Application of microRNA-29b in articular cartilage repair as drug target

A cartilage repair and drug technology, applied in the field of cartilage tissue engineering, to prevent cartilage fibrosis and heterotopic ossification, realize tissue engineered cartilage repair, and promote maintenance and stability

Active Publication Date: 2015-05-06
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on repairing defective articular cartilage from the perspective of inhibiting hypertrophy of MSCs-derived chondrocytes in the prior art

Method used

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  • Application of microRNA-29b in articular cartilage repair as drug target
  • Application of microRNA-29b in articular cartilage repair as drug target
  • Application of microRNA-29b in articular cartilage repair as drug target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Mesenchymal stem cell cell culture and hypertrophic chondrogenic differentiation induction experiment

[0036] (1) Acquisition and culture of mouse bone marrow mesenchymal stem cells

[0037] Under sterile conditions, use a 1 mL syringe to flush the bone marrow of the mouse femur, transfer it into a centrifuge tube containing 5 mL of DMEM medium, and gently pipette to make a cell suspension; transfer the cell suspension to a cell suspension containing 5 mL of lymphocytes. In the centrifuge tube of the separation liquid, centrifuge at 2000r / min for 20min; after centrifugation, collect the mononuclear cells of the cloudy buffy coat and place them in another centrifuge tube, wash twice with DMEM low-sugar medium, and centrifuge (1000r / min). min, 5min), discard the supernatant, resuspend the cells in DMEM basal medium containing 15% fetal bovine serum, and finally use the primary cells at 1×10 6 Cells / mL were seeded in culture flasks and placed at 37°C, 5% CO 2 ...

Embodiment 2

[0040] Example 2 The increased expression of microRNA-29b promotes the process of chondrocyte hypertrophy

[0041] (1) The cell clusters of 14d, 21d and 28d differentiated by cartilage induction were taken, total RNA was extracted and reverse transcribed into cDNA. The specific operation of reverse transcription is as follows: put the PCR tube on ice, and add 5 μL 5×RT Reaction Buffer, 1 μL 25mM dNTP mixture, 1 μL 25U / μL RNase inhibitor, and 1 μL 200U / μL M-MLV reverse transcription respectively. Enzyme and 4 μL of DEPC water, then the mixture of RNA and primers was added to make the total reaction volume 25 μL. After mixing the above components, water bath at 42 °C for 60 min, and finally heated to 85 °C for fire treatment for 5 min to obtain a cDNA solution. Store at -70°C for later use.

[0042] The mRNA sequences of type II collagen (Col II), type X collagen (Col X), Sox9 and internal reference gene GAPDH were obtained from the Nucleotide database, and the corresponding PC...

Embodiment 3

[0065] Example 3 Transfection of microRNA-29b inhibitor can significantly inhibit the expression of hypertrophic chondrocyte-related genes

[0066] Transfection experiment: using Lipofectamin TM 2000 kit, take the normal cultured mesenchymal stem cells for cell-forming micromass culture, use medium B (complete cartilage induction medium containing TGF-β3) for cartilage induction culture for the first 14 days, and change to medium from the 15th day C (hypertrophic cartilage induction complete medium) continued to be cultured, and materials were collected on the 14th, 21st, and 28th days of culture for subsequent experimental detection. The experiment was divided into two groups: the control group and the experimental group. The experimental group was transfected with microRNA-29b inhibitor, and the control group was transfected with microRNA-29b inhibitor negative control (bioinformatics analysis showed that it was related to the genomes of humans, mice, and rats, and All miR...

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Abstract

The invention discloses an application of microRNA-29b in articular cartilage repair as a drug target. According to the application disclosed by the invention, the research indicates that microRNA-29b is a key miRNA for regulating the hypertrophy change of MSCs after chondrogenic differentiation, therefore, the hypertrophy of MSCs-derived cartilage cells is inhibited by virtue of an microRNA-29b inhibitor, the phenotypes of the MSCs-derived cartilage cells are stabilized, and the fibrosis and the heterotopic ossification of tissue engineering cartilages are prevented, thus further realizing tissue engineering cartilage repair, and providing a new thought for tissue engineering cartilage construction, cartilage framework material design and cartilage repair.

Description

technical field [0001] The invention belongs to the technical field of cartilage tissue engineering, in particular to the application of MicroRNA-29b as a drug target in the repair of articular cartilage. Background technique [0002] Damage to the articular cartilage on the articular surface can be caused by various causes such as trauma, osteoarthritis, and tumors. It often presents progressive damage, which seriously affects the quality of life of patients. Due to the lack of self-repairing ability of articular cartilage, how to achieve functional repair of articular cartilage defects is a major problem in the medical field. Tissue engineering technology combines cell biology and biomaterials to perform physiological repair of damaged tissue, providing an ideal repair method for the repair of articular cartilage defects. Among them, the selection and optimization of suitable seed cells is an important proposition in this field. Mesenchymal stem cells (MSCs) are consider...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61P19/04
Inventor 董世武康菲苗莹杨波李建美曹震窦策赵春容
Owner ARMY MEDICAL UNIV
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