hp0169基因在幽门螺杆菌感染GES-1细胞中的作用研究
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摘要
目的构建幽门螺杆菌(Helicobacter pylori,Hp)hp0169基因的敲除突变株,研究其在Hp感染GES-1细胞中的作用。方法敲除质粒pSJHK为模板,通过同源重组双交换的方法构建hp0169基因敲除突变株(Δ0169),分析其与野生26695株的生长曲线及琼脂表面菌落扩散情况;通过FITC标记法检测两菌株对GES-1细胞的吸附率;以感染复数(MOI=200)构建Hp感染GES-1细胞体系,比较Δ0169突变株与野生株感染后致细胞凋亡及细胞活性变化的差异。结果通过质粒敲除、电击转化成功获得hp0169基因敲除突变株Δ0169。该突变株与野生菌株的生长曲线、琼脂表面菌落扩散情况基本一致,对GES-1细胞的吸附率差异无统计学意义(t值为1.102,P>0.05)。Δ0169突变株与野生株感染GES-1细胞活力分别为6h(0.74±9.56)%、(0.90±6.31)%,12h(0.53±7.89)%、(0.73±8.31)%,差异均有统计学意义(t值分别为-3.474,-2.880,P<0.05);野生组和突变组GES-1细胞凋亡率分别为6h(19.2±11.03)%、(20.4±8.67)%和12h(29.3±14.47)%、(28.6±10.32)%,差异均无统计学意义(t值分别为-0.995,0.218,P>0.05)。结论 Hp hp0169基因敲除后不影响其生长、运动以及对GES-1细胞的吸附,不影响细菌感染GES-1细胞致细胞凋亡的程度,但影响细菌感染致细胞活力下降的程度。这对研究Hp感染及致病机制有重要意义。
Objectives To construct an hp0169 gene knockout mutant strain and to analyze the function of the hp0169 gene in Helicobacter pylori infection of GES-1 cells. Methods The plasmid pSJHK was used as a template to generate an hp0169 knockout plasmid via double-crossover recombination.The knockout mutantΔ0169 was obtained via electroporation of the knockout plasmid.The growth curve and cell motility of wild-type H.pylori(WT)andΔ0169 were compared.Adhesion of two strains to GES-1 cells was detected using FITC labeling.In addition,H.pylori was co-cultured with GES-1 cells at an MOI of 200.The viability and apoptosis of infected GES-1 cells were determined and compared between WT H.pylori andΔ0169.Cell viability was determined by counting living cells using Cell Counting Kit 8(CCK-8).The apoptosis of infected GES-1 cells was determined using the Annexin V-FITC Apoptosis Detection Kit,and the relative number of apoptotic cells was detected using flow cytometry. Results An hp0169 knockout mutant,Δ0169,was constructed via electroporation of a plasmid and knock out of a gene.TheΔ0169 mutant had a growth curve and level of cell motility similar to the growth curve and level of cell motility of WT H.pylori,and cell adhesion did not differ significantly for WT H.pylori andΔ0169.When GES-1 cells were infected with WT H.pylori,their viability at 6 hwas0.74±9.56%;when those cells were infected withΔ0169,their viability at 6 hwas 0.90±6.31%.When GES-1 cells were infected with WT H.pylori,their viability at 12 hwas 0.53±7.89%;when those cells were infected withΔ0169,their viability at 12 hwas 0.73±8.31%.The viability of GES-1 cells differed significantly(t=-3.474,-2.880,P<0.05)when they were infected with WT H.pylori or the mutant.When GES-1 cells were infected with WT H.pylori,the rate of their apoptosis at 6 hwas 19.2±11.03%;when those cells were infected withΔ0169,the rate of their apoptosis at 6 hwas 20.4±8.67%.When GES-1 cells were infected with WT H.pylori,the rate of their apoptosis at 12 hwas29.3±14.47%;when those cells were infected withΔ0169,the rate of their apoptosis at 12 hwas 28.6±10.32.The rate of apoptosis did not differ significantly(t=-0.995,0.218,P>0.05)between WT H.pylori and the mutant. Conclusion Deletion of hp0169 did not affect bacterial growth,motility,adhesion to GES-1 cells,or apoptosis of infected GES-1 cells.However,GES-1 cells infected with theΔ0169 mutant had a higher cell viability compared to those infected with WT H.pylori,suggested that hp0169 plays a role in H.pylori infection.This finding is crucial to studying the pathogenesis of H.pylori.
Objectives To construct an hp0169 gene knockout mutant strain and to analyze the function of the hp0169 gene in Helicobacter pylori infection of GES-1 cells. Methods The plasmid pSJHK was used as a template to generate an hp0169 knockout plasmid via double-crossover recombination.The knockout mutantΔ0169 was obtained via electroporation of the knockout plasmid.The growth curve and cell motility of wild-type H.pylori(WT)andΔ0169 were compared.Adhesion of two strains to GES-1 cells was detected using FITC labeling.In addition,H.pylori was co-cultured with GES-1 cells at an MOI of 200.The viability and apoptosis of infected GES-1 cells were determined and compared between WT H.pylori andΔ0169.Cell viability was determined by counting living cells using Cell Counting Kit 8(CCK-8).The apoptosis of infected GES-1 cells was determined using the Annexin V-FITC Apoptosis Detection Kit,and the relative number of apoptotic cells was detected using flow cytometry. Results An hp0169 knockout mutant,Δ0169,was constructed via electroporation of a plasmid and knock out of a gene.TheΔ0169 mutant had a growth curve and level of cell motility similar to the growth curve and level of cell motility of WT H.pylori,and cell adhesion did not differ significantly for WT H.pylori andΔ0169.When GES-1 cells were infected with WT H.pylori,their viability at 6 hwas0.74±9.56%;when those cells were infected withΔ0169,their viability at 6 hwas 0.90±6.31%.When GES-1 cells were infected with WT H.pylori,their viability at 12 hwas 0.53±7.89%;when those cells were infected withΔ0169,their viability at 12 hwas 0.73±8.31%.The viability of GES-1 cells differed significantly(t=-3.474,-2.880,P<0.05)when they were infected with WT H.pylori or the mutant.When GES-1 cells were infected with WT H.pylori,the rate of their apoptosis at 6 hwas 19.2±11.03%;when those cells were infected withΔ0169,the rate of their apoptosis at 6 hwas 20.4±8.67%.When GES-1 cells were infected with WT H.pylori,the rate of their apoptosis at 12 hwas29.3±14.47%;when those cells were infected withΔ0169,the rate of their apoptosis at 12 hwas 28.6±10.32.The rate of apoptosis did not differ significantly(t=-0.995,0.218,P>0.05)between WT H.pylori and the mutant. Conclusion Deletion of hp0169 did not affect bacterial growth,motility,adhesion to GES-1 cells,or apoptosis of infected GES-1 cells.However,GES-1 cells infected with theΔ0169 mutant had a higher cell viability compared to those infected with WT H.pylori,suggested that hp0169 plays a role in H.pylori infection.This finding is crucial to studying the pathogenesis of H.pylori.
引文
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[10]季晓飞,赵慧琳,张莹,等.基因敲除质粒载体的构建及其在幽门螺杆菌基因敲除中的应用[J].中国病原生物学杂志,2016,11(12):1066-73.
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[13] Kavermann H,Burns BP,Angermuller K,et al.Identification and characterization of Helicobacter pylori genes essential for gastric colonization[J].J Exp Med,2003,197(7):813-22.
[2] Forman D.Helicobacter pylori infection and cancer[J].Br Med Bull,1998(54):71-8.
[3] Peek RM Jr,Blaser MJ.Helicobacter pylori and gastrointestinal tract adenocarcinomas[J].Nat Rev Cancer,2002(2):28-37.
[4] Giyama T,Asaka M.Helicobacter pylori infection and gastric cancer[J].Med Electron Microsc,2004(37):149-57.
[5]刘国栋,张建中.幽门螺杆菌致病因子研究进展[J].现代消化及介入诊疗,2010,15(1):33-7.
[6] Posselt G,Backert S,Wessler S.The functional interplay of Helicobacter pylori factors with gastric epithelial cells induces a multi-step process in pathogenesis[J].Cell Commun Signal,2013(11):77.
[7] Franco AT,Johnston E,Krishna U,et al.Regulation of gastric carcinogenesis by Helicobacter pylori virulence factors[J].Cancer Res,2008(68):379-87.
[8] Akhter Y,Ahmed I,Devi SM,et al.The co-evolved Helicobacter pylori and gastric cancer:trinity of bacterial virulence,host susceptibility and lifestyle[J].Infect Agent Cancer,2007(2):2.
[9]荣倩玉,李娇娇,季晓飞,等.幽门螺杆菌在两种液体培养基中生长规律的探讨[J].中国病原生物学杂志,2017,12(8):738-40.
[10]季晓飞,赵慧琳,张莹,等.基因敲除质粒载体的构建及其在幽门螺杆菌基因敲除中的应用[J].中国病原生物学杂志,2016,11(12):1066-73.
[11] Kato T,Takahashi N,Kuramitsu HK.Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene,which expresses a novel collagenase activity[J].J Bacteriol,1992,174(12):3889-95.
[12]Wu MT,Carlson SA,Meyerholz DK.Cytopathic effects observed upon expression of a repressed collagenase gene present in Salmonellaand related pathogens:mimicry of a cytotoxin from multiple antibiotic-resistant Salmonella enterica serotype Typhimurium phagetype DT104[J].Microb Pathog,2002,33(6):279-87.
[13] Kavermann H,Burns BP,Angermuller K,et al.Identification and characterization of Helicobacter pylori genes essential for gastric colonization[J].J Exp Med,2003,197(7):813-22.