Anti-tumor virus
A virus, enterovirus technology, applied in the field of tumor treatment
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Embodiment 1
[0049] Embodiment 1: Amplification and purification of CV-A10 virus
[0050] Coxsackievirus A10 (CV-A10) HB09-035 strain was isolated in Hebei Province in 2009. The virus strain was deposited in the China General Microorganism Culture Collection Management Center (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China) with the preservation number CGMCC NO.19294 on January 19, 2020. Human rhabdomyosarcoma cells (rhabdomyosarcoma cell, RDcell, ATCC CCL-136) were cultured in DMEM medium containing 10% fetal bovine serum, 5×10 7 Cells were seeded in 15 cm Petri dishes at 37 °C in 5% CO 2 Incubate for 24 hours. The culture medium was discarded, and CV-A10 virus seed was added according to the multiplicity of infection (MOI=0.1), and the virus seed was diluted with DMEM medium. Continue to cultivate for 36 hours, when the microscope observes 90% cytopathic effect (CPE), collect the supernatant, use TFF systems 300kD concentrates virus and removes cellular debris. ...
Embodiment 2
[0051] Example 2: CV-A10 inhibits the growth of human colon cancer cell line RKO
[0052] Culture RKO colon cancer cell line (ATCC CRL-2577), take 2×10 4 Cells were placed in a 96-well plate and cultured for 8 h. The culture medium was replaced with serum-free DMEM medium, infected with different titers of virus (MOI=0.001-10), continued to culture at 37°C, and measured cell viability at different time points. The viability was determined using the CellTiter-Glo Luminescent CellViability Assay assays (Promega) kit and detected according to the user manual. The result is as Figure 2A As shown, 12 hours after infection with a high virus titer (MOI=10), the cell viability began to decline, and almost all cells died after 48 hours. A lower virus titer (MOI=1) also resulted in the death of almost all cancer cells after 60 h. Other titers of virus produced gradient inhibitory effects at different times. The lowest virus titer (MOI=0.001) also showed inhibition of cell growth a...
Embodiment 3
[0053] Example 3: Intratumoral or intravenous injection of CV-A10 inhibits the growth of solid tumors in mice
[0054]A total of 15 4-week-old NCG mice (17-19g) were selected, and 5×10 6 RKO cells / only. After the tumors were planted, they were raised for 5 days to observe the tumor formation on their backs. When the tumor grows to 120mm 3 Intratumoral injection or intravenous injection of CV-A10 virus was carried out on the 5th, 8th, 11th, 14th, and 17th days, and a control group was set up, with 5 rats in each group. Among them: the intratumoral injection group received intratumoral multi-point injection, and the intravenous injection group received single-needle injection in the tail vein, with a therapeutic dose of 1×10 7 Virus particles / time. In the control group, PBS was used for intratumoral multi-point injection to exclude operational errors. The body weight of the mice was recorded every two days and the long and short diameters of the tumors were measured with a ...
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