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Anti cancer gene medicinal composition, micromolecule interfere RNA and its filtrate method

A small molecule interference and gene drug technology, applied in the field of gene therapy, can solve the problems of undiscovered liver cancer treatment and achieve the effect of a good screening platform

Inactive Publication Date: 2007-06-27
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of RNAi for liver cancer treatment

Method used

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  • Anti cancer gene medicinal composition, micromolecule interfere RNA and its filtrate method
  • Anti cancer gene medicinal composition, micromolecule interfere RNA and its filtrate method
  • Anti cancer gene medicinal composition, micromolecule interfere RNA and its filtrate method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Obtaining of antisense fup1 gene sequence

[0044] According to the coding gene sequence of fupl (SEQ ID NO: 1), the cDNA sequence (SEQ ID NO: 2) of the antisense fupl gene was obtained after processing with DNATool software. Using upstream and downstream primers with BamHI and EcoRI cleavage sites respectively, using the plasmid extracted from the human hepatocyte cDNA library as a template, the target gene fup1 and antisense fup1 gene were obtained by PCR amplification (according to the literature "Pan Wei et al., Acta Biochemistry and Biophysics, 2001, 33(2): 173-178"). After the PCR products of the above fup1 gene and antisense fup1 gene were treated with EcoRI and BamHI respectively, according to the difference in the enzyme cutting sites at both ends of the primers, they were ligated with double sticky ends, and inserted directionally into pcDNA3 eukaryotic cells treated with the same endonuclease in the expression vector.

Embodiment 2

[0045] Example 2 Determination of growth curves of eukaryotic cells transfected with fup1 gene and antisense fup1 gene

[0046] Transfect mouse fibroblast NIH3T3 with the pcDNA3 plasmid and empty vector integrated with the full-length fup1 and antisense fup1 cDNA by liposome method: NIH 3T3 cells were cultured in a 35mm culture dish, and when the fullness reached about 60%, According to each transfection reaction, use 2ug plasmid and 5ug liposome to operate: first mix the plasmid and liposome in 100ul DMEM basic medium, then mix the two with each other and place at room temperature for 30 minutes. Thereafter, 0.8 ml of DMEM basic medium was added to the sample and mixed evenly, and then the above mixture was added to the cells washed with DMEM basic medium to remove residual serum. After culturing at 37°C and 5% CO2 for 5 hours, add 1 ml of DMEM medium containing 20% ​​serum to the transfected cells, and continue culturing at 37°C and 5% CO2. 24 hours after the start of trans...

Embodiment 3

[0049] Example 3 Soft agar colony formation experiment of antisense fup1 gene transfection of human liver cancer cell BEL7404

[0050] Mix 0.5ml 1.2% agar and 0.5ml 2× medium to make a complete medium containing 0.6% agar, quickly add it to a 6-well culture plate, let it stand at room temperature to solidify, and use it as the lower medium; then mix 0.5ml 0.6% agar and 0.5ml 2× medium to make a complete medium containing 0.3% agar. BEL7404 cells stably transfected with fup1 antisense cDNA identified by RT-PCR and BEL7404 control cells transfected with pcDNA3 empty vector were counted at 5×10 per well 3 Cells were seeded in soft agar medium in a 6-well culture plate and added to the lower medium. After the upper layer culture was solidified at room temperature, incubate at 37°C, 5% CO 2 cultivated under conditions. After 2 weeks of culture, the cell morphology was observed and photographed under a phase-contrast microscope. The analysis results showed that the colonies form...

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PUM

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Abstract

The liver cancer resisting gene medicine composition includes antisense fup1 gene and / or small molecular interfering RNA and pharmaceutically acceptable supplementary material. The antisense fup1 gene has the DNA sequence of SEQ ID No. 2 in the sequence list, while the small molecular interfering RNA includes the RNA sequence of SEQ ID No. 3 in the sequence list. The present invention also provides the liver cancer resisting gene medicine screening method to design small molecular interfering RNA based on fup1 encoding area via adopting ambion web site provided RNAi design software. The present invention further provides liver cancer resisting small molecule include RNA sequence of SEQ ID No. 3 in the sequence list. The present invention adopts gene fup1 high expressed in liver cancer cell as medicine target to design the antagonistic molecule to provide powerful measure for the gene treatment of liver cancer and excellent platform for further screening liver cancer resisting gene medicine.

Description

technical field [0001] The invention relates to gene therapy, in particular to a gene therapy drug for liver cancer and a screening method thereof. Background of the invention [0002] Liver cancer is one of the most common malignant tumors in the world. my country is a country with a high incidence of hepatitis and liver cancer. 48% of the world's liver cancer patients are concentrated in my country. In the past 20 years, the incidence of liver cancer in my country has increased by nearly 40%, ranking third among malignant tumors. The early diagnosis of liver cancer is very difficult, and the recurrence rate after operation is as high as 50%. The mortality rate of liver cancer is second only to gastric cancer in our country. [0003] Existing research results show that the occurrence of liver cancer is closely related to hepatitis virus (HBV, HCV) infection, liver cirrhosis, aflatoxin B1 and some genetic factors. Similar to tumors derived from other tissues, the occurrenc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K31/7088A61P35/00
Inventor 周庆玮张倩朱俊潘巍金由辛甘人宝李载平
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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