Application of tetrandrine in preparation of leukemia multidrug resistance (MDR) reversal agent
A technology of tetrandrine and multidrug resistance, which is applied in the field of biomedicine, can solve the problems of no specific target, weak curative effect, low toxicity and side effects, etc., and achieve the reversal of tumor multidrug resistance and inhibition of multidrug resistance on the cell surface. Synthesis of the drug-resistant protein P-glycoprotein, which enhances the effect of chemotherapy
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Embodiment 1
[0015] Example 1: Effects of Tet combined with daunorubicin (DNR) on the proliferation of human chronic myelogenous leukemia acute erythroleukemia cell line K562 and its drug-resistant strain K562 / A02 in vitro.
[0016] The tetrandrine used is a commercially pure product (purity>98%).
[0017] Cells in the logarithmic growth phase were seeded in a 96-well plate, each well containing 2×10 4 160uL of cell suspension, add different concentrations of tetrandrine (0mg / L, 0.5mg / L, 1.0mg / L, 2.0mg / L) to each plate for 1 hour, then add different concentrations of soft red according to the set gradient Mycin (final concentration 50mg / L, 25mg / L, 12.5mg / L, 6.25mg / L, 3.125mg / L, 1.5625mg / L and 0.78125mg / L), each daunorubicin concentration set 5 complex hole. A control group without reversal agent, daunorubicin, and cells was also set up, and the volume of each well was adjusted to 200uL. Placed at 37°C, 5% CO 2 After 48 hours of incubation in the incubator, take it out, add 20ul of 5m...
Embodiment 2
[0020] Example 2 Detection of P-glycoprotein (P-glycoprotein, P-gp) expression on the surface of leukemia drug-resistant cells K562 / A02 by flow cytometry
[0021] Cells were divided into groups by adding Tetrandrine at the set concentration (0mg / L, 0.5mg / L, 1.0mg / L, 2.0mg / L), at 37°C, 5% CO 2 After incubation in the incubator for 48 hours, the cells were harvested. The cells of each experimental group were concentrated by centrifugation (the number of cells in each group was 1×10 6 ), washed 3 times with cold PBS, centrifuged at 1500 rpm for 5 minutes, discarded the supernatant, and added 5 μl of P-gp monoclonal antibody (labeled with fluorescein PE). Protect from light and store at room temperature for 20 minutes, then wash once with normal saline, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 0.5ml of normal saline, and perform flow cytometry detection.
[0022] The results showed that after Tetrandrine (0mg / L, 0.5mg / L, 1.0mg / L, 2.0mg / L) acted on the...
Embodiment 3
[0023] Example 3 Semi-quantitative reverse transcriptase polymerase chain reaction (reverse transcript polymase chain reaction, RT-PCR) method to detect multidrug resistance gene 1 (multidrug resistance 1, mdr1) mRNA expression level
[0024] Take K562 / A02 cells (final concentration 2×10 4 mL, 4mL for each group), add the set final concentration of Tetrandrine (0mg / L, 0.5mg / L, 1.0mg / L, 2.0mg / L) in groups, at 37℃, 5% CO 2Cells were cultured in an incubator and harvested after 48 hours. TRIzol (invitrogen) was used to extract total RNA in one step, and the mdr1 gene fragment (436 bp) and β-actin gene fragment (270 bp) were amplified according to the instructions of the RT-PCR kit (Dalian Bao Biological Engineering Co., Ltd.), and the upstream primer of mdr1 (SEQ ID NO:1) 5'-TGGTTTGATGTGCACGATGTTGGG-3'; mdr1 downstream primer (SEQ ID NO:2) 5'-AGATCAGCAGGAAAGCAGCACCTA-3'; β-actin upstream primer (SEQ ID NO:3) 5'-ACACTGTGCCCATCTACGAGGGG- 3'; β-actin downstream primer (SEQ ID NO...
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