C cholecystitis.CharacteristicsNo. of Patients n ( )H.pylori (+) in gallbladder mucosa (n = 67)Age (yr) Gender Male: Female ( Male) BMI (kg/m2) Symptom Mild Abdominal Pain Biliary Colic Loss of Appetite Acid Regurgitation Heartburn Preoperative Ultrasound Diagnosis Gallstone Title Loaded From File Disease No. of Gallstones Single: Multiple ( Single) Polypoid Lesion No. of Polypoid Lesions Single: Multiple ( Single) Gallbladder Wall Thickening Atrophic Gallbladder History of Other Gastrointestinal Diseases Chronic Gastritis Gastric Ulcer Duodenal Ulcer Reflux Esophagitis Chronic Enteritis H.pylori (+) in Gastric or Duodenal Mucosa Warthin-Starry Stain H.pylori Culture 16s rRNA gene PCR *p,0.01. m p,0.05. NS: not significant. N/A: not applicable. doi:10.1371/journal.pone.0070265.t002 39(58.21) 33(49.25) 42(62.69) 45(67.16) 11(16.42) 8(11.94) 12(17.91) 3(4.48) 8:4(66.67) 12(17.91) 15(22.39) 21:34(38.18) 44(65.67) 32(47.76) 12(17.91) 22(32.84) 16574785 20(29.85) 19:48(28.36) 23.1062.24 45.54612.H.pylori (2) in gallbladder p value mucosa (n = 259)47.82611.56 NS78:181(30.12) 22.7261.NS NS149(57.53) 102(39.38) 28(10.81) 39(15.06) 50(19.31)NS NS NS 0.001* NS104:108(49.06)NS18:31(35.29) 39(15.06) 35(13.51)NS NS NS 0.005* 0.042m 0.026m NS NS 0.011m 0.029m 0.008*124(47.88) 21(8.11) 12(4.63) 25(9.65) 8(3.09)106(40.93) 90(34.75) 115(44.40)PCR for Helicobacter 16s rRNA GeneThe DNA extracts were prepared from the paraffin specimens as the kit (TIAN amp Micro DNA kit, TIANGEN Biotech, Beijing, China) instructed. A semi-nested PCR assay specific for Helicobacter 16s rRNA gene (16s rDNA) was amplified as previously described [15], using primers 1F (59CTATGACGGGTATCCGGC39), 1R (59CTCACGACACGAGCTGAC39) and 2R (59TCGCCTTCGCAATGAGTATT39). Primers 1F and 1R were used in the first step, whereas primers 1F and 2R were used in the second step. The PCR reaction mixture contained 1 ml of 10 mM each Title Loaded From File primer (1F and 1R), 2 mL of 10 mM dNTP, 16PCR reaction buffer, 2.5 mM MgCl2, 0.05 casein, 0.05 formamid, 1.25 U rTaq DNA polymerase (Takara, Dalian, China), and 2 mL extracted DNA as templates. The amplification conditions for the first step were 94uC for 2 min; 30 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 30 s; and finally 72uC for 10 min. The reaction mixture of the second step (25 mL) contained 0.5 ml of 10 mM eachprimer each primer (1F and 2R), 0.2 mmol/L each dNTP, 16PCR reaction buffer, 2.5 mmol/L MgCl2, 1.0 U ExTaq DNA polymerase (Takara, Dalian, China), and 2 mL 106diluted PCR product from the first step. The 416-bp PCR products were visualized by 1.2 agarose gel electrophoresis.Sequence AnalysisThe PCR products were subsequently ligated into pMD-19T simple vector (Takara, Dalian, China) for sequencing. The product was sequenced after PCR amplification and double enzyme digestion. The sequences were compared with the 16S rRNA gene sequences of the strains that have been registered in the GenBank database. Products of the sequence reaction were aligned and the closest homologous sequence was identified by Standard Nucleotide BLAST analysis tool on the internet (BLASTn, http://blast.ncbi.nlm. nih.gov).Helicobacter pylori and Chronic CholecystitisFigure 7. ROS expression in gallbladder mucosa of chronic cholecystitis with H. pylori infection (A) and without H. pylori infection (B) (6100). doi:10.1371/journal.pone.0070265.gFigure 5. Metaplasia of Chronic Cholecystitis (hematoxylineosin stain,6100). doi:10.1371/journal.pone.0070265.gHistological Analysis of Chronic CholecystitisGal.C cholecystitis.CharacteristicsNo. of Patients n ( )H.pylori (+) in gallbladder mucosa (n = 67)Age (yr) Gender Male: Female ( Male) BMI (kg/m2) Symptom Mild Abdominal Pain Biliary Colic Loss of Appetite Acid Regurgitation Heartburn Preoperative Ultrasound Diagnosis Gallstone Disease No. of Gallstones Single: Multiple ( Single) Polypoid Lesion No. of Polypoid Lesions Single: Multiple ( Single) Gallbladder Wall Thickening Atrophic Gallbladder History of Other Gastrointestinal Diseases Chronic Gastritis Gastric Ulcer Duodenal Ulcer Reflux Esophagitis Chronic Enteritis H.pylori (+) in Gastric or Duodenal Mucosa Warthin-Starry Stain H.pylori Culture 16s rRNA gene PCR *p,0.01. m p,0.05. NS: not significant. N/A: not applicable. doi:10.1371/journal.pone.0070265.t002 39(58.21) 33(49.25) 42(62.69) 45(67.16) 11(16.42) 8(11.94) 12(17.91) 3(4.48) 8:4(66.67) 12(17.91) 15(22.39) 21:34(38.18) 44(65.67) 32(47.76) 12(17.91) 22(32.84) 16574785 20(29.85) 19:48(28.36) 23.1062.24 45.54612.H.pylori (2) in gallbladder p value mucosa (n = 259)47.82611.56 NS78:181(30.12) 22.7261.NS NS149(57.53) 102(39.38) 28(10.81) 39(15.06) 50(19.31)NS NS NS 0.001* NS104:108(49.06)NS18:31(35.29) 39(15.06) 35(13.51)NS NS NS 0.005* 0.042m 0.026m NS NS 0.011m 0.029m 0.008*124(47.88) 21(8.11) 12(4.63) 25(9.65) 8(3.09)106(40.93) 90(34.75) 115(44.40)PCR for Helicobacter 16s rRNA GeneThe DNA extracts were prepared from the paraffin specimens as the kit (TIAN amp Micro DNA kit, TIANGEN Biotech, Beijing, China) instructed. A semi-nested PCR assay specific for Helicobacter 16s rRNA gene (16s rDNA) was amplified as previously described [15], using primers 1F (59CTATGACGGGTATCCGGC39), 1R (59CTCACGACACGAGCTGAC39) and 2R (59TCGCCTTCGCAATGAGTATT39). Primers 1F and 1R were used in the first step, whereas primers 1F and 2R were used in the second step. The PCR reaction mixture contained 1 ml of 10 mM each primer (1F and 1R), 2 mL of 10 mM dNTP, 16PCR reaction buffer, 2.5 mM MgCl2, 0.05 casein, 0.05 formamid, 1.25 U rTaq DNA polymerase (Takara, Dalian, China), and 2 mL extracted DNA as templates. The amplification conditions for the first step were 94uC for 2 min; 30 cycles of 94uC for 30 s, 55uC for 30 s, and 72uC for 30 s; and finally 72uC for 10 min. The reaction mixture of the second step (25 mL) contained 0.5 ml of 10 mM eachprimer each primer (1F and 2R), 0.2 mmol/L each dNTP, 16PCR reaction buffer, 2.5 mmol/L MgCl2, 1.0 U ExTaq DNA polymerase (Takara, Dalian, China), and 2 mL 106diluted PCR product from the first step. The 416-bp PCR products were visualized by 1.2 agarose gel electrophoresis.Sequence AnalysisThe PCR products were subsequently ligated into pMD-19T simple vector (Takara, Dalian, China) for sequencing. The product was sequenced after PCR amplification and double enzyme digestion. The sequences were compared with the 16S rRNA gene sequences of the strains that have been registered in the GenBank database. Products of the sequence reaction were aligned and the closest homologous sequence was identified by Standard Nucleotide BLAST analysis tool on the internet (BLASTn, http://blast.ncbi.nlm. nih.gov).Helicobacter pylori and Chronic CholecystitisFigure 7. ROS expression in gallbladder mucosa of chronic cholecystitis with H. pylori infection (A) and without H. pylori infection (B) (6100). doi:10.1371/journal.pone.0070265.gFigure 5. Metaplasia of Chronic Cholecystitis (hematoxylineosin stain,6100). doi:10.1371/journal.pone.0070265.gHistological Analysis of Chronic CholecystitisGal.
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