A Fusobacterium nucleatum subsp. animal strain and its application
A Fusobacterium nucleatum, animal technology, applied in the direction of animal cells, non-animal cells, vertebrate cells, etc., can solve the problem of not being able to reflect the biological characteristics of Fusobacterium nucleatum, and achieve the effect of promoting proliferation
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Embodiment 1
[0031] To isolate Fusobacterium nucleatum subsp. animal strain THCT5A4, the specific steps are as follows:
[0032] Step 1: Take 1-2mm from the surgical resection specimen of rectal cancer 3 Tumor tissue blocks, placed in a sterile tube, transported and stored at 4°C under anaerobic conditions;
[0033] Step 2: Bacteria isolation was carried out within 4 hours. Place the tissue block in 500 μL trypticase soybean broth medium containing 0.05% cysteine, break it up mechanically quickly, dilute 100 times with the same medium, and take 100 μL Spread the diluted solution on a Columbia blood plate and incubate anaerobically at 37°C for 48 hours;
[0034] Step 3: Pick a single colony from the plate, streak and dilute it on a Columbia blood plate, culture it anaerobically at 37°C for 48 hours, pick a single colony from it and repeat the above steps to isolate and purify the strain;
[0035] Step 4: spread the obtained single colony on the Columbia blood plate, culture it anaerobical...
Embodiment 2
[0037] THCT5A4 was co-cultured with colorectal cancer HCT116 cell line, and the specific operation steps were as follows:
[0038] The cultured HCT116 cells were taken, digested with trypsin to prepare cell suspension, and seeded into 24-well plates (1×10 5 cells / well), cultured for 12-24 hours after seeding until the cells adhered to the wall. After adhering to the wall, wash with PBS and replace the medium without anti-antibody. The experimental group was added with freshly prepared THCT5A4 bacterial suspension, 1×10 8 CFU / well (use PBS to suspend bacteria); add an equal volume of PBS to the control group. Three replicate wells were made for each group, and cultured under normal conditions. After culturing for 6 hours, 24 hours, and 48 hours, the CCK8 method was used to detect the cell proliferation activity according to the steps given in the instructions. The bacterial suspension was replaced every 24 hours.
[0039] result( figure 1 ) showed that THCT5A4 could sign...
Embodiment 3
[0041] THCT5A4 was co-cultured with colorectal cancer LoVo cell line, and the specific operation steps were as follows:
[0042] The cultured LoVo cell suspension was prepared by trypsinization, counted and inoculated into 24-well plates (1×10 5 cells / well), cultured for 12-24 hours until the cells adhered. After adhering to the wall, wash with PBS and replace the medium without anti-antibody. Freshly prepared THCT5A4 bacteria were suspended in PBS, and the bacterial suspension was added to the experimental group (1×10 8 CFU / well); the control group was added with an equal volume of PBS. Three replicate holes were made for each group. cultured under normal conditions. The cell proliferation activity was detected by CCK8 method at 6 hours, 24 hours, 48 hours, and 72 hours of culture respectively. The bacterial suspension was replaced every 24 hours.
[0043] Depend on figure 2 It can be seen that the proliferation activity of LoVo cells at 72 hours was significantly h...
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