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Application of ORP4L (Oxsterol binding protein-related protein, 4L) in preparation of product for treating acute myeloid leukemia

A technology for acute myeloid leukemia, applied in the field of application in the preparation of products for the treatment of acute myeloid leukemia

Inactive Publication Date: 2015-11-18
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the function of ORP4L in tumor cells, especially leukemia cells, is still unknown, and the pharmacological mechanism of how OSW-1 kills tumor cells by targeting ORP4L still needs to be further studied

Method used

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  • Application of ORP4L (Oxsterol binding protein-related protein, 4L) in preparation of product for treating acute myeloid leukemia
  • Application of ORP4L (Oxsterol binding protein-related protein, 4L) in preparation of product for treating acute myeloid leukemia
  • Application of ORP4L (Oxsterol binding protein-related protein, 4L) in preparation of product for treating acute myeloid leukemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, clinical specimen collection and cell sorting:

[0052] 1. Collection of bone marrow from patients with AML

[0053] The first cases of acute myeloid leukemia (AML) who were hospitalized were collected, and 2 mL of bone marrow was extracted under sterile conditions, and heparin was added for anticoagulation. Bone marrow specimen collection requirements: heparin anticoagulation, no blood clots, bone marrow volume greater than 2mL, cell sorting should be performed within 24 hours after specimen collection.

[0054] 2. Collection of umbilical cord blood

[0055] Cord blood was collected from the obstetrics and gynecology department of the hospital, and heparin was added for anticoagulation. Cell sorting was performed within 24 hours after specimen collection.

[0056] 3. Mononuclear cell sorting

[0057] Sorting mononuclear cells from bone marrow and cord blood collected from AML patients involves the following steps:

[0058] A. Dilute whole blood with ...

Embodiment 2

[0075] Example 2, detection of ORP4L gene expression in leukemia stem cells and normal hematopoietic stem cells

[0076] 1. q-PCR detection of ORP4L gene expression

[0077] A. RNA extraction:

[0078] Wash the cells twice with PBS, add 1mL TriReagent, lyse and mix with a pipette several times; place at room temperature for 5 minutes to fully separate the nucleoprotein; add 200 μL of chloroform, cover the tube tightly, shake vigorously for 15 seconds, and place at room temperature for 5 minutes; Centrifuge at 12,000g for 15min, transfer about 400μL of the aqueous phase to another new 1.5mLEP; add 500μL of 1mL isopropanol, mix thoroughly, and place at room temperature for 5min; centrifuge at 12,000g for 10min at 4°C, discard the supernatant; Wash the RNA pellet with ethanol; centrifuge at 7,500g at 4°C for 5 minutes, discard the supernatant; air-dry the pellet for 3-5 minutes, add 40 μL DEPC-treated water to dissolve the RNA pellet, measure the RNA concentration and integrity,...

Embodiment 3

[0103] Example 3. Detection of the effect of siRNA-mediated ORP4L gene silencing on the calcium ion rhythm and ATP production of leukemia stem cells

[0104] 1. Design of ORP4LsiRNA and packaging of lentivirus containing encoding DNA

[0105] Three pairs of siRNAs were designed according to three fragments of ORP4L mRNA, the sequences are as follows:

[0106] siRNA1:

[0107] SEQ ID NO: 1: 5'-GCAAUGGUUUGCUCUCUUA-3';

[0108] SEQ ID NO: 2: 5'-UAAGAGAGCAAACCAUUGC-3';

[0109] siRNA2:

[0110] SEQ ID NO: 3: 5'-GCCAUGAUGUUUAGUAAAU-3';

[0111] SEQ ID NO: 4: 5'-CGGUACUACAAAUCAUUUA-3';

[0112] siRNA3:

[0113] SEQ ID NO:5:5'-UUGACACGGAGGACUCUUGUG-3';

[0114] SEQ ID NO:6:5'-AACUGUGCCUCCUGAGAACAC-3';

[0115] The siRNA2 sequence with the highest interference efficiency was selected to synthesize and construct the ORP4L siRNA expression vector, and the lentivirus was packaged to obtain the shORP4L lentivirus.

[0116] 2. To detect the effect of ORP4L gene silencing on the ca...

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Abstract

The invention discloses an application of ORP4L (Oxsterol binding protein-related protein, 4L) in preparation of a product for treating acute myeloid leukemia. According to specific expression of an ORP4L gene in a leukemia stem cell, the specific energy support mechanism of a leukemia stem cell is discovered, that is, oxidative phosphorylation depends on existence of the ORP4L gene. The novel application of the ORP4L gene is provided, that is, the ORP4L gene taken as a target site for selectively killing the leukemia stem cell is applied to new product development and treatment of acute myeloid leukemia. A novel ORP4L inhibitor is screened and has greater practical significance and a broad application prospect in leukemia treatment through blocking of LSC (leukemia stem cell) energy supply. Small interfering RNA (ribonucleic acid) has a remarkable inhibition effect on ORP4L gene expression, LSC energy generation and cell viability can be inhibited by silent ORP4L genes, and the ORP4L gene can be taken as a novel target site in drugs for treating leukemia and resisting leukemia.

Description

technical field [0001] The present invention relates to a new application of Oxsterol binding protein-related protein 4L (Oxsterolbinding protein-related protein 4L, ORP4L), in particular to the application of ORP4L as a target in the preparation of products for treating acute myeloid leukemia. Background technique [0002] Leukemia is a type of clonal malignant disease originating from hematopoietic stem cells (Mandavilli, A, Nature, 2013). According to statistics, about 350,000 people die of leukemia every year worldwide. Therefore, leukemia has become the main malignant tumor that threatens human health. Leukemia stem cells (leukemiastemcells, LSC) are a group of extremely small cell populations in leukemia patients, accounting for about 0.1% to 1% of all leukemia cells. LSC was first discovered in acute myeloid leukemia (AML) and has been widely recognized (Bonnet, D., et al. Nature medicine, 1997). Whether in long-term cell culture or in animal models, LSCs can cause ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K48/00A61K31/713A61P35/02C12Q1/68
Inventor 闫道广钟文彬
Owner JINAN UNIVERSITY
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