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Human leukemia hl-60 cell drug-resistant cell line hl-60/rs cell and preparation method thereof

A technology for drug-resistant cells and leukemia, applied in biochemical equipment and methods, animal cells, tumors/cancer cells, etc., can solve problems such as lack of arsenic resistance and arsenic sensitivity

Inactive Publication Date: 2016-11-16
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently commonly used leukemia multidrug-resistant cell lines induced by doxorubicin and overexpressing P-gp (such as K562 / ADM, etc.) are still sensitive to arsenic trioxide and have no arsenic resistance

Method used

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  • Human leukemia hl-60 cell drug-resistant cell line hl-60/rs cell and preparation method thereof
  • Human leukemia hl-60 cell drug-resistant cell line hl-60/rs cell and preparation method thereof
  • Human leukemia hl-60 cell drug-resistant cell line hl-60/rs cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Adriamycin was used as an inducer to induce HL-60 cells to establish multidrug-resistant cell lines. ① HL-60 was inoculated in RPMI 1640 culture medium containing 15% newborn bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, placed at 37°C, 5% CO 2, in a saturated humidity incubator. ②Add doxorubicin to the cell culture system, the initial impact concentration is 0.1mg / L, and when the surviving cells return to normal growth, add the same concentration of drugs to induce, and so on, until the cells can survive and proliferate normally at this concentration , then increase the impact drug concentration, and carry out the second round of impact induction, and the drug concentration increase range is 0.1mg / L. Such repeated intermittent shock induction, and with the increase in the number of shock inductions and drug resistance, the increase in drug concentration in each round of shock is gradually increased, the initial range is small, and the amplitude in the lat...

Embodiment 2

[0046] The induction establishment method of HL-60 / RS cells is the same as that in Example 1. One month after the stable growth of HL-60 / RS cells, the sensitivity to arsenic trioxide was determined. HL-60 / RS cells (choose HL-60 sensitive cells, K562 / ADM multidrug-resistant cells and their parental K562 cells as controls), press 1.0×10 5 cells / mL inoculated in 96-well culture plate, adding 0.1-5.0mg / L As 2 o 3 , 37℃, 5%CO 2 And cultured under saturated humidity conditions for 24-72h, MTT assay cell proliferation inhibition and IC50. HL-60 / RS cells are highly resistant to arsenic trioxide, 12.89 times that of parental HL-60 cells and 10.89 times that of K562 / ADM drug-resistant cells.

Embodiment 3

[0048] The induction establishment method of HL-60 / RS cells is the same as that in Example 1. HL-60 / RS cells and control HL-60 cells, K562 cells and K562 / ADM cells were extracted by TRIZOL method for total RNA and total protein by RIPA lysate, and real-time fluorescence quantitative RT-PCR method and Western blot were used respectively The expression of arsenic transport related proteins MRP1, MRP2 and ASNA1 was detected by the method. The expression of MRP (MRP1 and MRP2) in HL-60 / RS cells was significantly higher than that in HL-60 cells and K562 / ADM cells; the expression of ASNA1 in HL-60 / RS cells was significantly higher than that in HL-60 cells and K562 / ADM cells, respectively It is 7.34 times of K562 / ADM cells and 2.45 times of HL-60 cells. The expression level of K562 / ADM cells was 0.83 times that of K562 cells. Expression levels of MRP and ASNA1 were consistent with arsenic trioxide sensitivity.

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Abstract

The invention discloses the drug-resistant cell line HL-60 / RS of human leukemia HL-60 cells. The preservation number of the cell line HL-60 / RS is CCTCC NO: C201430, and provides its preparation method. The HL‑60 cell line is the parental cell, and the acquired leukemia multidrug resistant cell line HL‑60 / RS. The beneficial effect of the present invention is: the leukemia multidrug-resistant HL-60 / RS cell line induced by the present invention adopts the induction method of "gradually increasing the concentration shock + continuous induction", and the experiment proves that the established drug-resistant cell line has It has the characteristics of wide drug resistance spectrum and stable drug resistance; and has high drug resistance to arsenic trioxide, which can be used to study the mechanism of sensitivity, drug resistance and arsenic resistance of tumor cells to anticancer drugs, as well as to screen reliable Drug resistance reversal agents.

Description

technical field [0001] The invention relates to a drug-resistant human leukemia HL-60 cell strain HL-60 / RS cell and a preparation method thereof. Background technique [0002] Leukemia is one of the most important malignant tumors that endanger young adults. Chemotherapy is still the most important treatment for leukemia. However, drug resistance induced by anticancer drugs in chemotherapy, especially acquired multidrug resistance (MDR) ) phenomenon is the main obstacle hindering the therapeutic effect of leukemia, and it is also a difficult problem that has not been solved yet. Arsenic trioxide is an active ingredient of the traditional Chinese medicine arsenic, which has been widely used in the treatment of various types of leukemia and solid tumors, and has achieved good clinical efficacy. Studies have confirmed that arsenic trioxide is not a substrate of drug-resistant protein P-gp and inhibits the expression of P-gp, and does not cause cross-resistance with conventiona...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09C12R1/91
Inventor 魏虎来陈静程杰谢蓓易娟王蓓
Owner LANZHOU UNIVERSITY
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