Study Of Rabbit Gallstone Produce With Nanobacteria Infection

Objectives:Gallstone is a kind of patho-mineralization phenomenon in biliary tract system, which is also called cholelithiasis. The gallstones incidence is about 7% to 10% in the world population, and it is on the increase obviously. More and more studies have found that infection of biliary tract is intimate relative to cholecystolithiasis with considerable application of new techniques and methods in this field. Evidence shows that there are bacteria in most Gallstone sufferer's gallbladder. So more and more attention is focusing on the relationship between infection of biliary tract and cholecystolithiasis now. Nanobacteria is a kind of tiny-bulk and brady-growth microorganism with the unique capability of bio-mineralization, studies suggest that the infection of nanobacteria may play an important role in cholelithiasis.In order to testify the correlation between nanobacteria infection and cholecystolithiasis, we infected rabbits with nanobactria cultured from bile to lead to rabbit gallstone produce.Methods:1 Isolation, curture and identification of nanobacteria from bile.1.1 Six gallbladder bile samples were obtained from gallstone patients without acute cholecystitis symptoms and without antibiotic therapy before LC. Then,the bile samples were cultured for nanobacteria under cell culture conditions after being filtered with 0.22μm filter. At the same time, full-culture media DMEM, Hap and inactivated nanobacteria roasted for 4h at 180℃were cultured as negative control.1.2 The scanning electron microscope was used for energy dispersive x-ray microanalysis of the cultured nanobacteria and hydroxyapatite.1.3 The morphology of sub-cultured nanobacteria and hydroxyapatite was observed by transmission electron microscopy.1.4 Indirect immunofluorescence staining (IIFS) was used for specific identification of nanobacteria. Detected mineralizer on the surface of nanobacteria by Von KOSSA staining.1.5 Bisbennzimide H 33258 was used for staining DNA of nanobacteria.2 Infective experiment of nanobacteria from bile2.1 Collected blood 5ml from auricular vein of each rabbit for ELISA to eliminate positive rabbits.2.2 Animal infective experiment2.2.1 Group of experiment2.2.1.1 Infective experiment of nanobacteria cultured from the cholelithiasis patient's bile. Forty eight rabbits were randomly assigned to following 3 groups: Nb infected group (16 rabbits); DMEM control group (16 rabbits); Hap control group (16 rabbits).2.2.1.2 Infective experiment of nanobacteria cultured from the rabbit's bile. Forty eight rabbits were randomly assigned to following 3 groups: Nb infected group (16 rabbits); DMEM control group (16 rabbits); Inactivated Nb control group (16 rabbits).2.2.2 Animal infection2.2.2.1 After anesthetizing the rabbits, surgery was performed with a top-right incision as the protocol described bellow: briefly, sought the gallbladder, extracted gallbladder bile, injected the prepared solution, then conglutinated the hole in the gallbladder using fibrin adhesive and closed the incision at last. All the rabbits were injected with Penicillin intramuscular at a dose of 400,000 IU once a day for three days postoperatively and fed in separate cages for 3 weeks.2.2.2.2 Rabbits were euthanized under anesthesia after collecting blood 5ml from auricular vein. Bile samples were collected and then gallbladders were cut and gallstones were collected.?Gallbladder and liver samples were cut into small pieces, and then fixed with 10% formaldehyde and 2.5% separately.2.2.2.3 Obtained pre- and post-experiment bile samples for culture of nanobacteria. 3 Detection of rabbit tissue specimen3.1 HE staining and transmission electron microscopy were used to observe the pathological variation of gallbladder and liver specimen.3.2 Von KOSSA staining and indirect immunofluorescence staining were used to detect nanobacteria infection of gallbladder and liver specimen.3.3 Took the pre-execute blood serum samples for ELISA.4 Statistics methods: Statistically significant differences were assessed by Chi-square test. Statistical analysis of those results was carried out using SPSS software.Results:1. Results of culture for nanobacteria were positive in all the six patients'bile samples and rabbits'bile samples of two infection groups. Results of culture for nanobacteria were negative in all the control groups.2. Nanobacteria were successfully cultured in DMEM with 10%γ-FBS under mammalian cell culture conditions (37℃, 5%CO2). They growed very slowly. After about a two-weeks culture period,tiny particles could be observed making Brownian movement near the bottom of the culture vessel by using phase contrast microscope. After three weeks,nanobacteria started to attach to the bottom of the culture vessel and formed biofilm.At the same time, thin white-colored precipitates could be seen in the cultures by the naked eye. Four weeks later, there were much more white-colored precipitates could be seen. During the cause of culture, the changing of the pH value was not obvious. The sub-cultured nanobacteria take the same characteristic as the original.3. Observing with transmission electron microscopy, nanobacteria revealed 80～350 nm particles. They appeared bacilli or coccoid in shape, predominantly as clusters by attaching to each other with biofilms-like material. Elements of calcium, phosphor, magnesium, aluminum, oxygen, chrome, copper, iron could be detected by EDX microanalysis, and the calcium-phosphate ratio of nanobacteria was 1.62, which was similar to that of hydroxyapatite. 4. Calcified nanobacteria (in biofilms) could be stained as black dots by Von KOSSA method.5. The result of indirect immunofluorescence staining showed that nanobacteria could be binded with fluorescently-labeled antibody, and produce green fluorescence.6. Stained with Bisbenzimide H 33258, nanobacteria showed reaction to the dye by producing the characteristic blue fluorescence, which indicted that DNA exists in nanobacteria.7. In the infective experiment of nanobacteria cultured from the cholelithiasis patient's bile, the gallstone incidence of Nb infected group (81.25%) was significantly greater than that in DMEM control group (6.25%) and Hap control group (18.75%). In the infective experiment of nanobacteria cultured from the rabbit's bile, the gallstone incidence of Nb infected group (56.25%) was significantly greater than that in DMEM control group (6.25%) and Inactivated Nb control group (12.5%).8. HE staining of gallbladder and liver tissue specimens showed that inflammation infiltration occurred in most gallbladders of rabbits infected with nanobacteria (22/32), and the minor changes occurred in liver. Ultramicrostructure of Gallbladder and liver has changed in infected group with TEM. But the ultramicrostructure of control groups was normal.9. Von KOSSA staining results showed small black particles distributed in epithelial cells, smooth muscle cells of nanobacteria- infected gallbladders, while rabbits'gallbladders of DMEM control group, Hap control group and Inactivated Nb control group did not. Nanobacteria with bright green color were clearly detected in epithelial cells and smooth muscle cells of nanobacteria-infected gallbladders by IIFS method. But they were not detected in rabbits'gallbladders of DMEM control group, Hap control group and Inactivated Nb control group.10. Results of ELISA showed that the positive rate of Nb infected group (cultured from the patient's bile) was 100%, while the positive rate of 2 control groups was 0; The positive rate of Nb infected group (cultured from the rabbit's bile) was 100%, while the positive rate of DMEM control group and Inactivated Nb control group was 6.25% and 25% separately.Conclusions:1. Nanobacteria can be isolated from the cholelithiasis patient's bile, and can be subcultured in vitro.2. The size of nanobacteria is super small. They grow slowly under mammalian cell culture conditions and can produce calcified biofilms which are mainly composed of hydroxyapatite to escape damages.3. The culture in vitro of nanobacteria can lead to rabbit gallstone produce.4. Nanobateria can be isolated from gallstone produed rabbit's bile and can infect rabbit again after be subcultured and lead to rabbit gallstone produce.5. Anti-nanobateria antibody could be produced in rabbit's serum after that rabbit was infected with nanobacteria.6. Hyperplasy, atrophy, inflammatory infiltration could occur in rabbit gallbladder and liver which was infected with nanobacteria after 3 weeks.7. The biological characteristics and pathogenicity of nanobacteria cultured from the cholelithiasis patient's bile and infected rabbits'bile are elemental coincidence.