Application of ING4 and IL-24 double-gene coexpression vector as radiotherapy sensitizing agent

A technology of IL-24 and co-expression carrier, which is applied in the field of application of ING4 and IL-24 double-gene co-expression carrier as a radiotherapy sensitizer, which can solve the problems of not being able to act

Inactive Publication Date: 2010-12-15
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report about the application of human ING4 and IL-24 double gene co-expression vector as a tumor radiosensitizer, and it is known to those skilled in the art that chemosensitizing drugs may not be used as radiosensitizing drugs; radiosensitizing effect It is mainly proposed for the part of hypoxic cells that are resistant to radiation in tumors. It refers to certain chemical substances that can enhance the killing effect of radiation on hypoxic cells in tumors and cause less damage to normal aerobic tissues. These chemicals Substances are called radiosensitizers, and commonly used radiosensitizers mainly include misonidazole (MISO), glycididazole sodium (CMNa), flavone acetic acid and oxygen; some drugs can be used as both radiosensitizers and Chemosensitizers, such as misonidazole (MISO), glycidiazole sodium (CMNa), CMNa, cyclophosphamide, fluorouracil, VP-16, bleomycin, mitomycin C, (hydroxy)hi Phytidine, paclitaxel, doxorubicin, cis (carbo)platinum, etc.; however, some drugs can only be used as chemotherapy sensitizers but not radiosensitizers, such as sulfonine, decitabine, etc. can overcome drug resistance and make cancer more stable. good treatment
[0007] Moreover, there is no literature report about the human ING4 and IL-24 dual-gene co-expression vector and its dual-gene encoded protein combined with radiotherapy to play a radiosensitizing role.

Method used

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  • Application of ING4 and IL-24 double-gene coexpression vector as radiotherapy sensitizing agent
  • Application of ING4 and IL-24 double-gene coexpression vector as radiotherapy sensitizing agent
  • Application of ING4 and IL-24 double-gene coexpression vector as radiotherapy sensitizing agent

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] (1) According to the prior art, pAdTrack-CMV-ING4-polyA was obtained from Escherichia coli with the preservation number CCTCC M 208155 Δ296～298 +CMV-IL-24 (abbreviated as pAd-ING4-IL-24); (the Escherichia coli preservation information is: depository unit: China Center for Type Culture Collection; address: Wuhan University, China; preservation date: October 12, 2008 ; Deposit number CCTCC M 208155; Classification name: Escherichia coli DH5α / pAdTrack-CMV-ING4-polyA Δ296～298 +CMV-IL-24; Escherichia coli DH5α / pAdTrack-CMV-ING4-polyA Δ296～298 +CMV-IL-24. );

[0044] According to the prior art, human ING4 and IL-24 double gene co-expression vector plasmid pAdTrack-CMV-ING4-polyA was adopted Δ296～298 +CMV-IL-24 construction of adenovirus-mediated recombinant virus Ad-ING4-polyA Δ296～298 +CMV-IL-24 (referred to as Ad-ING4-IL-24);

[0045] (2) Cell culture: SPC-A1 cells, MDA-MB-231 breast cancer cells use RPMI1640 complete medium (10% FCS) respectively, at 37 ° C, 5% CO 2 ...

Embodiment 2

[0050] Example 2: Ad-ING4-IL-24 recombinant adenovirus infection of SPC-A1 cells

[0051] The SPC-A1 cell line in the logarithmic growth phase was digested with 0.25% trypsin, and made into a cell suspension with RPMI1640. After counting, the cell concentration was adjusted to 1×10 5 / ml, 100μl per well, inoculated in 96-well culture plate, 37°C, 5% CO 2 Incubate overnight. On the next day, the AdV empty vector adenovirus was used to infect SPC-A1 cells in vitro with different doses of 1, 10, 25, 50, 100, and 200 MOI to screen and determine the optimal infectious dose and infect SPC-A1 cells with the optimal infectious dose. The results were as follows: : In the field of view of ordinary light microscope, different doses of AdV empty vector adenovirus infection of SPC-A1 group can be seen at 1, 10, 25, 50, 100 MOI. There was no difference, but the cells in the SPC-A1 group infected with 200 MOI doses shrank and fell off, showing obvious cytotoxicity caused by the adenovirus ...

Embodiment 3

[0052] Example 3: RT-PCR identification of target gene transcription after infection of SPC-A1 lung adenocarcinoma cells by recombinant adenoviruses with different genes

[0053]The cells in the PBS group and the SPC-A1 lung cancer cells were infected with 50 MOI of AdV, Ad-ING4-IL-24, Ad-IL-24, and Ad-ING4 respectively for 72 hours, and the cells were collected by centrifugation at 1500r / min, and washed with PBS for 2 hours. -3 times, total RNA was extracted according to the instructions of the RNA extraction kit, and the first strand of cDNA was obtained by reverse transcription. Amplify the above cDNA template and upstream and downstream primers in a PCR machine. The upstream and downstream primers of IL-24 and ING4 genes are shown in Table 1-1. The PCR conditions are 94°C for 4min, 94°C for 30s, 55°C for 45s, and 72°C for 1min , 30cycle, 72°C 10min. Finally, take 10 μl of the product and perform agarose gel electrophoresis together with DNA Marker. After agarose gel elect...

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Abstract

The invention relates to genomics, molecular biology, cell biology, gene engineering and clinical medicine, in particular to application of a human ING4 and IL-24 double-gene coexpression vector as a radiotherapy sensitizing agent. Before the radiotherapy, the human ING4 and IL-24 double-gene coexpression vector is led in tumor cells so that the human ING4 and IL-24 genes are expressed in the tumor cells, and then the radiotherapy is carried out. The Ad-ING4-polyA<delta 296-298>+CMV-IL-24 can retard lots of tumor cells in the proliferative stage in the G2/M stage and is beneficial to enhancing the irradiation sensitivity. The MTT and the FCM detection results further show that the Ad-ING4-IL-24 combined radiotherapy has the effect of obviously inhibiting the growth of the SPC-A1 lung adenocarcinoma cells, MDA-MB-231 breast carcinoma cells and the transplanted tumor thereof and inducing the cell apoptosis, exceeds the Ad-ING4-IL-24 single genome and the radiotherapy single genome, and presents obvious radiotherapy sensitization synergistic effect. Therefore, the human ING4 and IL-24 double-gene coexpression vector can be used for enhancing the sensitivity of the tumor cells to the radiotherapy.

Description

technical field [0001] The invention relates to genomics, molecular biology, cell biology, genetic engineering and clinical medicine. The invention specifically relates to the application of human ING4 and IL-24 double gene co-expression vector as a chemotherapeutic drug sensitizer. Background technique [0002] Radiation therapy is one of the routine means of tumor treatment, and 70% to 80% of malignant tumor patients need to receive radiotherapy during the course of treatment. Tumor insensitivity or resistance to radiation is one of the main reasons why radiation therapy is difficult to cure malignant tumors. In recent years, with the in-depth research on the molecular mechanism of tumor pathogenesis and the mechanism of cell growth regulation, some exciting results have been achieved in tumor gene therapy, but the transgene therapy targeting a single link often cannot achieve satisfactory therapeutic effects. Weichselbaum et al. proposed a new idea of ​​tumor gene-radiat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/00
Inventor 杨吉成凌春华盛伟华黄锦宏谢宇锋赵大国李正祎
Owner SUZHOU UNIV
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