Method for establishing HT-29 cell inflammatory model and application
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A technology of HT-29, an inflammation model, applied in the biological field, can solve the problem that the mechanism of action needs to be studied, and achieve a good therapeutic effect and a long time-consuming effect.
Inactive Publication Date: 2019-01-25
FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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A large number of animal model experiments have shown that the probiotic agent VSL#3 can alleviate DSS (dextran sodium sulfate)-induced ulcerative colitis in rats, but the specific mechanism of action remains to be studied
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Embodiment 1
[0023] A kind of establishment method of HT-29 cell inflammation model
[0024] 1. Cell culture
[0025] HT-29 cell culture medium: DMEM high glucose medium added with 10% FBS; culture conditions: carbon dioxide concentration 5%, temperature 37°C.
[0026] 2. Cell Processing
[0027] Inoculate HT-29 cells in a 24-well plate and culture overnight. After the cells grow on the wall, replace the medium and add human TNF-α (20mg / ml). After co-cultivating for 12 hours, add LPS to the medium (1 μg / ml), cultivated for 15 hours.
[0028] 3. Result detection
[0029] 3.1 Real-time fluorescent quantitative PCR (real-time PCR) detection of TLR4, NF-κB, TNF-α and IL-1β mRNA expression in HT-29 cells
[0030] 3.1.1 Extraction of total cellular RNA
[0031] (1) After digesting the cells with trypsin, transfer the cell suspension together to a nuclease-free centrifuge tube, centrifuge at 500g, 4°C for 5 minutes, discard the supernatant, and collect the precipitated cells;
[0032] (2) A...
Embodiment 2
[0094] Effect of probiotic agent VSL#3 on HT-29 cell inflammation model
[0095] 1. Implementation steps
[0096] The cells were plated in T-25 flasks, and after the cells adhered to the wall, they were processed according to the following groups:
[0097] Group A: Blank control group: 90% DMEM high glucose medium + 10% FBS cultured for 24 hours;
[0098] Group B: model control group: after giving Human TNF-α (20mg / ml) for incubation for 12 hours, then giving LPS (1ug / ml) for incubation for 15 hours;
[0099] Group C: VSL#3 treatment group: after the same treatment as the model group, probiotic agent VSL#3 (1×10 5 cfu / ml) were co-incubated for 7 days.
[0100] 2. Test results
[0101] According to the detection steps of Example 1, the mRNA and protein of HT-29 cells were extracted respectively, and the expression levels of TLR4, NF-κB, TNF-α and IL-1β in cells were detected and compared. The result looks like this:
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Abstract
The invention relates to the field of biotechnology, in particular to a method for establishing an HT-29 cell inflammatory model and application. The invention comprises two steps: cell treatment andresult detection. The cell processing step comprises the following steps: adding HT-29 cells are seeded on 24-well plates and cultured overnight, after the cells adhere to the wall, the culture mediumis changed, the human source TNF-alpha is added, the final concentration is 20mg/ml, after 12 hours of co-culture, LPS is added into the culture medium, the final concentration is 1 [mu]g/ml, and theculture time is 15 hours. The result detection includes expressing detection of protein and mRNA of TLR4, NF-kB, TNF-alpha and IL-1beta in treated HT-29 cells.. The invention establishes HT-29 cell inflammatory model, which provides a screening method at that drug cell level for the treatment or symptom relief of ulcerative colitis, and provides a method step for the treatment mechanism researchof the drug with therapeutic effect on the animal ulcerative colitis model.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for establishing an HT-29 cell inflammation model and its application. Background technique [0002] Ulcerative colitis (Ulcerative colitis, UC) is a chronic non-specific colonic inflammation, which belongs to a kind of inflammatory bowel disease (Inflammatory bowel disease, IBD), mainly manifested as abdominal pain, diarrhea, blood in the stool, etc. Most of the lesions develop from the distal segment of the colon to the proximal segment, and in severe cases, the entire colon and the terminal ileum may be involved. Ulcerative colitis cannot be completely cured at present, and the repeated episodes of colon inflammation seriously affect the quality of life of patients, and it can further develop into colon cancer. The etiology and pathogenesis of UC are unclear, but it involves at least three interacting elements: genetic susceptibility, abnormal immune regulation, and chan...
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