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An antitumor medicine screening kit and a using method thereof

A technology of anti-tumor drugs and kits, which is applied in the field of biomedicine to achieve the effects of objective and accurate data, simple operation and short cycle.

Active Publication Date: 2016-07-06
甘肃药业集团科技创新研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some difficulties in the use of C. elegans at this stage. The main reason is that for ordinary people, the modeling, cultivation, and testing of C. elegans still require systematic technical training and practice in order to complete drug screening. Therefore, , we are in urgent need of an anti-tumor drug screening kit using Caenorhabditis elegans as a model

Method used

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  • An antitumor medicine screening kit and a using method thereof
  • An antitumor medicine screening kit and a using method thereof
  • An antitumor medicine screening kit and a using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Confirmatory Experiment of the Staining Effect of Caenorhabditis elegans

[0052] (1) Synchronization of Caenorhabditis elegans

[0053] ① Select mature Caenorhabditis elegans into a 2ml centrifuge tube filled with 1ml M9 solution, centrifuge at 4000rpm for 5 minutes, and remove the supernatant;

[0054] ② Add 500 μL of 1M sodium hydroxide and 500 μL of 6.4% (V / V) sodium hypochlorite to the centrifuge tube, shake and lyse;

[0055] ③Remove the supernatant, add 1.5μL M9 solution to the precipitate, shake well, centrifuge at 4000rpm for 5 minutes, repeat this step three times;

[0056] ④ Add 500 μL of M9 solution to the washed eggs, and hatch at 18°C ​​for 24 hours to form L1-stage Caenorhabditis elegans.

[0057] (2) Staining and detection method of Caenorhabditis elegans

[0058] ①Staining and detection of Caenorhabditis elegans

[0059] Mix the above synchronized L1 stage Caenorhabditis elegans MT2124, count the number of Caenorhabditis elegans per unit l...

Embodiment 2

[0067] Example 2 The effect of anti-tumor candidate drugs on Caenorhabditis elegans CB1275 (lin-1 deletion mutation)

[0068] lin-1 is a gene downstream of ras, a nuclear transcription factor that can be phosphorylated by MAPK, a negative regulator of vulva development, and can regulate the signaling pathway of let-60 downstream of vulva development. lin-1 and lin-31 form a complex, lin-1 encodes ETS family members, and lin-31 encodes a winged helix transcription factor, when the ras signal is not activated, lin-1 and lin-31 interact to form a complex, This complex binds to the promoters of the ovipositor functional genes and prevents their expression. After the lin-1 deletion mutation, the complex cannot be formed, the complex cannot be formed, and it cannot be combined with the promoter part of the ovipositor function gene, and the ovipositor gene is expressed, and then the pseudovulva develops to form a polyvulva phenotype.

[0069] If the results of Example 1 show that th...

Embodiment 3

[0071] The assembly of embodiment 3 antitumor drug screening kits

[0072] Antitumor drug screening kit of the present invention, the kit includes the following reagents:

[0073] ① Caenorhabditis elegans wild-type N2;

[0074] ② Caenorhabditis elegans mutant MT2124;

[0075] ③ Caenorhabditis elegans CB1275;

[0076] ④ NGM medium;

[0077] ⑤ Escherichia coli OP50;

[0078] ⑥Dye: 2',7'-dichlorodihydrofluorescein diethyl ester.

[0079] Wherein, the concentration of Caenorhabditis elegans is 100 / 20 μl, the concentration of Escherichia coli OP50 is 10 mg / ml, and the final concentration of the dye is 50 μg / mL.

[0080] Dispense the above reagents into suitable containers and put them into an outsourcing kit, in which M9 buffer, S buffer and LB medium are described in detail in the instruction manual.

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Abstract

The invention discloses an antitumor medicine screening kit and a using method thereof, and belongs to the technical field of biomedicine. The kit comprises caenorhabditis elegans, a nematode culture medium, escherichia coli and dye. An objective of screening effective antitumor medicines rapidly, efficiently and accurately with simple operation and a high throughout is achieved by utilizing different dyeing effects of the dye that is 2',7'-dichlorofluorescein diacetate on mutants and wild type caenorhabditis elegans, thus saving time and the cost for personnel researching and developing antitumor medicines.

Description

technical field [0001] The invention relates to an antitumor drug screening kit and a method for using the kit, belonging to the technical field of biomedicine. Background technique [0002] It is well known that the RAS signaling pathway is a signaling pathway related to tumorigenesis. Abnormal function of RAS proteins is ubiquitous in human tumors. According to reports, about 50% of lung and intestinal cancers, 95% of pancreatic cancers have ras mutations. If the RAS protein is continuously activated, it can bind to the downstream effector protein and transmit the signal to the downstream signaling element, which may cause abnormal proliferation of cells and lead to the occurrence of tumors. Therefore, the signaling pathway of RAS protein has become a new target for screening anti-tumor drugs. [0003] Tumor cell lines, immortalized cells, and primary cultured cells are important models for studying tumor etiology and pathogenesis. However, many drug candidates have po...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 不公告发明人
Owner 甘肃药业集团科技创新研究院有限公司
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