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Method for inhibiting proliferation and inducing apoptosis of cancer cells and use thereof

A cancer cell, apoptosis technology, applied in inhibiting the proliferation of cancer cells and inducing their apoptosis and its application fields, can solve the problems of not being suitable for biological missiles, not biological missiles, affecting the therapeutic effect, etc., to reduce the impact, reduce side effects, Improves the effect of gene silencing effects

Active Publication Date: 2010-12-01
SUN YAT SEN UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] For breast cancer, what is the ideal bio-missile that can bind to the Her2 receptor on the surface of breast cancer cells and carry RNAi? Although the Her2 ligand can bind to its receptor, it activates the receptor after binding, transmits the proliferation signal, and stimulates the division and metastasis of cancer cells, so it is not an ideal biological missile
Anti-Her2 monoclonal antibody will not activate the receptor, but it is a macromolecular protein, it is difficult to complex with anticancer drugs or genetic material, and it has strong immunogenicity, causing an immune response against the antibody itself, affecting treatment effect, so it should not be used as a biological missile

Method used

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  • Method for inhibiting proliferation and inducing apoptosis of cancer cells and use thereof
  • Method for inhibiting proliferation and inducing apoptosis of cancer cells and use thereof
  • Method for inhibiting proliferation and inducing apoptosis of cancer cells and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Cell recovery and subculture

[0043] Cell recovery is performed as follows:

[0044] 1. Take out the cryopreservation tubes of breast cancer SKBR3, BT 474 and MDA-MB 231 cells from liquid nitrogen or cryogenic refrigerators and put them directly into warm water at 37°C. Keep the top of the cryovials above the water surface to avoid any contamination and shake gently Let its contents melt as soon as possible.

[0045] 2. Take out the cryopreservation tube from the water bath at 37°C, centrifuge at 800rpm×2min, disinfect it with alcohol and open it, suck out the supernatant with a dropper, inject 1ml of basic culture medium and mix well, then transfer it to a graduated centrifuge tube to make a cell suspension Centrifuge at 800 rpm for 2 min at low speed, remove the supernatant, and wash with culture medium again if necessary.

[0046] 3. After appropriate dilution with RPMI 1640, L15 and DMEM / F12 culture medium containing 10% FBS, transfer the inoculated ce...

Embodiment 2

[0053] Example 2: Transfection of cell seed plate for MTT experiment

[0054] The steps of MTT experiment for cell plate transfection are as follows:

[0055] 1. Human breast cancer cells SKBR3, BT 474 and MDA-MB 231 covered 95% of the bottom of the bottle and then digested with 0.25% trypsin;

[0056] 2. Count the cell suspension by blowing it evenly, and calculate the number of cells per milliliter of the suspension;

[0057] 3. Plant the cells in a 96-well plate, the number of cells per well is about 7000 MDA-MB 231 cells and 10000 BT-474 and SKBR3 cells;

[0058] 4. Carry out transfection experiment after 24 hours;

[0059] 5. Replace with RPMI 1640, L15 and DMEM / F12 fresh medium containing 10% FBS (fetal bovine serum);

[0060] 6. Set each processing factor and add corresponding reagents;

[0061] Control (optimized medium), liposomal Lipofectamine 2000+PLK1siRNA, F5-P1+PLK1siRNA, F5-P2+PLK1siRNA, F5-P3+PLK1siRNA, F5-P3, PLKsiRNA and F5-P3+NC-siRNA

[0062] Wherein, ...

Embodiment 3

[0074] Example 3: Cell seed plate transfection 3 H thymidine incorporation assay

[0075] The operation steps are as follows:

[0076] 1. Human breast cancer cells SKBR3, BT 474 and MDA-MB 231 covered 95% of the bottom of the bottle and then digested with 0.25% trypsin;

[0077] 2. Count the cell suspension by blowing it evenly, and calculate the number of cells per milliliter of the suspension;

[0078] 3. Seed the cells in a 96-well plate, the number of cells per well is about 7000 MDA-MB 231 cells and 10000 BT-474 and SKBR3 cells;

[0079] 4. Carry out transfection experiment after 24 hours;

[0080] 5. Replace with fresh medium;

[0081] 6. Set each processing factor and add corresponding reagents;

[0082] Control (optimized medium), Lipofectamine2000+PLK1siRNA, F5-P1+PLK1siRNA, F5-P2+PLK1siRNA, F5-P3+PLK1siRNA, F5-P3, PLK1siRNA and F5-P3+NC-siRNA

[0083] 7. After adding the corresponding reagents to each group, the mixture was incubated on ice for 30 minutes;

[...

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Abstract

The invention provides a method for inhibiting proliferation and inducing apoptosis of cancer cells, which inhibits the proliferation and induces the apoptosis of the cancer cells by using the siRNA of the targeted transportation gene of a protamine polypeptide fusion protein which is a single-chain fragment antibody. The invention also provides the protamine polypeptide fusion protein and a medicament for inhibiting the proliferation and inducing the apoptosis of the cancer cells. The fusion protein can inhibit the proliferation and induce the apoptosis of the cancer cells. The medicament contains an effective amount of the fusion protein and the siRNA of a tumor gene. In the invention, the siRNA of the tumor gene can be transferred to a specific cell population needing gene silencing to improve treatment effect, the RNAi has high pertinence, and the gene silencing effect is improved with the reduction in side effects. It is expected to block the expression of a cancer gene on a genetic level, destruct the proliferation of the cancer cells and a transfer signal transmission mechanism radically, and improve the sensitivity of the cancer cells to chemotherapeutics.

Description

technical field [0001] The present invention relates to a method for inhibiting the proliferation of cancer cells and inducing its apoptosis and its application, in particular to a method for using a fusion protein of a single-chain fragment antibody-protamine polypeptide to target delivery of tumor gene siRNA and its application. Background technique [0002] Traditional antisense gene methods include specific antisense oligonucleotides and ribozymes (ribozymes), but because these traditional antisense gene methods are weak in inhibiting gene expression, they cannot meet the needs of clinical applications. [0003] In 2001, Tuchl et al. introduced artificially synthesized exogenous siRNA with a length of about 19-23 base pairs into mammalian cells, which can induce RNA interference (RNAi) effect that specifically inhibits the expression of complementary sequence genes. After the report was published, there was an upsurge in the study of RNAi. Since then, siRNA has not only...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/42A61P35/00A61P15/14
Inventor 宋尔卫姚燕丹王均张佩琢
Owner SUN YAT SEN UNIV
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