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Application of OPTN gene in identification of dental pulp stem cells for identifying dental pulp stem cells and method thereof and product

A technology of dental pulp stem cells and periodontal ligament stem cells, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of no effective tools, cumbersome process, high identification cost, etc. low effect

Active Publication Date: 2020-11-13
KYBIOSTEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The proliferation, differentiation and clinical application of different stem cells are different. However, there is currently no effective tool for the identification of these stem cell subsets from different locations in the oral cavity. The cost of identification by flow cytometry in the prior art is high and The process is cumbersome

Method used

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  • Application of OPTN gene in identification of dental pulp stem cells for identifying dental pulp stem cells and method thereof and product
  • Application of OPTN gene in identification of dental pulp stem cells for identifying dental pulp stem cells and method thereof and product
  • Application of OPTN gene in identification of dental pulp stem cells for identifying dental pulp stem cells and method thereof and product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Detection of Cell Proliferation Ability

[0069] Simultaneously culture human dental pulp stem cells (DPSCs) and gingival stem cells (GMSCs) until the fusion rate is above 80%, digest and centrifuge, count, and count according to 10 4 Cells / mL were inoculated into 96-well plates, and MTT was added every 24 hours for incubation for 4 hours, and the OD value was measured at 490 nm in order to measure cell viability and proliferation ability.

[0070] The results are attached figure 1 As shown, compared with GMSCs, DPSCs showed stronger value-added ability.

Embodiment 2

[0071] Example 2 Detection of protein expression level of OPTN in dental pulp stem cells

[0072] To search for molecules associated with proliferation, we investigated the differences in the protein composition of the two DPSCs and GMSCs cells by quantitative proteomics. We removed the supernatant from the overgrown two kinds of cells, washed them three times with PBS, added cell lysate, centrifuged at 12000rpm for 5 min, carried out reduction, alkylation and peptide fragmentation respectively, and added TMT tag for labeling, It was then quenched with 0.5% hydroxylamine at room temperature and detected on LC-MS / MS after drying.

[0073] The results are attached figure 2 As shown, it was found that the protein OPTN molecule was highly expressed in DPSCs.

Embodiment 3

[0074] Example 3 Detection of OPTN gene expression in stem cells by qPCR

[0075] Remove the supernatant (cells of the same generation) from the DPSCs and GMSCs confluent in the culture dish, wash with PBS three times, then add Trizol for cell lysis, then add chemical reagents chloroform, isopropanol and 75% absolute ethanol, Total RNA was extracted from the cells after distribution by centrifugation. Then the concentration was measured, quantified and reverse transcribed into cDNA, and the following primers were added to measure the expression of mRNA levels of OPTN genes in DPSCs and GMSCs.

[0076] OPTN-F: 5'-AGCAAAACCATTGCCAAGC-3' (SEQ ID NO.1);

[0077] OPTN-R: 5'-TTTCAGCATGAAAATCAGAACAG-3' (SEQ ID NO. 2).

[0078] The result is as image 3 As shown, OPTN gene was highly expressed in DPSCs compared with GMSCs.

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Abstract

The invention relates to the technical field of biology, and particularly provides an application of an OPTN gene in identification of dental pulp stem cells for identifying the dental pulp stem cells, and a method thereof and a product. The biomarker OPTN gene capable of being used for identifying the dental pulp stem cells is discovered for the first time through tests, and the high-purity dental pulp stem cells can be identified and separated through differential expression of the gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, to an OPTN-based detection method for identifying dental pulp stem cells. Background technique [0002] Dental pulp mesenchymal stem cells (DPSCs), a type of mesenchymal stem cells (MSCs), are adult stem cells isolated from the pulp tissue of adult teeth. Studies have proved that DPSCs are a kind of mesenchymal stem cells with high proliferation and multi-lineage differentiation ability. The isolated DPSCs can be differentiated into osteogenic, chondrogenic, adipogenic and neuroblast cells under certain culture conditions in vitro. In addition to promising applications in dental tissue engineering, DPSCs are also widely used in the field of regenerative medicine. Compared with embryonic stem cells, DPSCs have no ethical issues, no immune rejection, no tumorigenicity and other issues. So DPSCs is a good cell material with potential clinical application. [0003] Oral stem cells have...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/158
Inventor 雷童王建张晓霜刘彦彦王治世杜宏武
Owner KYBIOSTEM CO LTD
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