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Double-chain siRNA molecule and application thereof

A molecular and antisense chain technology, applied in the field of biomedicine, can solve the problems of tumor treatment that plagues the medical field and the lack of a radical cure, and achieve the effects of inhibiting tumor proliferation and metastasis, realizing gene silencing, and obtaining gene silencing efficiency

Inactive Publication Date: 2014-04-02
首都儿科研究所 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The treatment of tumors is a worldwide problem that has plagued the medical field for a long time. Although various treatment methods continue to emerge, there is no really effective radical cure at present.

Method used

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  • Double-chain siRNA molecule and application thereof
  • Double-chain siRNA molecule and application thereof
  • Double-chain siRNA molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Detection experiment of chemically synthesized siRNA on survivin gene silencing effect

[0044] 1. Main instruments, reagents and materials.

[0045] 1.1 Instruments: Nucleic acid synthesizer (GE Company), PCR instrument (ABI Company); real-time quantitative PCR instrument (Bio-Rad); cell incubator (Thermo), etc.

[0046] 1.2 Materials and reagents: RFect siRNA transfection reagent (BIO-TRAN), DMEM medium (Gibco), TurboCapture mRNA kit (QIAGEN), SensiMix TM one-step kit (Quantace), etc. Other biochemical reagents were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0047] 1-3PCR primers:

[0048] survivin forward primer: 5′-cagtgtttcttctgcttcaagg-3′,

[0049] survivin reverse primer: 5′-CTTTTTATGTTCCCTCTATGGGGTC-3′,

[0050] GAPDH forward primer; 5′-CATCAGCAATGCCTCCTGCAC-3′,

[0051] GAPDH reverse primer: 5'-TGAGTCCTTCCACGATACCAAAGTT-3'.

[0052] 2. Preparation of siRNA by chemical synthesis

[0053] Design small molecule...

Embodiment 2

[0065] Example 2: Inhibition of tumor cells by survivn gene silencing

[0066] According to the following steps, the inhibition of tumor cells by the siRNA molecule s-siRNA-1 prepared in Example 1 was detected by the MTT method:

[0067] Cell growth was detected by MTT assay. Divide the cells into 1×10 4 Inoculate / well into a 24-well cell culture plate at 37°C, 5% CO 2 Cells were cultured in an incubator for 24 hours, in DMEM medium containing 10% FBS without double antibody, and transfected according to RNAiMAX TM instructions for transfection. After incubation at 37°C for 48h, 30 μL of MTT (5 mg / mL) was added to each well, and incubation was continued at 37°C for 4h. Aspirate the medium containing MTT and add 200 μL dimethylsulfoxide (DMSO) to dissolve the formazan crystals. Then, its absorbance value was measured at 492 nm.

[0068] The small interfering RNA (s-siRNA-1) acts at a concentration of 25nM to 100nM for 48 hours, and the inhibition rate of the tumor cells d...

Embodiment 3

[0073] Example 3: Western blot detection of siRNA inhibition of survivin expression

[0074] According to the following steps, the inhibitory rate of the siRNA molecule s-siRNA-1 prepared in Example 1 to tumor cells was detected by Western blot method:

[0075] 48 hours after the cells (MGC-803, Hep G2 and SMMC-7721) were transfected, the 6-well plate was taken out, the culture medium was blotted dry, and 200 μL of trypsin solution was added. After the cells were digested, 1 mL of PBS solution was added, and the cells were washed down by repeated pipetting. Transfer the cell suspension into a 1.5mL centrifuge tube, centrifuge at 2,000r / min for 3min to collect the cell pellet, add 50μL of pre-cooled cell lysate, mix the cells thoroughly by pipetting repeatedly, place in an ice bath for 40min, and centrifuge at 10,000r / min at 4°C for 10min , take the supernatant and store it at -80°C. The protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), 15% separating...

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Abstract

The invention discloses a double-chain siRNA molecule and application thereof, wherein, the double-chain siRNA molecule is composed of a sense strand and an antisense strand with following nucleotide sequences, and the sense strand is 5'-GCAUCUCUACAUUCAAGAA-3' (SEQ ID NO:1), and the antisense strand is 5'-UUCUUGAAUGUAGAGAUGC-3'(SEQ ID NO:2). The double-chain siRNA molecule can interfere with the post-transcriptional expression of survivin gene targetedly, thereby inducing apoptosis of tumor cells, inhibiting mitosis of tumor cells, inhibiting blood vessel formation during tumor metastasis and reducing survivability of tumor cells during radiotherapy and chemotherapy, and the product is suitable for preparation of antitumor drug.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to double-stranded siRNA molecules and applications thereof, more specifically, the present invention relates to two double-stranded siRNA molecules, plasmids and their applications in the preparation of antitumor drugs, Survivin gene silencing kits and drug. Background technique [0002] The treatment of tumors is a worldwide problem that has plagued the medical field for a long time. Although various treatment methods are emerging, there is no really effective radical cure. Therefore, this also forces scientific researchers to constantly look for new technologies and methods to try to inhibit tumor proliferation and metastasis, and then fundamentally overcome cancer. Contents of the invention [0003] The present invention has been accomplished based on the following findings of the inventors: [0004] The treatment of tumors is a worldwide problem that has plagued...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85A61K48/00A61P35/00A61P35/02
Inventor 王天有余波唐锁勤张晓敏张棪曲奎尧
Owner 首都儿科研究所
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