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Dowble-stranded RNA and use thereof

A technology of tumor cells and cationic liposomes, which is applied in the direction of medical preparations containing active ingredients, sugar derivatives, gene therapy, etc., can solve the problems of unreported tumor treatment, achieve the goal of inhibiting tumor growth and increasing sensitivity Effect

Inactive Publication Date: 2003-08-06
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the application of RNAi technology to anti-virus (such as HIV virus) and cell signal transmission system is in the ascendant, but there is no report on the application of RNAi technology in tumor treatment

Method used

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  • Dowble-stranded RNA and use thereof
  • Dowble-stranded RNA and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0053] siRNA-EGFR was synthesized in vitro, mimicking siRNA duplex (dsRNA).

[0054] Synthesize siRNA-EGFR according to known in vitro chemical synthesis method, its sequence is: 5'AAGGAGCUGCCCAUGAGAAAU...mRNA, GGAGCUGCCCAUGAGAAAUdTdT...siRNA, length: 21, molecular weight: 6732.9 (g / mole), ODU260: 31.9 (ug / ODU260), Quantity: 50nmol / tube×2 tubes, AUUUCUCAUGGGCAGCUCCdTdT……siRNA complement length: 21, molecular weight: 6583.7(g / mole), ODU260: 33.4(ug / ODU260), quantity: 50nmol / tube×2 tubes,

[0055] Separately package each RNA single strand synthesized above, dilute to 50uM with RNase-inactivated water, mix 30ul sense RNA and 30ul antisense RNA, add 15ul 5× fitting buffer, the final volume is 75ul, incubate at 90°C for 1 minute, centrifuge After 15 seconds, collect the liquid at the bottom of the tube, incubate at 37°C for 1 hour, and the final concentration of dsRNA double strands is 20uM. The composition of the fitting buffer is: 100mM KOAc, 30mM HEPES-KOH pH7.4, 2mM MgOAc. Af...

Embodiment 2

[0056] Example 2 RNA interference technology used in non-small cell lung cancer cells

[0057] Preparation of adherent cells: Inoculate A549 or SPC-A-1 cells in 12-well culture plates one day before transfection, so that the cell density reaches 40-50% (0.5-2×10 5 cells / well / 12-well culture plate), replace the culture medium (DMEM+10% FBS), volume 1ml.

[0058] To prepare the dsRNA-Lipofectamine complex:

[0059] 1) Dilute dsRNA in 100ul serum-free DMEM and mix well.

[0060] 2) Mix Lipofectamine before use, then mix 4ul Lipofectamine with 100ul serum-free DMEM. Incubate at room temperature for 5 minutes.

[0061] 3) Mix the diluent of dsRNA and the diluent of Lipofectamine to a total volume of 200ul, incubate at room temperature for 20min, and store at room temperature.

[0062] 4) Add to the 12-well culture plate, mix well, and incubate in the incubator for 24-48 hours.

[0063] The number of receptors was detected by flow cytometry, and the results were:

[0064] The ...

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Abstract

The present invention relates to a double-stranded RNA and its application for inhibiting mammalian tumor cell. Said invention adopts the RNA interference technique to introduce the externally-synthetic double-stranded RNA into mammalian tumor cell by means of combination of non-viral carrier and / or viral carrier, and can inhibit the expression of epidermal growth factor receptor so as to change NSCLC cell biological character and raise the sensitivity of mammalian tumor cell to radiation therapy and chemical therapy. Said invented double-strand RNA can be used for preparing antitumor biological preparation and antitumor medicine to make direct injection in tumor body in travenous medication for effectively inhibiting tumor growth.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a double-stranded RNA and its anti-tumor application. It specifically relates to a 21-nt double-stranded RNA and its use in inhibiting the expression of epidermal growth factor receptor of non-small cell lung cancer cell lines. Background technique [0002] Malignant tumor is one of the main diseases that threaten human health at present, and its incidence rate is showing an increasing trend year by year. According to statistics, cancer has become the second cause of death in China and the first cause of death in urban population. Taking lung cancer as an example, its incidence rate has risen sharply in the past 20 years. In developed countries in Europe and America, the incidence rate of lung cancer has taken the first place in common tumors, and it is increasing at a rate of 0.5% per year. In my country's large and medium-sized cities such as Beijing and Shanghai, lung cancer has ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61K48/00A61P35/00C07H21/02
Inventor 白春学张新张敏陈杰
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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