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Molecular marker combination and application thereof

A technique for using and labeling molecules, which is applied in the fields of cell biology and medicine, and can solve problems such as inability to distinguish megakaryocytes

Pending Publication Date: 2022-05-31
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, traditional transcriptomic methods cannot effectively distinguish functionally distinct megakaryocytes
[0006] Therefore, there are great challenges in exploring the functional heterogeneity of megakaryocytes

Method used

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  • Molecular marker combination and application thereof
  • Molecular marker combination and application thereof
  • Molecular marker combination and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] Example 1-Single-cell analysis found four megakaryocyte subpopulations and corresponding markers

[0161] Concrete experimental procedure, experimental result of the present embodiment are as follows:

[0162] 1. Experimental materials

[0163] (1) Experimental animals

[0164] C57BL / 6 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and bred at the Institute of Genetics and Development, Chinese Academy of Sciences.

[0165] (2) Donor bone marrow samples

[0166] Human bone marrow samples were obtained from 4 healthy male volunteers at the Institute of Hematology, Peking University People's Hospital. The acceptance of bone marrow samples from healthy volunteers was approved by the Ethics Committee of Peking University People's Hospital, in compliance with the regulations of the Declaration of Helsinki of the World Medical Association.

[0167] (3) Experimental reagents

[0168] Anti-mouse CD41 APC and anti-human CD41 APC antib...

Embodiment 2

[0184] Example 2 - Molecular markers can be used to label megakaryocyte subpopulations

[0185] Concrete experimental procedure, experimental result of the present embodiment are as follows:

[0186] 1. Experimental materials

[0187] (1) Experimental reagents

[0188] Anti-mouse CD105 PE and anti-mouse CD41 APC were purchased from eBioscience; rabbit polyclonal ARNTL antibody was purchased from Bio-Techne; anti-LSP1 antibody was purchased from Abcam; anti-MYLK4 polyclonal antibody was purchased from Cloud-Clone; MYLK4 rabbit polyclonal antibody was purchased from From Proteintech; goat anti-rabbit IgG AF488 and goat anti-rabbit IgG AF555 were purchased from Invitrogen; donkey anti-rabbit IgGDyLight 550 was purchased from Pierce; biotin anti-mouse Lineage Panel, biotin anti-mouse CD48 and BrilliantViolet 421 Streptavidin were purchased from BioLegend; 4% Paraformaldehyde (PFA) was purchased from YEASEN; sucrose was purchased from Amresco; EDTA was purchased from GIBCO; OCT...

Embodiment 3

[0209] Example 3 - Megakaryocytes marked by molecular markers have functions suggested by the transcriptome

[0210] 1. Experimental materials

[0211] Anti-mouse CD53 FITC was purchased from BioLegend; lipopolysaccharide LPS was purchased from Sigma; rabbit polyclonal ARNTL antibody was purchased from Bio-Techne; anti-LSP1 antibody was purchased from Abcam; anti-MYLK4 polyclonal antibody was purchased from Cloud-Clone; MYLK4 rabbit polyclonal antibody Purchased from Proteintech; goat anti-rabbit IgG AF488 and goat anti-rabbit IgG AF555 were purchased from Invitrogen; donkey anti-rabbit IgG DyLight 550 were purchased from Pierce; cell proliferation detection kit Cell Trace CFSE CellProliferation Kit and bacterial particles pHrodo Red E.coli BioParticles were purchased from Invitrogen.

[0212] 2. Experimental method

[0213] (1) LPS stimulation experiment

[0214] C57BL / 6 mice were injected intraperitoneally with 4mg / kg LPS, and the bone marrow of the mice treated for 0h,...

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Abstract

The invention relates to three megakaryocyte subgroups with different functional heterogeneity possibly existing in mouse megakaryocyte (MK), and specific molecular markers of the subgroups are respectively identified, including an MK molecular marker MYLK4 as a microenvironment supporting cell of HSC; the MK molecular marker for performing the immune regulation function is LSP1, CD53 or CD52; and the MK molecular marker with the plate production function is ARNTL. The molecular markers can be used for separating or marking MKs with different functions so as to promote the MKs to perform different functions, including HSC resting supporting, immunoregulation, plate production and the like.

Description

technical field [0001] The invention belongs to the field of cell biology and medicine, and specifically relates to a megakaryocyte group and a method for identifying the megakaryocyte group. Further, the present invention also relates to the use of a reagent for detecting marker molecules or a combination thereof in the preparation of a kit, and the use of an antibody or its composition in the preparation of a reagent. Background technique [0002] Megakaryocytes (Megakaryocytes, MK) are the largest and least numerous hematopoietic cells in the bone marrow. The diameter of MK can reach 50-100 μm, and the number of MK only accounts for 0.05-0.1% of bone marrow cells. MK is developed and differentiated from hematopoietic stem cells (HSC). Hematopoietic stem cells first differentiate into megakaryotic progenitor cells. Megakaryotic progenitor cells undergo polyploidization through endokaryotic division and mature gradually. Eventually, the number of chromosomes in megakary...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N5/078C12Q1/6888A61K35/19A61P7/00A61P37/02A61P35/00A61P35/02A61P7/06A61P19/02A61P29/00A61P25/00A61P19/08A61P21/00A61P19/06A61P17/06
CPCG01N33/6872G01N33/68G01N33/56966C12N5/0644C12Q1/6888A61K35/19A61P7/00A61P37/02A61P35/00A61P35/02A61P7/06A61P19/02A61P29/00A61P25/00A61P19/08A61P21/00A61P19/06A61P17/06C12N2509/00C12Q2600/158
Inventor 王前飞李玥莹孙舒金晨
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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