency. In addition, the expressions of PCNA , K I‐67, caspase‐3, and MMP ‐9 were detected by western blot and immunofluorescence assay, respectively. Results The CCK ‐8 assay revealed that cell proliferation was increased in a time and dose‐dependent manner in AGS + rS lyD group compared with that of AGS or AGS + PBS group ( p < .05). There are significant difference of PCNA and K I67 expressions among AGS , AGS + PBS , AGS + rS ly D groups ( p < .05). Soft agar colony formation assay revealed the colony number (foci>100 μm) in AGS + rS ly D group was 26.3 ± 7.09, whereas 5.6 ± 1.15 in AGS and 5.0 ± 1.0 in AGS + PBS groups, respectively ( p < .01). Colorimetric enzyme assay revealed the activity of caspase‐3 was decreased to 31.45 ± 0.49 after treatment with rS ly D , whereas 55.5 ± 0.43 in AGS and 55.1 ± 0.25 in AGS + PBS group, respectively ( p < .001). Similar caspase‐3 expression also was confirmed by Western blot. The number of invasive cells in transwell chambers assay is 196.66 ± 40.41 in AGS + rS ly D group higher than 85 ± 22.9 in AGS or 81.66 ± 15.27 in AGS + PBS group, respectively ( p < .001). The MMP ‐9 expression in AGS + rS ly D group was also higher than that of AGS or AGS + PBS group. Conclusion These results suggest that the H p S ly D may play an important role in disturbing cell proliferation, apoptosis, and enhancing cell transformation and invasion in the AGS cell line. H p S ly D might contribute to gastric pathogenicity in H .pylori‐associated diseases.