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Sorting method of human osteoblasts

An osteoblast and sorting technology, applied in the field of biomedicine, can solve the problem that the separation scheme of human osteoblasts cannot meet the requirements of single-cell sequencing, and achieve the effect of reducing cell impurities, high cell activity, and large number of cells

Inactive Publication Date: 2020-08-21
HUNAN NORMAL UNIVERSITY
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Problems solved by technology

The human osteoblast isolation protocol proposed by Fujita et al. (1) cannot meet the requirements of single-cell sequencing

Method used

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  • Sorting method of human osteoblasts
  • Sorting method of human osteoblasts
  • Sorting method of human osteoblasts

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Embodiment Construction

[0036] The technical solution of the present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

[0037] Bone samples and flow cytometry results such as figure 1 shown. The cell data obtained after sorting can be clustered into 6 clusters according to the uniform manifold approximation and projection (UMAP) clustering method ( figure 2 ), the osteoblast-specific marker gene RUNX2 was only highly enriched in C1 and C2 ( image 3 ), were not expressed in other clusters, indicating the presence of other contaminating cells in our dataset. Contaminating cells are (1) two nucleated erythrocyte clusters C3 and C4 ( image 3 ); (2) a smooth muscle cell cluster C5, in which ACTA2 and CNN1 genes encode smooth muscle α-2 actin, which is specific for differentiated mature smooth muscle cells ( image 3 ); (3) a neutrophil cluster C6 ( image 3 ). The above analysis shows that based on ALPL + / CD45 / 31 / 34 - The human ...

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Abstract

The invention discloses a sorting method of human osteoblasts, which comprises the following steps: performing digestion twice by using endotoxin-free collagenase with the digestion concentration of 1.0 mg / m, cell suspension centrifugal force being 400; after the twice digestion is finished, filtering out bone fragments by using a 70-micron filter screen, collecting the supernatant, adding PBS to50 ml, and centrifuging the liquid; carrying out lysis for multiple times by using an erythrocyte lysis solution, wherein the lysis cannot last for excessive long time, until no obvious red substanceexists in cell precipitate; filtering the liquid by using a 40-micron filter screen to obtain a supernatant after the erythrocyte splitting is finished; and washing the red-split filtered cells with PBS (phosphate buffer solution) for multiple times, centrifuging the liquid, and removing impurities. The FACS sorting employs ALPL and CD45 / 34 / 31 antibodie for labeling and separating human osteoblasts, so that the activity of the human osteoblasts obtained through the method is higher, the number of cells is larger, and cell impurities are greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a method for sorting human osteoblasts, in particular to a method for sorting human osteoblasts used in single-cell sequencing technology. Background technique [0002] Osteoblasts (OB) are mainly differentiated from mesenchymal progenitor cells in the inner and outer periosteum and bone marrow stroma. They can specifically secrete a variety of biologically active substances, regulate and affect the process of bone formation and reconstruction. At different stages of maturation, osteoblasts exhibit four different morphologies in vivo, namely, preosteoblast, osteoblast, osteocyte, and bonelining cell. Preosteoblasts are the precursors of osteoblasts, differentiated from stromal stem cells, develop along the osteoblast lineage, and are located on the outside of the osteoblasts covering the bone-forming surface. Mature osteoblasts are a single layer of cells located on the bone su...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0654C12N2509/00C12N2509/10
Inventor 谭丽君周翠陈玉刘莹李小华张会洗肖红梅陈湘定邓红文
Owner HUNAN NORMAL UNIVERSITY
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