Combination of epac direct or indirect agonists with oncolytic viruses
A technology of oncolytic virus and agonist, which is applied in the field of joint application of Epac direct or indirect agonist and oncolytic virus, which can solve the problems of slow clearance, restriction of virus replication, narrow anti-tumor spectrum of oncolytic virus, etc.
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Embodiment 1
[0093] Example 1 Epac agonists significantly increase the replication of oncolytic viruses.
[0094] 1) Epac agonists significantly increase the expression of oncolytic virus proteins.
[0095] Material:
[0096] Human colorectal cell carcinoma HCT116, human pancreatic cell carcinoma Capan-1, human immortalized normal liver cell line L-02, M1 virus, high-glucose DMEM medium, db-cAMP (Sigma, USA).
[0097] Western bolt: cell total protein extract ( Mammalian Protein Extraction Reagent, Thermo), E1 antibody (homemade), NS3 antibody (homemade), GAPDH antibody (Bioworld USA);
[0098] method:
[0099] a) Cell culture: human colorectal cancer cell line HCT116, human pancreatic cancer cell line Capan-1, human immortalized normal liver cell line L-02, grown in a medium containing 10% FBS, 100 U / ml penicillin and 0.1 mg / ml streptomycin in DMEM complete medium or primary cell culture medium; all cell lines were placed in 5% CO 2 , cultured and subcultured in a closed incubator wi...
Embodiment 2
[0144] Example 2 Epac agonist and M1 virus selectively cause tumor cell death
[0145] 1) Epac agonist and M1 virus selectively cause tumor cell lesions
[0146] Material:
[0147] Human colorectal cancer HCT116, human immortalized normal liver cell line L-02, M1 virus, high-glucose DMEM medium, inverted phase-contrast microscope.
[0148] method:
[0149] a) Cell culture: human colorectal cancer HCT116, human immortalized normal liver cell line L-02, grown in DMEM complete medium containing 10% FBS, 100 U / ml penicillin and 0.1 mg / ml streptomycin; All cell lines were placed in 5% CO 2 , cultured and subcultured in a closed incubator with a constant temperature of 37°C (95% relative humidity), and observed the growth with an inverted microscope. The cells were subcultured every 2-3 days, and the cells in the logarithmic growth phase were taken for formal experiments.
[0150] b) Observation under the cell microscope: cells in the logarithmic growth phase were selected, and...
Embodiment 3
[0163] Example 3 Epac agonists enhance the oncolytic effect of M1 through Epac1, not PKA
[0164] 1) Interference with PKA cannot cancel the enhanced oncolytic effect of cAMP activation
[0165] Material:
[0166] Human colorectal cancer HCT116, M1 virus, high glucose DMEM medium, MTT, siRNA, RNAiMAX, inverted phase contrast microscope.
[0167] method:
[0168] a) Cell culture: human colorectal cancer HCT116, grown in DMEM complete medium containing 10% FBS, 100U / ml penicillin and 0.1mg / ml streptomycin; cells placed in 5% CO 2 , cultured and subcultured in a closed incubator with a constant temperature of 37°C (95% relative humidity), and observed the growth with an inverted microscope. The cells were subcultured every 2-3 days, and the cells in the logarithmic growth phase were taken for formal experiments.
[0169] b) RNA interference: select logarithmic growth phase cells, DMEM complete culture medium (containing 10% fetal bovine serum, 1% double antibody) to make cell...
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