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Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent

A leukemia and drug resistance technology, applied in pharmaceutical formulations, gene therapy, genetic material components, etc., can solve problems such as the inability to effectively predict chemotherapeutic agent tolerance, and achieve significant effects, high efficiency, and improved sensitivity.

Active Publication Date: 2013-03-27
广州医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The first aspect of the present invention is to overcome the shortcoming that conventional drug treatment of leukemia patients cannot effectively predict their chemotherapeutic agent tolerance, and use peripheral blood or bone marrow samples of patients to conduct in vitro culture and drug effect measurement to determine the expression level of ALC1 gene before and after treatment. Changes are used as drug tolerance evaluation indicators, and combined with the growth characteristics of leukemia cells after drug treatment to screen out sensitive chemotherapy or targeted therapy drugs

Method used

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  • Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent
  • Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent
  • Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Detection of ALC1 in peripheral blood and bone marrow samples

[0026] Peripheral blood and bone marrow were collected from normal people and patients with various types of acute and chronic leukemia (the bone marrow of normal people was obtained from bone marrow donors who matched). In order to compare the expression of ALC1 in the normal and abnormal proliferation of white blood cells, the experiment also selected infection after general surgery. Peripheral blood (total white blood cells > 10×109 / L) was used as a control. Total RNA was extracted, total RNA was quantified, and cDNA was synthesized by reverse transcription according to the kit instructions. The cDNA product was stored at -20°C or used immediately. PCR primers were synthesized by Shanghai Sangong Company: ALC1 (800bp) Sence: 5′-CCTCCTTCTATGATCATGTTG-3′, Antisence: 5′-TGTTCTCATCCCAGGCCTTG-3′; GAPDH (310bp) Sence: 5′-GCCTCAAGATCAGCAAT-3′, Antisence: 5 '-AGGTCCACCACTG ACACGTT-3'. The PCR reaction...

Embodiment 2

[0028] Example 2 Semi-quantitative analysis of ALC1 expression in acute and chronic leukemia drug-resistant cells and non-drug-resistant parental cells

[0029] Human chronic myeloid leukemia acute erythroleukemia cell line K562 and its drug-resistant cell line K562 / ADR (doxorubicin-resistant cell line, 1.0μg / ml doxorubicin culture system maintains drug resistance), human acute myeloid leukemia cells Strain HL-60 and multidrug-resistant cell line HL-60 / ADR (doxorubicin-resistant cell line, 0.5 μg / ml doxorubicin culture system to maintain drug resistance) in RPMI1640 culture medium containing 15% fetal bovine serum at 37°C with 5% CO 2 Cultured in an incubator with saturated humidity, subcultured once every 2-3 days, and cultured for 4 generations before the experiment, all experiments were carried out with cells in the exponential growth phase. Extract total RNA from logarithmic growth phase cells (K562 and K562 / ADR cells, HL-60 and HL-60 / ADR cells) with RNA extraction kit (R...

Embodiment 3

[0031] Example 3 Reversal effect of ALC1 siRNA interference on drug resistance of leukemia cells

[0032] K562 cells and K562 / ADR cells, HL-60 cells and HL-60 / ADR cells were selected for pair verification.

[0033] (1) The half maximal inhibitory concentration (IC) of doxorubicin (ADR) was detected by MTT assay 50 ): K562 cells and K562 / ADR cells in the logarithmic growth phase (1×10 5 / ml), HL-60 cells and HL-60 / ADR cells (2×10 5 / ml) were inoculated in a 96-well culture plate, and each strain of cells was divided into 4 groups: control group (blank group without drug addition), drug group, ALC1 siRNA interference group, (siRNA+drug) group. For siRNA interference, the adenoviral vector (ad-ALC1 siRNA) carrying the ALC1 siRNA fragment that has been successfully constructed is preferred. The cells in each group were added with Ad-mock (MOI 100, blank group, ADR group) and ad-mock diluted with serum-free RPMI-1640. -ALC1 siRNA (MOI 100, siRNA interference group, siRNA+drug gr...

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Abstract

The invention relates to the field of leukaemia drug resistance and discloses application of an ALC1 inhibitor to preparation of a leukaemia drug resistance reversing agent and application of an ALC1 gene to preparation of a drug-resistant leukaemia diagnosing reagent. The expression of the ALC1 gene in a peripheral blood sample or a bone marrow sample of a patient is detected; and based on a characteristic that the high expression of the gene is related to the increase of the resistance of a cell to chemotherapeutic medicines, sensitive chemotherapeutic agents can be screened. The invention further discloses an application of the ALC1 gene to preparation of the leukaemia drug resistance reversing agent. By inhibiting the expression of the ALC1 gene, the drug resistance of the cell can be reversed, the sensitivity of a leukemia cell to the medicine can be improved, and the cell inhibiting effects and the apoptosis promoting effects of various chemotherapeutic medicines can be improved, so that the ALC1 inhibitor can be applied to the preparation of acute and chronic leukaemia multidrug resistance reversing agents and a new method for treating the drug-resistant leukaemia is provided.

Description

technical field [0001] The invention relates to the field of leukemia drug resistance, in particular to the application of ALC1 gene in the diagnosis of drug-resistant leukemia, the screening of sensitive chemotherapeutic agents and the preparation of leukemia drug resistance reversal agent. Background technique [0002] Leukemia is a clonal disease of hematopoietic stem cells, which is characterized by uncontrolled proliferation and differentiation disorder of leukemia cells in clones, blocked apoptosis, and stagnation at different stages of cell development. The treatment of leukemia is mainly chemotherapy, targeted therapy and bone marrow transplantation. Bone marrow transplantation is limited by many factors such as donor source and patient age. Therefore, only a small number of patients can actually receive bone marrow transplantation, and most patients need chemotherapy. and targeted drug therapy. In the process of drug treatment of leukemia, combined chemotherapy has...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00
Inventor 马宁芳关新元黄振倩熊喜峰
Owner 广州医学院
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