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Application of miR-99a as acute myelogenous leukemia stem cell molecular marker

A technology of leukemia stem cells and mir-99a, which is applied in the measurement/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of neglecting leukemia refractory and relapse, and achieve the effect of simple operation, low cost and good repeatability

Active Publication Date: 2016-08-10
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although some studies have used miRNA as a marker of acute myeloid leukemia to evaluate the prognosis and progression, these studies have ignored the root cause of leukemia refractory and relapse-leukemic stem cells

Method used

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  • Application of miR-99a as acute myelogenous leukemia stem cell molecular marker
  • Application of miR-99a as acute myelogenous leukemia stem cell molecular marker
  • Application of miR-99a as acute myelogenous leukemia stem cell molecular marker

Examples

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Embodiment 1

[0029] 1. For 13 patients with acute myeloid leukemia bone marrow and 5 normal umbilical cord blood controls, 3ml of Ficoll was preset in a 15ml centrifuge tube, and the bone marrow fluid was slowly added to it without damaging the interface. Obtain leukemia stem cells and normal hematopoietic stem cells by flow cytometry; centrifuge at 2000 rpm for 20 minutes at 20°C; aspirate and discard the supernatant and carefully collect the mononuclear cell layer (buffy coat) in a centrifuge tube containing 10ml of PBS, centrifuge (same as above); discard Supernatant, resuspended, counted, centrifuged, every 10 7 Resuspend the cells in 100 μl of PBS buffer, add 2 μl each of FITC-CD45 and PE-CD34 antibodies, and keep away from light for 30 minutes; wash the cells and sort CD34 on the machine + cells and CD34 - cell.

[0030] 2. Use 1ml Trizol to lyse the cells, add 0.2ml chloroform, shake vigorously at 15-30°C for 15s, and let stand for 3mins; centrifuge at 12000g for 15 minutes at 4°C...

Embodiment 2

[0048] An auxiliary diagnostic kit for the prognosis and progress of acute myeloid leukemia, the kit comprising:

[0049] (1), bone marrow mononuclear cell isolation reagent Ficoll (purchased from Sigma Company), leukemia stem cell enrichment markers FITC-CD45, PE-CD34 antibody (purchased from BD Biosciences Company);

[0050] (2), specific primers and probes of miR-99a (purchased from Invitrogen TM company);

[0051] (3), specific primers and probes of U6 internal reference marker (purchased from Invitrogen TM company);

[0052] (4), reverse transcriptase, qPCR Mix, 10mM dNTPs, reverse transcription buffer, RNase inhibitor (purchased from Invitrogen TM ), DEPC water;

[0053] (5), total RNA extraction reagent Trizol (purchased from Invitrogen TM ), isopropanol, absolute ethanol, chloroform, PBS buffer.

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Abstract

The invention provides an application of miR-99a as an acute myelogenous leukemia stem cell molecular marker and also an application thereof in evaluation of prognosis, disease progression and treatment of the acute myelogenous leukemia. In the application, with flow sorting and real-time fluorescent quantitative PCR technologies as bases and bone marrow miR-99a of leukemia patients as detection objects, through comparison of the expression level between leukemia stem cell and leukemia non-stem cell and the expression level between leukemia stem cell and normal hemopoietic stem cell in the leukemia patients, abnormal high expression of the miR-99a in the leukemia stem cell in relapsed and stubborn patients is found and is related to bad prognosis of the patients, so that the miR-99a, as the marker and molecular target of the leukemia stem cells, is a new molecular marker for prognosis evaluation of the leukemia, and also provides the new molecular target for individual treatment. The application is used for preparing a diagnosis therapy product on the basis of detection on expression of the miR-99a, has important significance and clinical application value for tumor therapy and has wide application prospect.

Description

technical field [0001] The present invention relates to the field of prognosis diagnosis of acute myeloid leukemia, and specifically relates to a molecular marker of acute myeloid leukemia stem cell miR-99a, which can be applied to acute myeloid leukemia prognosis assessment, disease progression and targeted therapy to provide patients with accurate Diagnosis and treatment, in order to achieve complete remission and long-term survival. Background technique [0002] Leukemia is a kind of malignant hematopoietic cell abnormal proliferation disease, which is listed as one of the top ten high-incidence malignant tumors in my country, and ranks first among children and adults under the age of 35. Among them, acute myeloid leukemia (AML) has the highest incidence rate in my country, and it has an increasing trend year by year. With the development of various treatment methods in recent years, the complete remission rate (complete remission, CR) of leukemia chemotherapy has increa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 周圆司晓慧张孝云高瀛岱白洁杨逢春邢文
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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