Construction method of colorectal cancer cisplatin-resistant strain

A construction method, cisplatin technology, applied in the field of bioengineering, can solve the problems of affecting the therapeutic effect of tumors, unstable cell drug resistance, difficult cell state, etc., and achieve stable cell state, stable drug resistance, and high success rate Effect

Active Publication Date: 2021-12-10
湖南丰晖生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) The process of inducing drug resistance in cells by the concentration gradient method is very long, usually 6-8 months;
[0007] (2) The main purpose of constructing drug-resistant cell lines is to explore the drug resistance process of tumor cells to clinical chemotherapy drugs to achieve the purpose of optimizing treatment, but the action process of the gradient method is different from the basis of high-dose and short-course chemotherapy drugs in clinical practice. There are differences in principle;
[0008] (3) High-concentration impact method Due to the high concentration of drugs impacting cells, it is difficult to adjust the cell state, and the drug resistance of cells is unstable
[0009] Chemotherapy is one of the methods to treat malignant tumors. It mainly uses chemical drugs to kill tumor cells. However, during chemotherapy, some tumor cells will develop drug resistance, which greatly affects the therapeutic effect of tumors. Therefore, exploring the drug resistance of tumors The mechanism is very important for improving the effect of chemotherapy and choosing the correct chemotherapy method.

Method used

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  • Construction method of colorectal cancer cisplatin-resistant strain
  • Construction method of colorectal cancer cisplatin-resistant strain
  • Construction method of colorectal cancer cisplatin-resistant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: Construction of HCT116 cisplatin-resistant strain (HCT116 / DDP)

[0083] 1. Recovery of HCT116 cells

[0084] 1) Take out the frozen cells from the liquid nitrogen tank, put them into a 37°C water bath and shake them quickly, add 5mL DMEM complete medium (90%DMEM+10%FBS+1%P / S) and mix well.

[0085] 2) Centrifuge at 1000rpm for 4min, discard the supernatant, add 1mL DMEM complete medium, add the cell suspension to a 10cm culture dish, add 7mL medium, put in 5% CO 2 , cultured in a 37°C incubator.

[0086] 2. The IC50 value of cisplatin-resistant HCT116 parental cells determined by MTT method was 10.55 μg / mL, so 10 μg / mL cisplatin was selected to pretreat the cells.

[0087] 3. Expand the cells into 10 dishes, add 10 μg / mL cisplatin to treat them for 24 hours, remove the drug, replace the normal medium, and culture them for 24 hours, then passage them at a rate of 1:1.

[0088] 4. After the cells grow to the logarithmic growth phase, collect the cells for...

Embodiment 2

[0097] Embodiment 2: Construction of HCT15 cisplatin-resistant strain (HCT15 / DDP)

[0098] 1. Recovery of HCT15 cells

[0099] 1) Take out the frozen cells from the liquid nitrogen tank, put them in a 37°C water bath and shake them quickly to melt, add 5mL DMEM complete medium (90% 1640+10%FBS+1%P / S) and mix well.

[0100] 2) Centrifuge at 1000rpm for 4min, discard the supernatant, add 1mL DMEM complete medium, add the cell suspension to a 10cm culture dish, add 7mL medium, put in 5% CO 2 , cultured in a 37°C incubator.

[0101] 2. The IC50 value of cisplatin-resistant HCT15 parental cells determined by MTT method was 10 μg / mL, so 10 μg / mL cisplatin was selected to pretreat the cells.

[0102] 3. Expand the cells into 10 dishes, add 10 μg / mL cisplatin to treat them for 24 hours, remove the drug, replace the normal medium, and culture them for 24 hours, then passage them at a rate of 1:1.

[0103] 4. After the cells grow to the logarithmic growth phase, collect the cells for...

Embodiment 3

[0112] Embodiment 3: Construction of HCT8 cisplatin-resistant strain (HCT8 / DDP)

[0113] 1. Recovery of HCT8 cells

[0114] 3) Take out the frozen cells from the liquid nitrogen tank, put them into a water bath at 37°C and shake them quickly to melt, add 5 mL of DMEM complete medium (90% 1640+10% FBS+1% P / S) and mix well.

[0115] 4) Centrifuge at 1000rpm for 4min, discard the supernatant, add 1mL DMEM complete medium, add the cell suspension to a 10cm culture dish, add 7mL medium, put in 5% CO 2 , cultured in a 37°C incubator.

[0116] 2. The IC50 value of cisplatin-resistant HCT8 parental cells determined by MTT method was 9.2 μg / mL, so 10 μg / mL cisplatin was selected to pretreat the cells.

[0117] 3. Expand the cells into 10 dishes, add 10 μg / mL cisplatin to treat them for 24 hours, remove the drug, replace the normal medium, and culture them for 24 hours, then passage them at a rate of 1:1.

[0118] 4. After the cells grow to the logarithmic growth phase, collect the c...

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Abstract

The invention relates to a construction method of a colorectal cancer cisplatin-resistant strain, and belongs to the technical field of bioengineering. The invention provides a novel drug-resistant strain construction method, which comprises the steps: combining a magnetic bead sorting method with a conventional drug-resistant strain screening method, selecting P-glycoprotein (P-gp) as a drug-resistant target, labeling cells by using an antibody of a coding gene ABCB1 of the P-glycoprotein, and separating a cell population with drug resistance from a parent strain through magnetic bead sorting. The method is short in construction period, high in success rate, and stable in drug resistance of the constructed strain.

Description

technical field [0001] The invention relates to a method for constructing a cisplatin-resistant strain of colorectal cancer, belonging to the technical field of bioengineering. Background technique [0002] At present, there are mainly two methods for the screening of drug-resistant strains: the concentration gradient method and the high concentration shock method. [0003] Concentration gradient increasing method: measure the IC50 of the parental cells by MTT, select 1 / 10 of the IC50 concentration as the initial concentration to shock the cells, resume the culture after 24 hours of treatment, and shock the cells several times at the same concentration until the cells can grow stably at this concentration Then gradually increase the drug concentration and repeat the above steps to treat the cells until the drug resistance index of the cells reaches the target value and then stop the impact. The cultured cells grow normally at the maintenance concentration and the IC50 value ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N5/0679C12N2501/06C12N2509/00Y02A50/30
Inventor 胡清云刘彩云许澎
Owner 湖南丰晖生物科技有限公司
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