| Background and ObjectiveGenus Helicobacter is composed of the Gram-negative, microaerophilic, spiral curved bacteria. In accordance with the the main planting sites and 16S rRNA/23S rRNA, can Helicobacter spp. can be divided into two types:gastric Helicobacter (e.g. H. pylori) and enterohepatic Helicobacter (e.g. H. hepaticus, H. bilis). Although the colonization sites of enterohepatic Helicobacter, mainly intestine, liver or biliary system of humans, other mammals and birds, is different from that of gastric Helicobacter, enterohepatic Helicobacter and gastric Helicobacter share similar ultra structure and biochemical functions. In 1983, Warren and Marshall for the first time isolated H. pylori from the human gastric mucosa, at that time some scholars denied its pathogenicity.Nowadays H. pylori has been proved to be the major virulence factor of chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma and other diseases. In 1992, Lee et al. first reported one kind of enterohepatic Helicobacter, H. muridarum, which colonized in the intestinal mucosa of mice and rats. During the past two decades, other types of enterohepatic Helicobacters has been gradually isolated from animals or humans, such as H. hepaticus, H. bilis, H. muridarum, H. rodentium, and H. typhlonicus. Domestic and foreign researchers found that the enterohepatic Helicobacter infection was prevalent in experimental animals, wild animals and pets by epidemiological survey. Like H. pylori, enterohepatic Helicobacters used to be considered as normal flora; with further research, researchers have found that part of enterohepatic Helicobacters, possibly even all of them, could be causative of diseases in immunocompetent or immunodeficient rodent.H. hepaticus is one of the most well studied enterohepatic Helicobacters, while H. bilis isn't fully recognized and the examination methods still need to be optimized. Animal experiments have confirmed the correlation between enterohepatic Helicobacter infection and typhlocolitis, hepatitis, hepatocellular carcinoma, cholecystitis and other disease in mice, while the association of the infection and the human digestive system diseases remains unclear. To solve these problems, this project is divided into two parts. In part one, we induce different strains of the laboratory mice with H. bilis standard strain (ATCC51630), then monitor infection by faces culture, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), and compare the three methods. In part two, we collect the bile samples of cholelithiasis, gallbladder polyps, cholangiocarcinoma, hepatocellular carcinoma and liver metastases patients, and investigate the positivity of enterohepatic Helicobacter-16S rRNA gene amplification and antibody, and discuss the possible pathogenic role of enterohepatic Helicobacter in the development of these hepatobiliary diseases.Materials and MethodsPart One:H. bilis-free SPF male BALB/C, C57BL/6, KM, NIH, and SCID mice (n= 12, respectively) were equally divided into two groups:experimental group and control group. The experimental group was orally inoculated with 0.4mL (1×108 CFU/mL) H. bilis standard strains (ATCC51630) suspension for three times at an interval of 48 hours. The control group was fed with equal volume of PBS similarly. The feces of both groups were collected at 1,3,5,7,9,11,13,15 weeks after the last gavage, and were processed differently as follows.?The samples were continuously cultured in the selective Columbia blood agar medium (contained vancomycin, amphotericin B,polymyxin, TMP, and defibrinated sheep blood) at 37?, high humidity, and microaerophilic (5%O2,5%H2,10%CO2,80% N2) condition for three day, observing whether the colony grow or not for ten days.(2)DNA of the samples were extracted by genomic DNA extraction kit, the primer for the H. bilis-specific 16S rRNA was synthesized, the extracted DNA was amplified by PCR, observed and photographed under ultraviolet imaging system after gel electrophoresis. The positive PCR products underwent DNA fragments sequencing, and was compared with H. bilis gene sequenc in the Genebank.?The samples were tested for anti-H. bilis antibody by ELISA.15 weeks after the administration, all mice were sacrificed by cervical dislocation to obtain the stomach, liver, pancreas, jejunum, ileum, cecum and colon tissue for histopathological examination.Part Two:From January 2011 to January 2013, a total of 223 patients (134 male, 89 female, with an average of 49.8 years old) with hepatobiliary diseases who treated in Nanfang Hospital were concluded in the study. All the cases were confirmed by histopathological diagnosis, including 95 cases of cholelithiasis,11 gallbladder polyps,27 cholangiocarcinomas,59 hepatocellular carcinomas and 31 liver metastases. The bile samples of these patients obtained by surgery or ERCP were divided into three pieces equally. One piece was cultured to isolate Helicobacter. The second piece was examined by PCR using primers for Helicobacter-specific 16S rRNA, H. hepaticus-specific 16S rRNA, H. bilis-specific 16S rRNA, and H. pylori-specific UreaseA. The positive PCR products underwent DNA fragments sequencing, and was compared with known Helicobacter gene sequences in the Genebank. Some of these samples underwent TA clone sequencing randomly. The third piece was tested for anti-H. hepaticus antibody, anti-H. bilis antibody and anti-H. pylori antibody by ELISA. At least one positive in the three detections was considered as H. hepaticus (or H. bilis, H. pylori) infection in the bile. The statistics were analyzed by x2 test or Fisher exact test.ResultsPart One: ?H. bilis detection rate by feces culture:BALB/C mice and SCID mice of the experimental group were H. bilis-positive at the 3rd week, much earlier than KM mice (7th week), NIH mice (7th week) and C57BL/6 mice (9th week). The culture positive rate of SCID mice was maintained between 66.7% and 100% during the 7th?15th week, the rate of BALB/C mice remained in the range of 66.7%?83.3% during the 11th?15th week, the rate of KM mice and NIH mice maintained at a low level (33.3%-50%), the rate of C57BL/6 was lowest and failed to cultivate H. bilis at the 15th week. The feces samples of the PBS control group in each period were H.bilis-negative.?H. bilis-specific 16S rRNA gene detection rate by feces PCR: BALB/C mice and SCID mice of the experimental group were tested H.bilis-positive at the 1st week, much earlier than KM mice, NIH mice and C57BL/6 mice (7th week). The 16S rRNA gene positive rate of SCID mice was maintained between 66.7% and 100% during the 3rd?15th week, the rate of BALB/C mice remained in the range of 66.7%?100% during the 7th?15th week. The gene positive rate of KM mice and NIH mice was significantly higher than the culture positive rate of these mice, maintained at 66.7%?100% during the 7th?15th and the 9th?15th week respectively. The rate of C57BL/6 was 100% at the 11th week, but dropped to 33.3% at the 15th week. The feces samples of the PBS control group in each period were H. bilis-negative.?H. bilis antigen detection rate by feces ELISA:BALB/C mice and SCID mice of the experimental group were tested H. bilis antigen-positive at the 1st week, much earlier than KM mice, NIH mice and C57BL/6 mice (7th week).The antigen positive rate of SCID mice was maintained between 83.3% and 100% during the 5th?15th week, the rate of BALB/C mice remained in the range of 83.3%?100% during the 9th?15th week. The antigen positive rate of KM mice and NIH mice was significantly higher than the culture positive rate of these mice, maintained at 66.7%-83.3% during the 9th?15th and the 11th?15th week respectively. The rate of C57BL/6 was 66.7% at the 11th week, but dropped to 33.3% at the 15th week. The feces samples of the PBS control group in each period were H. bilis-negative.?Histopathological examination of digestive organs:the severity of inflammation were SCID >BALB/C>KM>NIH>C57BL/6.The digestive organs of mice in the PBS control group had no inflammation.Part Two:All of the bile samples were not cultured Helicobacter.28 out of 95(29.5%) bile samples of patients with cholelithiasis were H. hepaticus positive,23 (24.2%) were H. bilis positive,63 (66.3%) were H. pylori positive.1 out of 11 (9.1%) bile samples of patients with gallbladder polyps were H. hepaticus positive,4 (36.4%) were H. pylori positive.12 out of 27 (44.4%) bile samples of patients with cholangiocarcinoma were H. hepaticus positive,14(51.9%) were H. bilis positive,20 (74.1%) were H. pylori positive.16 out of 59 (27.1%) bile samples of patients with hepatocellular carcinoma were H. hepaticus positive,7 (11.9%) were H. bilis positive, 37 (62.7%) were H. pylori positive.3 out of 31 (9.7%) bile samples of patients with liver metastases were H. hepaticus positive,2 (6.5%) were H. bilis positive,15 (48.4%) were H. pylori positive. The H. hepaticus positive rate and H. bilis positive rate of patients with cholelithiasis, cholangiocarcinoma or hepatocellular carcinoma were significantly higher than the rate of patients with gallbladder polyps or liver metastases (P<0.05).ConclusionPart One:SCID mice are the most susceptible to H. bilis while C57BL/6 mice are resistant to H. bilis. Exact results of the susceptibility to H.bilis are listed as follows:SCID>BALB/C>KM>NIH>C57BL/6. Feces culture, feces H.bilis-specific 16S rRNA gene PCR and feces H. bilis antigen ELISA are able to detect the H.bilis infection. Susceptibility of these methods is listed as follows: PCR>ELISA>culture.Histopathological examination of digestive organs:the severity of inflammation were SCID>BALB/OKM>NIH>C57BL/6.Part Two:H. hepaticus, H. bilis and H. pylori gene or antibody exist in bile of patients with cholelithiasis, gallbladder polyps, cholangiocarcinoma, hepatocellular carcinoma or liver metastases. The infection rates were different according to the disease types. Enterohepatic Helicobacter infection may be associated with hepatobiliary disease pathogenesis. |
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