Method for sifting tumour chemotherapy medicament sensitive gene with RNA interference library

A technology of RNA interference and gene screening, applied in the field of biomedicine, can solve problems such as the limitation of tumor chemotherapy effects, the inability to conduct research, development and utilization of drug-sensitive genes, and the reduction of expression means

Inactive Publication Date: 2009-05-13
黄行许
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AI Technical Summary

Problems solved by technology

Since there is no reliable means of reducing expression, only a limited number of drug-sensitive genes have been identified for a long time,

Method used

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Examples

Experimental program
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Effect test

specific Embodiment approach

[0050] 1. Using RNA interference library DNA to transfect Phoenix retrovirus packaging cells

[0051] Day 1: Preparing Phoenix Retroviral Packaging Cells

[0052] 1. 18-24 hours before transfection, press 3×10 6 / 10 cm plate inoculation of Phoenix cells onto the plate;

[0053] 2. Let the cells adhere to the wall for 10-24 hours;

[0054] 3. When the cell density reaches 2 / 3, a 10 cm plate has about 5×10 6 cell. At this time, the packaging cells are most easily transfected and produce the highest titer of virus at 37°C;

[0055]*Medium: DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine.

[0056] Day 2: Transfection

[0057] 1. Prepare the DNA for transfection and dissolve it in HEPES solution;

[0058] 2. About 5 minutes before transfection, add 25 μM chloroquine to the plate (the storage concentration of chloroquine is 50 mmol);

[0059] * Chloroquine inhibits DNase in lysosomes by neutralizing the pH inside intracellular transport microvesicles; ...

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Abstract

The invention relates to a method for screening sensitive genes to tumor chemotherapy medicament by using an RNA interference library. The method comprises the step of using the RNA interference library with reduced gene expression to reduce medicament sensitive gene expression of tumor cells, so that the medicament sensibility of the tumor cells is weakened, but the medicament tolerance is raised; therefore, the tumor cells survive in high-concentration medication condition and form cell clone; finding and screening the medicament sensitive gene through gene clone; and extracting genome DNA from survived cells, amplifying a DNA fragment inserted from the RNA interference library by RCR, using a TA cloning method to clone the DNA fragment amplified by the RCR, selecting the clone, purifying the DNA, and finally measuring the sequence of the gene to determine screened genes. The method makes first screening of the medicament sensitive genes of liver cancer cells, first determines a batch of real medicament sensitive genes in the world, and is used for (1) guiding medicament selection of tumor chemotherapy, that is, when the sensitive genes aiming at certain medicament expresses normally, recommending the medicament, and when the sensitive genes aiming at certain medicament reduces expression or undergoes mutation, avoiding using the medicament, and (2) developing and applying the genes as biological markers of tumor for evaluating risk and preliminarily surveying pathogeny before diagnosis, classifying and defining the level of the tumor at the diagnosis, and evaluating treatment effect and detecting recurrence after the diagnosis.

Description

technical field [0001] The invention relates to a method for screening tumor chemotherapeutic drug sensitive genes by using an RNA interference library, and belongs to the technical field of biomedicine. Background technique [0002] The means of tumor treatment are mainly divided into surgical resection, chemotherapy, radiotherapy and other methods. Cancer chemotherapy is the treatment of tumors with chemical drugs. In recent years, chemotherapy has developed the fastest in tumor treatment, and it has become one of the most important means of clinical treatment of tumors today, belonging to the systemic treatment of tumors. With the gradual increase in the types of chemical drugs, the continuous improvement of drug use methods, and the continuous accumulation of clinical experience, its curative effect is increasing day by day. Chemotherapy has transitioned from the previous palliative treatment to the radical treatment. [0003] The principle of chemotherapy is to use dr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 黄行许
Owner 黄行许
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