Characteristics Of Nanobacteria And Their Possible Role In Gallbladder Stone Formation

Objectives: Nanobacteria and Escherichia coli in the stones werecultured and identified. Statistical analysis was carried out in the results.Observation of their interaction after mixed culture was done. Thepurpose of this study is to discover the evidence of nanobacteria andEscherichia coli co-existence, interaction and coordination of stoneformation in cholecystolithiasis patients and to offer a new research fieldabout the formation of the gallbladder stone.Methods: The samples of stone from 45 Cholecystolithiasis patientswere divided into two groups.The samples in Group one were culturedand observed by optic microscope. And the samples were identified bymonoclonal antibody immunofluorescent staining, Dahl McGec-Russelcalcium-alizarin red staining and checked by electronic microscope, tofind the morphological evidence of nanobacteria and judge the infection.The samples of stone in Group two were cultured and identified to judgethe Escherichia coli infection. Calculate the infection rate of nanobacteriaand Escherichia coli from the samples of stone in 45 cholecystolithiasispatients, and use the Kappa consistency method to find the symbiotic evidence of these two bacterias in the formation of gallbladder stone.Nanobacteria and Escherichia coli were mixed cultured and thenidentified by Dahl McGec-Russel calcium-alizarin red staining, observedby electronic microscope and determined byβ-glucuronidase, to find outthe path-physiologic mechanism of their interaction.Results: The infective rate of nanobacteria was 40% when the 45stone samples identified by immunofluorescence staining and checked bymicroscope. There were 21 positive samples when identified by calcificstaining, the positive rate of which is 46.7%. The infective rate of theEscherichia coli was 53.3% in the 45 stone samples of thecholecystolithiasis patients. 16 of the 45 gallstone samples of thecholecystolithiasis patients were found infected with both nanobacteriaand Escherichia coli. Kappa consistency check revealed a high consistentrate of co-infection. The mixed culture of the nanobacteria and theEscherichia coli led to the mineralization of the Escherichia coli whichwere positive with calcific staining. The mixed culture could not lead toan extra excretion ofβ-glucuronidase in Escherichia coli.Conclusions: There are nanobacteria infections in the gallbladderstones. There are Escherichia coli infections in the gallbladder stones.The incidence of nanobacteria and Escherichia coli co-infection is highand they may both participate in the formation of gallstones. Nanobacteria is a novel apatite mineral-forming bacteria which can leadto the mineralization of Escherichia coli in mixed culture. There is nosign that nanobacteria can promote the secretory of enzymeβ-glucuronidase in Escherichia coli, which is helpful to the formation ofgallbladder stone. Objectives: Make the research on nanobacteria's invasion towardhuman gallbladder epithelial cells after successful separation and cultureof human gallbladder epithelial cells.Methods: The human gallbladder epithelial cells were isolated,cultured and identified in suitable method and appropriate vitro condition.Then they were divided into two groups, the nanobacteria Se90 andhuman gallbladder epithelial cells were mixed cultured in the first group,compared with the control group of gallbladder epithelial cells as thesecond group. Through the optical microscope the morphologicalchanges of human gallbladder epithelial cells were observed after theattack of nanobacteria in different concentrations. The cells' survival andgrowth after the attack was assayed through tetrazolium colorimetric. Thespatial relations between nano-bacteria and epithelial cells were observedat different time through immunofluorescence. The gallbladder epithelialcells' ultrastructural changes after the attack were observed throughTransmission Electron Microscope (TEM).Results: Through blunt layering, we successfully conducted of thehuman gallbladder epithelial cells in primary culture which were put inDMEM culture medium supplemented with 20% FBS and placed in CO₂incubator at 37℃nd 5% CO₂ under saturated humidity. After the mixedculture of nanobacteria Se90 and human gallbladder epithelial cells, the gallbladder epithelial cells attacked were observed through opticalmicroscope along with the process of time, which began to collapse,edema, separate and then broke. More over, the nuclear dissolved anddisappeared, and the degree of damage was worsened with the increasingconcentration and time. Immunofluorescence staining showed thosenanobacteria could enter the gallbladder epithelial cells in a relativelyshort period of time, and as time goes by, the nanobacteria into thegallbladder epithelial cells increased, until accumulated into the nuclear.With the electron microscope we could observe the ultrastructural andfind that the rich microvilli on the gallbladder epithelial cells' surfacereduced and the mitochondrial were in edema obviously after the attackof nanobacteria, more over, it induced to the breakdown of gallbladderepithelial cells.Conclusion: Under appropriate method, the gallbladder epithelialcells can be effectively primarily cultured. And the nanobacteria canquickly enter the cells and injure them, causing a series of changes fromultrastructural morphology to the whole. The higher concentration ofbacteria was, the more serious the injury was. The mode of death of thegallbladder epithelial cells during a relatively short period of time (48hours) is more like necrosis rather than apoptosis. Objectives: The purpose of this study is to explore the damagemechanisms of Nanobacteria on human gallbladder epithelial cells fromthe perspective of inflammatory cytokines and free radicals.Methods: The concentration level of nanobacteria was 0.5Mcfarland. Using kit instruction, RT-PCR or ELISA, the LDH, MDA,IL-1β,IL-6 levels were measured in the medium at different time, afterNanobacteria attacked human gallbladder epithelial cells.Results: The activity of LDH was increased with time and there issignificant difference between each group in 72 hours (P＜0.01). Thelevel of MDA had no remarkable change between each group in 72 hours(P＞0.05).The levels of IL-6 was increased with time and there issignificant difference between each group in 72 hours (P＜0.01). Theexpression of IL-1βmRNA increased significantly after cells beingattacked. There is significant difference between each group (P＜0.01).Conclusions: A large number of inflammatory mediators can beproduced after Nanobacteria strain Se90 attacked gallbladder epithelialcells. This damage may be not relevant to free radicals but to thesecretory activation of a large number of inflammatory cytokines.