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Fusobacterium nucleatum subsp.animalis strain and application thereof

A technology of Fusobacterium nucleatum and strain, applied in animal cells, non-animal cells, vertebrate cells, etc., can solve problems such as the inability to reflect the biological characteristics of Fusobacterium nucleatum, and achieve the effect of promoting proliferation

Active Publication Date: 2020-05-29
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the strains currently used for colorectal cancer research may not reflect the actual biology of F. nucleatum in the gut
However, Fusobacterium nucleatum isolated from colorectal cancer tumors, especially animal subspecies, has rarely been reported.

Method used

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  • Fusobacterium nucleatum subsp.animalis strain and application thereof
  • Fusobacterium nucleatum subsp.animalis strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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, and the tissue block was placed in 500 μL trypticase soybean broth medium containing 0.05% cysteine, quickly crushed mechanically, diluted 100 times with the same medium, and 100 μL was taken 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 ...

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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Abstract

The invention provides a fusobacterium nucleatum subsp.animalis strain THCT5A4 separated from human rectal cancer tumor tissue. A classification name is Fusobacterium nucleatum subsp.animalis THCT5A4,a preservation number of the fusobacterium nucleatum subsp.animalis strain THCT5A4 separated from the human rectal cancer tumor tissue is CCTCC NO: M 2019366, the preservation date is May 17th, 2019,the preservation organization is China General Microbiological Culture Collection Center, and a 16SrRNA gene sequence of the fusobacterium nucleatum subsp.animalis strain THCT5A4 separated from the human rectal cancer tumor tissue is shown in SEQ ID NO:1 as shown in the description. A separation method of the fusobacterium nucleatum subsp.animalis strain is provided. A result of co-culture of a colorectal cancer cell line and the THCT5A4 shows that the THCT5A4 can significantly promote proliferation of colorectal cancer cells, therefore, the THCT5A4 can provide diverse in-vitro or in-vivo experimental conditions for simulating an intestinal environment for studies of colorectal cancer, and a colorectal cancer disease model can be built for screening medicines for treating colorectal cancer.

Description

technical field [0001] The invention relates to the field of Fusobacterium nucleatum subsp. animal, in particular to a strain of Fusobacterium nucleatum subsp. animal and application thereof. Background technique [0002] Colorectal cancer is a common malignant tumor in clinic. According to the data of global cancer cases in 2012, the World Health Organization pointed out that among all malignant tumors, the incidence of colorectal cancer ranks second among women and third among men; the overall mortality rate ranks fourth among the causes of cancer death. [0003] Studies have found that intestinal flora imbalance is associated with the occurrence and development of colorectal cancer. Some bacteria, such as Streptococcus bovis, Helicobacter pylori, Bacteroides fragilis, Enterococcus faecalis, Clostridium spp, Fusobacterium nucleatum, Escherichia coli, etc., have been found to be significantly correlated with the development of colorectal cancer. Among them, Fusobacterium ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12N5/09C12Q1/02C12R1/01
CPCC12N1/02C12N5/0679C12N5/0693C12N2502/70C12N2503/02C12Q1/025C12N1/205C12R2001/01G01N33/5011G01N2333/195G01N2500/10
Inventor 秦环龙蔚青毕德玺李豪
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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