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Application of miR-106a-5p simulant in preparation of bone defect repair medicine

A 1. mir-106a-5p, mimic technology, applied in the field of bone repair materials, can solve the problem of miR-106a-5p bone defect repair drugs and other problems

Pending Publication Date: 2021-07-27
THE FIRST AFFILIATED HOSPITAL OF 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 the study of vesicle-mediated miR-106a-5p in intercellular communication, and its role in the skeletal system is rarely reported. Fix any reports of drugs

Method used

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  • Application of miR-106a-5p simulant in preparation of bone defect repair medicine
  • Application of miR-106a-5p simulant in preparation of bone defect repair medicine
  • Application of miR-106a-5p simulant in preparation of bone defect repair medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Effects of miR-106a-5p mimics on osteogenic differentiation of bone marrow mesenchymal stem cells

[0028] The miR-106a-5p (SEQ ID NO.1) mimic was synthesized, and the miR-106a-5p mimic was transferred into mouse bone marrow mesenchymal stem cells (MSCs). The specific method is as follows:

[0029] 24 hours before transfection, an appropriate amount of MSCs cells were seeded in a 24-well culture plate, and the adherent cells were transfected when the cells grew to 50%. For transfection, dissolve miR-106a-5p mimic (5ng / ml) in serum-free medium, and at the same time, disperse Lipofectamine2000 in an equal volume of serum-free medium, shake gently for 5 minutes, mix the two solutions, and place 20 minutes. Aspirate the original cell culture medium, replace with fresh culture medium (with or without 10% calf serum), slowly add the transfection solution into the culture medium, and incubate at 37°C for 6-8 hours. Finally, the transfection solution was discarded, and the c...

Embodiment 2

[0033] sEVs protect miR-106a-5p mimics from RNase degradation

[0034] Differential centrifugation to extract sEVs, the specific steps are as follows: collect the supernatant of MSCs cells in a 15ml centrifuge tube, centrifuge at 300g, 4°C for 10min to remove dead cells; centrifuge at 1,000g for 15mins, discard the pellet, and remove cell debris; 3,000g, Centrifuge for 15mins, discard the pellet, and remove apoptotic bodies; centrifuge at 18,000g for 30mins, discard the pellet, and remove large extracellular vesicles; ultracentrifuge at 110,000g, 4°C for 70mins, take the pellet; resuspend in PBS, 110,000g, 4°C , ultracentrifugation for 70mins, and the precipitate is sEVs. The relative expression of miR-106a-5p in sEVs was engineered using the Exo-Fect Exosome Transfection Kit (System Bio, USA). The sEVs transfected with the miR-106a-5p mimic were again pelleted using the differential centrifugation method described above. Resuspended in PBS, sEVs transfected with miR-106a-5p...

Embodiment 3

[0040] MiR-106a-5p mimics induce the expression of osteogenic differentiation genes

[0041] (1) Effect of miR-106a-5p mimic inhibitor on osteogenic differentiation

[0042] The sEVs were extracted according to the differential centrifugation method described in Example 2, and the relative expression of miR-106a-5p in the sEVs was designed using the Exo-Fect exosome transfection kit (System Bio, USA). The sEVs transfected with miRNA mimic control fragment, miR-106a-5p mimic, miRNA inhibitor control fragment and miR-106a-5 inhibitor were obtained by precipitation again using the above centrifugation method.

[0043] Group 1: control group, sEVs of miRNA mimic control, miR-NC-sEVs;

[0044] Group 2: experimental group, miR-106a-5p overexpressed sEVs, miR-106a-5p-sEVs;

[0045] Group 3: control group, sEVs of miRNA inhibitor control, si-NC-sEVs;

[0046] Group 4: experimental group, miR-106a-5p low expression sEVs, si-miR-106a-5p-sEVs;

[0047] BMSCs were divided into 1×10 5...

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Abstract

The invention particularly relates to application of a miR-106a-5p simulant in preparation of a bone defect repair medicine, and belongs to the field of bone repair materials. According to the miR-106a-5p simulant provided by the invention, by down-regulating Fam134a, inhibition of Fam134a on gene expression of Alp, Sp7, Col1a1 and Runx2 is relieved, osteoblast differentiation and maturation are promoted, bone defect repair is realized, and a new research direction is provided for promoting bone defect repair.

Description

technical field [0001] The invention belongs to the field of bone repair materials, in particular to the application of miR-106a-5p mimics in the preparation of bone defect repair medicines. Background technique [0002] However, a shortage of bone due to trauma, inflammation, infection, bone disease, or surgery is called a bone defect. Due to the existence of bone defects, bone nonunion, delayed union or nonunion, and local dysfunction are often caused. Bone defect has a high incidence rate in clinical practice and is difficult to treat, which is one of the clinical problems in orthopedics. [0003] Bone is an organ with mechanical and metabolic functions. The maintenance of bone homeostasis mainly depends on the balance of functions between osteoblasts (OB) and osteoclasts (OC) and the coordination of the two cells. , bone homeostasis favors bone formation when osteoblasts are overactive, and bone resorption when osteoclasts are overactive. [0004] The process of bone ...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K47/46A61L27/36A61L27/54A61P19/08
CPCA61K31/7088A61K47/46A61P19/08A61L27/54A61L27/3608A61L2300/258A61L2300/64A61L2430/02
Inventor 马秦雨吴雨桐梁蒙蒙许建中
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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