Radioresistant lung cancer whole-cell vaccine, and preparation method and application thereof
A whole-cell vaccine and lung cancer cell technology, which is applied in the field of radiation-resistant lung cancer whole-cell vaccine and its preparation, can solve the problem of not being able to improve the immune function of T lymphocytes in C57BL/6 mice, achieve a durable and specific anti-tumor effect, reduce Protein expression, the effect of promoting cell apoptosis
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Embodiment 1
[0043] Example 1 Establishment of the radioresistant lung cancer cell line of the present invention
[0044] 1 Cell culture and irradiation method
[0045] LLC Lewis lung cancer cell line (abbreviated as LLC cells, purchased from ATCC) was placed in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 The cells were cultured in an incubator with saturated humidity, and the cells were subcultured to the exponential growth phase. 6MV X-rays were irradiated with a linear accelerator, the source-to-skin distance was 100cm, and the surface was covered with 1cm-thick tissue compensation glue.
[0046] 2 Establishment of radioresistant cell line R-LLC
[0047] Take LLC cells in the exponential growth phase, digest them into a single cell suspension and inoculate them on 25cm 2 In the culture flask, after the cells grow to the exponential growth phase, the pre-irradiation field is 7cm×7cm, 6MV X-rays, the absorbed dose rate is 200cGy / min, 5Gy is irradiated, and then th...
Embodiment 2
[0068] Example 2 Preparation of the radioresistant lung cancer whole cell vaccine of the present invention
[0069] For the R-LLC cells obtained by repeated radiation irradiation for routine passage>10 passages, the R-LLC cells in the exponential growth phase were collected, the culture supernatant was collected, counted on the counting plate, and the cell concentration was adjusted to 5.0×10 with the culture supernatant. 6 cells / ml, first inactivate the cells by repeated freezing and thawing (freeze in liquid nitrogen for 10 min, then quickly transfer to a 37°C water bath for 10 min, repeat four times), and crush them with an ultrasonic cell pulverizer on ice (power 130W / 20KHz, time 3s , 10-15 times) cells, the vortex shaker fully oscillated to prepare tumor seedlings, and the final concentration of tumor seedlings was 1.0×10 6 Individual tumor cells / 0.2ml.
Embodiment 3
[0070] Example 3 Anti-tumor effect of CpG ODN1826 combined with radioresistant lung cancer whole cell vaccine of the present invention
[0071] 1 method
[0072] 1.1 Immunization method and experimental grouping
[0073] There were 20 mice in each group. The mice in each group were first immunized with CpG ODN1826 (purchased from Shanghai Sangon Bioengineering Co., Ltd., product number: 9103288756) three times, and then immunized with tumor vaccine three times. Repeated immunization every day, divided into the following six groups according to different immunization methods:
[0074] (a) Negative control group (Control): Each mouse was subcutaneously injected with 0.15ml of PBS as the CpGODN1826 drug control, and then subcutaneously injected with 0.2ml of serum-free culture medium at multiple points as the tumor vaccine control;
[0075] (b) CpG ODN group (CpG): Each mouse was subcutaneously injected with CpG ODN1826 0.15 mg each time, and then subcutaneously injected with s...
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