| [Objective]As an important component of human intestinal microflora, intestinal fungi play an important role in the regulation of intestinal health. Intestinal tract has a close relationship with liver in respect of anatomy and physiology, and the function of them affects each other directly. In this study, we aim to find out the characteristics of molecular ecological structure of intestinal fungi in hepatitis B patients by using molecular techniques, and to reveal the diversity of fungi in the intestine of patients with hepatitis B infection, as well as the qualitative and quantitative results of intestinal fungi in different states of hepatitis B infection. We intend to recognize the relationship between ecological structure of fungal flora and the stage of hepatitis B infection, to explore the relationship between fungal flora imbalance, endogenous intestinal infection and degree of hepatitis B infection. We try to lay the experimental foundation for the further studying the main mechanisms which affect the process of liver disease, and to look for the measures to block the process of liver disease development.[Methods]1. To study the diversity of intestinal fungal flora of patients with hepatitis B infection, Four groups of HBV-liver cirrhosis patients, chronic hepatitis B patients, HBV carriers, and healthy volunteers were selected as research subjects. Method of QIAamp?DNA Stool Mini Kit-glass beads was used to extract the total DNA from stool of each subject. DNA was amplified using fungal 18S rDNA universal primer, and PCR products were transformed into Escherichia coli cell for the construction of 18S rDNA clone libraries. Technology of restriction fragment length polymorphism (RPLP) was used to select positive clones, followed by sequence analysis. Software such as MEGA4.0 was adopted to construct phylogenetic tree, diversity and structural characteristics of intestinal fungal flora in each group was then analyzed.2. An accurate and reliable real-time quantitative PCR method was set up to monitor intestinal fungi. Fungus-specific primers were designed for the quantitative analysis of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Saccharomyces cerevisiae in intestine, then quantitative results of various fungal species in different group were compared and analyzed.[Results]1. A total of 27 operational taxonomic units (OTUs) of intestinal fungal flora in all research subjects was acquired after RFLP analysis and sequencing of positive clones. All positive clones belonged to three fungal taxa:Zygomycetes (3.7%), ascomycetes (81.5%) and Basidiomycetes (14.8%). The largest clone number of fungal species detected was Candida spp.( 44.7% of the total number of clones), then was Saccharomyces spp. (14.0% of the total number of clones) and uncultured fungi (16.8% of the total number of clones). The proportion of clones which belonged to Candida spp. and Saccharomyces spp. was higher in patients with hepatitis B infection than that in healthy volunteers, while the proportion of clones which belonged to uncultured fungi was lower in patients with hepatitis B infection than that in healthy volunteers. The OTUs which belonged to patients with HBV-liver cirrhosis, chronic hepatitis B patients, HBV carriers, and healthy volunteers was 18,17,12,13 respectively. Diversity of intestinal fungal flora in patients with HBV-liver cirrhosis and chronic hepatitis B patients was greater than that in HBV carriers and healthy volunteers.2. The quantity of various common fungal species in intestine changed in patients with different stage of hepatitis B infection. Except Candida parapsilosis, the quantity of intestinal Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Saccharomyces cerevisiae was significant different among four groups. The quantity of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Saccharomyces cerevisiae in patients with HBV-liver cirrhosis was significantly increased than that in healthy volunteers (P <0.05). The quantity of Candida albicans, Candida krusei, Candida tropicalis and Saccharomyces cerevisiae in patients with chronic hepatitis B was significantly increased than that in healthy volunteers (P <0.05).[Conclusion]1. There is an abundant fungal flora in the human intestine. Diversity of intestinal fungal flora appears to be various in patients with different stage of hepatitis B infection. Higher diversity of intestinal fungal flora was found in patients with HBV-liver cirrhosis and chronic hepatitis B compared with that in patients with HBV carriers and healthy volunteers. Intestinal fungal flora is dominated by Candida spp. and Saccharomyces spp. in all research subjects. The results indicate the close relationship between the alteration of ecological structure of fungal flora and the stage of hepatitis B infection.2. Real-time quantitative PCR technology can be utilized accurately and rapidly for quantitative analysis of common fungal species in the human intestine. That provides a new means of detecting and monitoring intestinal fungi for the prevention of fungal infections in patients with liver disease.3. The ecological balance of intestinal fungal flora is disrupted in patients with hepatitis B infection. There exists a certain correlation between the imbalance of intestinal fungi flora and degree of hepatitis B infection. |
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