摘要
目的:
本实验通过检测PS患者胆管黏膜、胆汁和胆石中Hp,以探讨Hp感染与PS形成之间的关系及可能的致石形成机制。
方法:
对45例(其中35例PS患者及10例对照组患者)胆汁做需氧菌、厌氧菌及Hp培养,分别采用PCR的方法检测胆(囊)管黏膜、胆汁和胆石核心中的Hp-DNA和Western-blot的方法检测胆汁中Hp感染相关蛋白,所有粘膜用Warthin-Starry银染法染色。
结果:
1.实验组(35例PS患者)中胆汁细菌培养15/35(42.86%)需氧菌和/或厌氧菌阳性,对照组未培养出细菌,所有胆汁标本培养未发现Hp生长。
2.实验组胆石核心、胆汁和黏膜的Hp-DNA扩增阳性率分别为14.29 %、31.43 %和56.67 %。而对照组中胆汁和黏膜Hp-DNA为阴性,实验组黏膜的Hp-DNA扩增阳性率与对照组比较,差异有显著性(P<0.01),实验组胆汁的Hp-DNA扩增阳性率与对照组比较,差异有显著性(P<0.05),实验组间黏膜与胆汁胆石Hp-DNA扩增阳性率比较,差异均有统计学意义(P <0.05, P <0.01)。并且在胆石核心Hp-DNA阳性标本中其黏膜和胆汁的Hp-DNA均为阳性。
3.在实验组12例Hp-DNA阳性胆汁中,细胞毒素相关抗原(Cag A)检出7例,细胞空泡毒素(Vac A )检出6例,同时检出Hp Vac A相关亚单位37kD、30kD糖蛋白、尿素酶B(Ure B)和尿素酶A(UreA) ,在实验组23例Hp-DNA阴性胆汁中,仅1例检测到Cag A、Ure B、UreA三条蛋白。对照组胆汁中未检出Hp感染相关蛋白。
4.实验组中,30例胆管黏膜W-S染色有7例(23.33%)镜下观察到类似Hp的菌体。并且这7例胆管黏膜Hp- DNA均为阳性,而对照组中黏膜W-S染色后镜下未观察到类似Hp的菌体。结论:
PS患者胆道系统中存在Hp感染依据,并存在多种Hp感染相关蛋白,Hp可能定植于胆管黏膜内,进而参与PS的形成。
Objective:
This experiment explored the relationship between Hp infection and the formation of pigmented calculus and the possible mechanism of gallstone formation through the study on Hp in the center of gallstones , bile and bile duct mucosa.
Methods:
Regular, anaerobic and Helicobacter pylori cultures were performed for all bile (include 35 samples of bile pigment calculus and 10 samples of the controls). Helicobacter pylori DNA in bile, bile duct mucosa and pigmented calculus were detected by PCR , and Western- blot was performed to detect Helicobacter pylori associated protein in all bile samples. The bile duct mucosa was stained by Warthin-Starry.
Results:
1.The regular, anaerobic and Helicobacter pylori cultures were found 15 positive of regular and anaerobic in 35 bile samples of bile pigment calculus and negative of regular in the controls. Helicobacter pylori cultures were found no bacteria growing in all bile samples.
2.In 35 samples of bile pigment calculus ,the positive rate of Hp -DNA PCR amplification in the center of gallstones , bile and bile duct mucosa was 14.29% , 31.43 % and 56.67 % respectively. The Hp-DNA in the bile and bile duct mucosa of control group were negative. There were significant differences between the positive rates of Hp -DNA PCR amplification in bile(p<0.01) and bile duct mucosa(p<0.05) of experimental group and those of control group. In experimental group, there were significant differences between the positive rate of Hp -DNA PCR amplification in bile duct mucosa and those in bile(p<0.05) and center of gallstones(p<0.01), and the Hp–DNA in bile and bile duct mucosa of all the samples of which Hp–DNA in center of gallstones were postive were postive.
3.PCR showed positive in 11 of 35 bile samples,of which 7contained anti-CagA antibodies,6 contained Vac A. Vacuolating cytotoxin associated 35kD and 30 kD glycoprotein ,Ure B and UreA were also found in the bile. PCR showed negative in 23 of 35 bile samples ,of which 1 contained anti-CagA antibodies, Ure B and UreA found in the bile .The associated protein of Helicobacter pylori infection was not found in the controls
4.Bacteria resembling Helicobacter pylori were found in 7(23.33 %) of 30 bile duct mucosa samples stained by Warthin-Starry and Hp–DNA of these samples was positive, however the results in the control group were negative.
Conclusions :
The presence of Helicobacter pylori DNA in biliary tract system and associated protein of Helicobacter pylori infection in some bile samples may be represent risk factors for the formation of pigmented calculus
引文
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[2] Neri V, Margiotta M, Ambrosi A, et al. DNA sequences and proteic antigens of H. pylori in cholecystic bile and tissue of patients with gallstones[J].Aliment Pharmacol Ther. 2005 Oct15; 22(8):715-720.
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[4] Silva CP, Pereira -Lima JC, Oliverira AG, et al. Association of the presence of helicobacter in gallbladder tissue with cholelithiasis andcholecvtitis[J]. J Clin Microbiol. 2003 Dec; 41(12): 5615 - 5618.
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