Nanovesicles derived from bacteria of genus sphingomonas and uses of same
A technology of sphingomonas and vesicles, which is applied in the direction of bacterial medical raw materials, cosmetic preparations, skin care preparations, etc., and can solve problems such as diagnosis and treatment that have not been reported
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Embodiment 1
[0102] Example 1. Analysis of in vivo absorption, distribution and excretion patterns of bacteria and bacteria-derived vesicles
[0103] To assess whether bacteria and vesicles derived from bacteria are absorbed systemically through the gastrointestinal tract, the following experiments were performed. First, a dose of 50 μg each of fluorescently labeled bacteria and bacteria-derived vesicles was orally administered to the stomach of mice, and fluorescence was measured after 0 min, 5 min, 3 h, 6 h and 12 h. Observe the whole image of the mouse as Figure 1A As shown, the results showed that the bacteria were not absorbed systemically, but the vesicles derived from the bacteria were absorbed systemically at 5 minutes after administration, and obvious fluorescence was observed in the bladder 3 hours after administration, so it can be seen that the vesicles is excreted into the urethra. Furthermore, it can be seen that vesicles are present in vivo until 12 hours after administrat...
Embodiment 2
[0105] Example 2. Evaluation of Bacteria and Bacteria-Derived Vesicles Penetrating the Protective Membrane of the Intestinal Mucosa
[0106] To assess whether bacteria and bacteria-derived vesicles cross the protective mucosal membrane to infiltrate tissues, after direct administration of bacteria and bacteria-derived vesicles to the intestine, the ability to pass through the protective mucosal membrane was evaluated by immunohistochemistry. Permeability of intestinal tissue. To assess the presence of bacteria and vesicles in mucosal tissues, antibodies against bacteria and vesicles were prepared, attached to green fluorescent protein (GFP) and used, and in combination with 4,6-diamidino 2-phenyl After indole (DAPI) staining, it was observed under a microscope.
[0107] As a result, it was demonstrated that bacteria failed to pass through the protective membrane of the mucosa, whereas vesicles derived from bacteria passed through the mucosa and penetrated into the intestinal ...
Embodiment 3
[0108] Example 3. Metagenomic Analysis of Bacterial-Derived Vesicles in Clinical Samples
[0109] Blood or urine was first placed in a 10 ml tube and the suspension was pelleted by centrifugation (3,500 xg, 10 min, 4°C), then only the supernatant was transferred to a new 10 ml tube. After removing bacteria and impurities using a 0.22-μm filter, transfer them to a Centriprep tube (50kD centrifugal filter) and centrifuge at 1,500×g and 4°C for 15 minutes, discard the material smaller than 50kD, and the residue Concentrate to 10ml. After bacteria and impurities were removed again using a 0.22-μm filter, ultracentrifugation was performed at 150,000 × g and 4 °C for 3 h using a 90Ti type rotor, the supernatant was discarded, and the aggregated precipitate was dissolved in saline ( PBS).
[0110] Internal DNA was extracted from lipids by boiling 100 μl of vesicles isolated by the method described above at 100 °C, followed by cooling on ice for 5 min. Then, in order to remove the ...
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