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Application of KDM3B gene in mesenchymal stem cell osteogenic/odontogenic differentiation, proliferation and migration chemotaxis

A technology of stromal stem cells and directed differentiation, applied in the field of bioengineering, can solve the problem that the function regulation of odontogenic mesenchymal stem cells has not been explored yet.

Active Publication Date: 2020-11-24
BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the functional regulation of KDM3 family on odontogenic mesenchymal stem cells has not been excavated

Method used

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  • Application of KDM3B gene in mesenchymal stem cell osteogenic/odontogenic differentiation, proliferation and migration chemotaxis
  • Application of KDM3B gene in mesenchymal stem cell osteogenic/odontogenic differentiation, proliferation and migration chemotaxis
  • Application of KDM3B gene in mesenchymal stem cell osteogenic/odontogenic differentiation, proliferation and migration chemotaxis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0232] Example 1 Effect of KDM3B on SCAP osteogenic / odontogenic differentiation ability

[0233] 1. Determination of alkaline phosphatase activity:

[0234] KDM3Bsh, Scramsh, HA-KDM3B and Vector cells in the logarithmic growth phase were digested to make a single cell suspension, and 2×10 5 The concentration of each well was inoculated in a 6-well plate, placed in an incubator overnight, and the growth of the cells was observed under an inverted microscope to 80%, and the original medium was replaced with a commercially available osteogenic induction medium STEMPRO (Invitrogen, USA). After 3 days of osteogenesis induction, alkaline phosphatase activity kit was used to detect.

[0235] The operation steps are as follows:

[0236] (1) Preparation of alkaline phosphatase lysate

[0237] Table 1

[0238]

[0239] (2) Prepare Stock substrate Sol gold + silver flakes and add 5ml of pure water

[0240] (3) Preparation of stop solution: 0.5N NaOH

[0241] (4) Make a standard ...

Embodiment 2

[0331] Example 2 The effect of KDM3B on the proliferation ability of SCAP

[0332] 1. CFSE proliferation assay:

[0333] (1) Take the KDM3Bsh, Scramsh, HA-KDM3B and Vector cells in the logarithmic growth phase, trypsinize and count them, and adjust the cell density to 1×10 with PBS 6 a / mL;

[0334] (2) Add 1 μL of CFSE working solution (1 μM) to each mL of cell suspension, and incubate at 37° C. in the dark for 20 minutes.

[0335] (3) Incubate with 5 times the volume of normal medium at 37°C for 5 minutes to remove excess dye in the solution.

[0336] (4) After centrifugation and washing with PBS, add common medium to resuspend, press 1×10 5 Inoculate in a 6-well plate at a density of one per well, with 3 wells in each group, and culture in an incubator for 3 days;

[0337] (5) Digest with 0.25% trypsin, wash by centrifugation with PBS, discard the supernatant, add 300 μL of 1% paraformaldehyde to fix, and protect from light at 4°C;

[0338] (6) Flow cytometry was used t...

Embodiment 3

[0349] Example 3 Effect of KDM3B on SCAP Migration and Chemotaxis

[0350] 1. Scratch test:

[0351] (1) KDM3Bsh, Scramsh, HA-KDM3B and Vector cells were mixed with 4×10 5 The density of cells / well is seeded in a 6-well plate, and cultured in the culture medium reaches 90% to 95% confluence;

[0352] (2) Use a 1000 μl pipette tip to draw a scratch on the bottom of the dish compared to a ruler;

[0353] (3) Wash twice with PBS, remove the underlined cells, and culture in DMEM fresh medium without FBS and double antibody;

[0354] (4) Record the images of the same position after injury induction at 0h, 24h, and 48h under a microscope, and measure the degree of wound closure;

[0355] (5) Image Pro 1.49 software was used to analyze the numerical values, and the void area (VA) and height (H) were calculated to obtain the relative width (RW), with the formula RW=VA / H.

[0356] 2. Transwell chamber experiment:

[0357] (1) Take KDM3Bsh, Scramsh, HA-KDM3B and Vector cells in a g...

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Abstract

The invention relates to the technical field of bioengineering, in particular to application of a KDM3B gene in mesenchymal stem cell osteogenic / odontogenic differentiation, proliferation and migration chemotactic processes. It is found that the KDM3B gene has the effect of positively regulating the function of the mesenchymal stem cells, a basis is provided for application of KDM3B in the processof promoting osteogenic / odontogenic differentiation and proliferation and migration chemotaxis of the mesenchymal stem cells, and a pathway for mediating significant changes of the function of the differentially expressed gene is enriched based on a KEGG database. 14 significant pathways related to differential gene expression are identified in total, and the pathways play a key role in functionregulation of KDM3B. An interaction library based on remarkably regulated GOs and pathways is constructed through signal network analysis, so that 12 core genes are identified according to the degreeof gene interaction, and an important theoretical basis is laid for studying the reconstruction and regulation mechanism of tooth root regeneration seed cells.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the application of the KDM3B gene in the process of osteotropic / odontogenic differentiation, proliferation, migration and chemotaxis of mesenchymal stem cells. Background technique [0002] Caries, deformity, trauma and other reasons can lead to irreversible pulp inflammation in young permanent teeth, and then lead to complete necrosis of the pulp. In severe cases, it can cause periapical inflammation, and ultimately affect the normal development of the root. Young immature permanent teeth have short roots, apical foramen opening in a bell-shaped shape, and root canal walls are weak. The clinical treatment of this kind of teeth is difficult, and root fracture or loosening is very easy to occur, and the tooth loss rate is high, and this kind of patients are young, and premature tooth loss will lead to the disorder of full-mouth occlusal function, affect pronunciation and fa...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0654C12N2506/1361C12N2501/73C12N2501/15C12N2501/58C12N2501/50C12N2501/40
Inventor 张建鹏张琛刁树范志朋杨昊清
Owner BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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