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Application of mesenchymal stem cell in preparation of medicine used for treating M5 type leukaemia

A kind of mesenchymal stem cell and leukemia technology, applied in the field of medicine and biology, can solve the problems of poor prognosis, patients' intolerance, easy relapse, etc., achieve the effect of improving the ability of apoptosis, avoiding toxic and side effects, and meeting clinical needs

Active Publication Date: 2016-07-20
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most M5 patients are clinically manifested as leukocytosis, prone to extramedullary infiltration (including liver, spleen, lymph nodes, gums, skin, central nervous system, etc.), high rate of adverse chromosomal karyotype, and complete remission (CR) rate Low, easy to relapse, poor prognosis, etc.
The heterogeneity and complexity of M5 determine that it still lacks specific tumor-related markers for molecular diagnosis and targeted therapy
[0003] At present, chemotherapy is still the main treatment for leukemia in China, but chemotherapy drugs often have toxic damage to normal cells and tissues while killing leukemia cells; in addition, gastrointestinal reactions, bone marrow suppression, Heart, liver, kidney and other organ damage, bleeding, infection and other toxic side effects make a considerable number of patients intolerable
In addition, allogeneic hematopoietic stem cell transplantation (Hematopoietic Stem Cell Transplantation, HSCT) shows effective anti-leukemia effect, but because of the difficulty of matching, the cost is expensive, and more importantly, there are some life-threatening complications in the body, such as Graft Versus Host Disease (GraftVersusHostDisease). , GVHD), making this treatment plan only suitable for a small number of young and suitable patients, and most patients cannot accept this treatment

Method used

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  • Application of mesenchymal stem cell in preparation of medicine used for treating M5 type leukaemia
  • Application of mesenchymal stem cell in preparation of medicine used for treating M5 type leukaemia
  • Application of mesenchymal stem cell in preparation of medicine used for treating M5 type leukaemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Isolation and subculture of umbilical cord mesenchymal stem cells

[0037] 1) Take the umbilical cord tissue of a newborn baby, put the umbilical cord in a petri dish and clean it with PBS containing 0.1% double antibody in the ultra-clean workbench, then put it into α-MEM medium, and cut the umbilical cord along the outer wall , strip three blood vessels, and cut the Wharton's jelly and umbilical cord wall into 1-2mm 3 tissue blocks, waiting to be digested;

[0038] 2) Prepare tissue-digesting enzymes (type II collagenase 250U / ml, dispase 100U / ml, hyaluronidase 10U / ml), dissolve in α-MEM medium at 37°C, filter and sterilize with a 0.22μm filter spare;

[0039] 3) Mix the shredded tissue pieces in 1) and the tissue-digesting enzyme solution prepared in 2) in a 1:1 volume ratio in a 50ml centrifuge tube, place in a shaker at 37°C, vibrate at 200rpm, and digest for about 3 hours. Wait until the tissue block is basically completely digested;

[0040] 4) The ...

Embodiment 2

[0043] Example 2: Identification of umbilical cord mesenchymal stem cells

[0044] 1) Identification of MSCs cell surface markers by flow cytometry: Take MSCs grown to 90% confluence at passages P4-P6, digest with cell digestion solution, resuspend and centrifuge, and resuspend the cell pellet with PBS to 1-2×10 6 / ml of single cell suspension, 0.1ml in each flow tube; add fluorescently labeled antibodies CD14-FITC, CD34-FITC, CD45-FITC, CD73-FITC, CD90-FITC, CD105-FITC, IgG1, κ-FITC, IgG2α, κ-FITC, incubated on ice in the dark for 40 minutes; PBS was washed to remove unbound antibodies, then resuspended in 500ulPBS, and tested on the machine; CD14-FITC used IgG2α, κ-FITC as the isotype control, and the rest IgG1, κ-FITC were used as the isotype control; the identification results can be found in figure 1 , the expression of markers CD73, CD90, and CD105 is above 90%, and the expression of CD14, CD34, and CD45 is below 5%;

[0045] 2) MSCs osteogenic differentiation identifi...

Embodiment 3

[0046] Embodiment 3: Real-timequantitativePCR experiment detects the expression of TRAIL

[0047] 1) Take the MSCs grown to 90% fusion in the P4-P6 generation, digest the cells, and re-inoculate into a 6-well cell culture plate, 2×10 5 / per well, 2ml of α-MEM medium containing 10% fetal bovine serum and 1% double antibody per well, placed at 37°C, 5% CO 2 1. After adherent culture in a saturated humidity incubator for 12 hours, inflammatory factor stimulation was added, and 4 groups were set up, TNF-α stimulation group alone, IFN-γ stimulation group alone, TNF-α and IFN-γ combined stimulation group , and an unstimulated control group, wherein the final concentrations of TNF-α and IFN-γ were 40ng / ml and 100ng / ml, respectively;

[0048] 2) After being stimulated by inflammatory factors for 24 hours, remove the medium, add 1ml Trizol to each well of the 6-well plate, pipette repeatedly, lyse the cells for 5min, and transfer the Trizol solution to 1.5ml RNasefree EP tube;

[004...

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Abstract

The invention belongs to the medical and biotechnical fields and in particular relates to application of a mesenchymal stem cell in preparation of a medicine used for treating M5 type leukaemia. The mesenchymal stem cell provided by the invention is obtained by stimulating specific inflammatory factors TNF-alpha and IFN-gamma; the capability of the mesenchymal stem cell for inducing tumor cell apoptosis is realized by a tumor necrosis factor-related apoptosis inducing ligand TRAIL; and the mesenchymal stem cell is in vitro. By virtue of stimulation on TNF-alpha and IFN-gamma, the TRAIL expression capability of the mesenchymal stem cell is obviously improved, and anti-tumour capability is obviously enhanced; compared with chemical therapy, toxic and side effects in mesenchymal stem cell transplantation are lower; due to low immunogenicity of the mesenchymal stem cell, morbidity of graft versus host disease is also greatly reduced, clinical requirement is met, and a new thought is provided for stem cell transplantation therapy. The mesenchymal stem cell can be applied to preparation of the medicine used for treating the M5 type leukaemia.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and in particular relates to the application of mesenchymal stem cells in the preparation of medicines for treating M5 leukemia. Background technique [0002] Acute myeloid leukemia (AML) is a common malignant tumor of the hematopoietic system, characterized by rapid progression, severe disease, and high mortality, which seriously threatens people's health. Acute monocytic leukemia (Acute Monocytic Leukemia, M5) is a more common type of AML. In my country, the incidence of M5 in AML is second only to M2, accounting for 23.2%-26.9% of AML. Most M5 patients are clinically manifested as leukocytosis, prone to extramedullary infiltration (including liver, spleen, lymph nodes, gums, skin, central nervous system, etc.), high rate of adverse chromosomal karyotype, and complete remission (CR) rate Low, easy to relapse, poor prognosis, etc. The heterogeneity and complexity of M5 determine t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/51A61K9/08A61P35/02
CPCA61K9/0019A61K35/51C12N5/0668
Inventor 沈萍萍姚永芳陈兵黄亚红
Owner NANJING UNIV
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