Osteosarcoma stem cell molecular marker CD24 and application thereof

A molecular marker, CD24 technology, applied in the field of biomedicine, can solve the problems of rare osteosarcoma stem cell system research, and there is no report on the correlation between CD24 gene and protein and osteosarcoma stem cells, achieving good specificity and high sensitivity , the effect of strong proliferation and invasion ability

Inactive Publication Date: 2019-04-16
SECOND AFFILIATED HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although osteosarcoma is the most common malignancy, systematic studies on osteosarcoma stem cells are r

Method used

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  • Osteosarcoma stem cell molecular marker CD24 and application thereof
  • Osteosarcoma stem cell molecular marker CD24 and application thereof
  • Osteosarcoma stem cell molecular marker CD24 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Osteosarcoma CD24 + Expression of stemness genes in subpopulation cells

[0046] experimental method:

[0047] (1) Cell sorting: immunomagnetic bead sorting and flow analysis

[0048] Four osteosarcoma cell lines MG-63, MNNG / HOS, U-2OS and OSC228 parental cells and corresponding spheroid cells were subjected to immunomagnetic bead separation. The magnetic bead separation steps were performed in accordance with the operating instructions provided by the manufacturer with Miltenyi Biotec MACSCell Separation Kit is performed. The cells are repeatedly passed through the sorting column at least three times to achieve a purity of more than 95% of the sorted cells. Finally, the four parent cells of osteosarcoma cell lines and the corresponding spheroid cells are sorted to obtain high-purity CD24 + .

[0049] Flow cytometric sorting was performed using BD FACSAria. Collect the cells to remove the culture medium, digest with trypsin for about 3 minutes, digest until the cells...

Embodiment 2

[0059] Example 2 Osteosarcoma CD24 + Subgroups have stronger proliferation, invasion and migration capabilities

[0060] experimental method:

[0061] (1) Cell sorting

[0062] Same as Example 1

[0063] CD24 was sorted from MG-63 and MNNG / HOS cell lines + Subgroups and CD24 - Subpopulation of cells.

[0064] (2) Cell proliferation ability experiment

[0065] Add 100 μL of CD24+ subgroup and CD24- subgroup cell suspension to 96-well plates. The culture plate was pre-cultured in an incubator for 24 hours (37°C, 5% CO2). PBS can be optionally added to the periphery of the 96-well plate to prevent excessive liquid evaporation during the culture process. Incubate the culture plate in the incubator for different periods of time (1, 2, 3, 4, 5 days). Discard the medium and wash the cells twice with medium, then add new 100ul medium, add 10μLCCK8 solution to each well (it is best to mix CCK8 reagent and medium according to 1:10 before adding). Incubate the culture plate in the incubator fo...

Embodiment 3

[0073] Example 3 CD24 as a target affects the biological function of osteosarcoma cells

[0074] experimental method:

[0075] (1) Lentiviral vector construction, lentiviral packaging and cell infection

[0076] The CD24 sequence was amplified by PCR from human genomic DNA and cloned into the lentiviral expression vector PLVX (restriction endonuclease sites are BamH and EcoRI), and the recombinant plasmid was named PLVX-CD24. The recombinant plasmid that interfered with CD1 was named PLVX-CD24-shRNA. In this embodiment, two sh sequences are designed as CD24sh-1: TACTGGAACATCTAAGCAT; CD24sh-2: TTAATATTGGCATCCATCA. During lentivirus packaging, PLVX-CD1 / PLVX-CD1-shRNA, packaging plasmid psPAX2 and envelope plasmid pMD2.G were co-transfected into 293T cells with Lipofectamine2000 (Invitrogen) to obtain virus particles. The titer of virus was determined in HEK 293T cells by serial dilution method. Infect the cells with the virus after measurement, add 6μg / mL polybrene, and then determ...

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Abstract

The invention relates to an osteosarcoma stem cell molecular marker CD24 and application thereof. The molecular marker comprises a CD24 gene or CAD24 protein. The molecular marker CD24 is used as an osteosarcoma stem cell molecular marker, a product for detecting CD gene expression is used for detecting the CD24 gene expression level in a to-be-measured osteosarcoma tissue sample, the CD24 gene expression level in the to-be-measured osteosarcoma tissue sample is compared with that in the normal para-carcinoma tissue, the progression of the osteosarcoma disease is diagnosed, assessment and prognosis are conducted, the marker has good specificity and high sensitivity, and a brand-new approach is provided for osteosarcoma diagnosis and/or treatment.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and relates to an osteosarcoma stem cell molecular marker CD24 and its application. Background technique [0002] Osteosarcoma is one of the most common bone malignant tumors, accounting for about 20-35% of primary malignant bone tumors, and the age of occurrence is 10-25 years old. Osteosarcoma is extremely malignant. Hematogenous dissemination can occur in the early stage. About 10% of patients have metastasis at the first visit, especially lung metastasis. In addition, osteosarcoma is sensitive to various antitumor drugs. The sex is poor, the prognosis is also poor, the 5-year survival rate is low, the disability rate is high, and the quality of life is low. Generally speaking, the average time from diagnosis to lung metastasis of osteosarcoma is 10 months, and the time from metastasis detection to death is 6 months. Most of them die within 1-2 years after diagnosis. Although with the progress of osteo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886G01N33/68G01N33/574
CPCC12Q1/6886G01N33/57407G01N33/68G01N2333/70503
Inventor 周振华肖建如胡硕王旭东张薇薇匡牧宇龚德军贾奇李焱曹佳实
Owner SECOND AFFILIATED HOSPITAL SECOND MILITARY MEDICAL UNIV
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