Application of cdc20 in osteogenic differentiation of mesenchymal stem cells

A technology of 1. CDC20 and mesenchymal stem cells, applied in the field of stem cells, can solve problems such as increased expression and unclear effect of CDC20 on bone formation

Active Publication Date: 2022-05-17
PEKING UNIV SCHOOL OF STOMATOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many studies have reported the biological functions of CDC20 in brain development, cilia disassembly and regulation of apoptosis, and the expression of CDC20 is elevated in many cancers, which are associated with poor prognosis in lung, bladder, colon, breast cancer and oral squamous cell carcinoma Related, but role of CDC20 on bone formation is unclear

Method used

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  • Application of cdc20 in osteogenic differentiation of mesenchymal stem cells
  • Application of cdc20 in osteogenic differentiation of mesenchymal stem cells
  • Application of cdc20 in osteogenic differentiation of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Changes in the expression level of CDC20 during the osteogenic differentiation of human bone marrow mesenchymal stem cells

[0032] Cell culture:

[0033] Human bone marrow mesenchymal stem cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Human bone marrow mesenchymal stem cells were collected from three different donors and were approved by the Ethics Committee of Peking University Stomatological Hospital. Proliferation medium is α-MEM medium (including 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin). Osteogenic differentiation medium includes proliferation medium plus 0.2mM vitamin C, 10mM sodium β-glycerophosphate and 100nM dexamethasone. Cells were seeded in culture dishes at 37°C, 5% CO 2 Cultured under conditions, the medium was changed once every 3 days. When the cells grew to 80% confluent state under an inverted microscope, they were digested and passaged with 0.25% trypsin, and added to the mediu...

Embodiment 2

[0062] Effects of inhibiting or overexpressing CDC20 on the osteogenic differentiation of human bone marrow mesenchymal stem cells

[0063] Plasmid expression, siRNA and lentiviral transfection:

[0064] The overexpression plasmid vector Vector and the overexpression CDC20 plasmid WT-CDC20 were purchased from ADDGENE Company. When the cells grew to 70%-80% confluence, the plasmid was transferred into the mesenchymal stem cells using the transfection reagent Lipo3000 according to the instructions, and the cells were collected after 24-36 hours for subsequent experiments. siRNA (small interfering RNA) was used to silence the expression of genes. The control siRNA and CDC20si were purchased from Shanghai Gemma Company. For mesenchymal stem cells, cells were collected at an appropriate time for subsequent experiments. The lentivirus can be integrated into the genome for gene interference. The knockdown control and CDC20sh were purchased from Shanghai Gemma Company. 72h after tr...

Embodiment 3

[0096] Knockdown of CDC20 regulates downstream genes of NF-κB pathway

[0097] Experiments were performed using qRT-PCR methods as described above. The primer information used for amplification is as follows:

[0098]

[0099]

[0100] image 3 It shows that the low expression of CDC20 provided by the embodiment of the invention promotes the high expression of NF-κB downstream genes. image 3 A-3D shows that after TNF-α was treated for 0.5 hours, the qRT-PCR results showed that the expression levels of NF-κB downstream genes IL-8, IL-6, and ICAM1 in the CDC20 knockdown group were significantly increased.

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Abstract

The invention relates to the application of CDC20 in the osteogenic differentiation process of mesenchymal stem cells. It was found that CDC20 can positively regulate the osteogenic function of mesenchymal stem cells, which provided the basis for the application of CDC20 in promoting the osteogenic differentiation of mesenchymal stem cells, and identified its effect on NF‑κB active subunit p65 through proteasome Mechanism of action for pathway degradation. It provides a new theoretical and experimental basis for the targeted use of small molecular compounds to inhibit the activity of target proteins in the molecular therapy of bone regeneration.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to the application of CDC20 in the osteogenic differentiation of mesenchymal stem cells. Background technique [0002] Bone defects caused by trauma, congenital malformations, infection, and tumors require bone tissue regeneration and reconstruction. As the population ages, there is an increasing need for regenerative repair of bone defects. In recent years, the application of mesenchymal stem cells combined with tissue engineering technology to promote tissue regeneration has become a frontier and important scientific issue. Human mesenchymal stem cells are an attractive source of adult stem cells in bone tissue engineering technology, and have important application value and good application prospects in the repair of various bone defects and bone regeneration in the oral and maxillofacial region. Human mesenchymal stem cells have the potential to differentiate into bone, car...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0654C12N2501/405C12N2506/1353
Inventor 周永胜张萍杜杨格刘云松吕珑薇张晓张敏
Owner PEKING UNIV SCHOOL OF STOMATOLOGY
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