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Application of miR-27 or analog thereof to in vitro restraining of differentiation of mesenchymal stem cells to hypertrophic chondrocyte

A bone marrow mesenchymal and mir-27 technology, applied in the field of cartilage damage drugs, can solve problems such as prone to ossification and affecting the effect of regenerated cartilage repair

Inactive Publication Date: 2019-06-04
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, bone marrow mesenchymal stem cells have their own differentiation shortcomings in the differentiation of chondrocytes: the differentiation of bone marrow mesenchymal stem cells into chondrocytes undergoes a process similar to that of embryonic endochondral ossification, and the differentiated chondrocytes have the characteristics of growth plate hypertrophic chondrocytes. Express type X collagen and MMP13, prone to ossification, seriously affecting the effect of regenerated cartilage repair

Method used

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  • Application of miR-27 or analog thereof to in vitro restraining of differentiation of mesenchymal stem cells to hypertrophic chondrocyte
  • Application of miR-27 or analog thereof to in vitro restraining of differentiation of mesenchymal stem cells to hypertrophic chondrocyte
  • Application of miR-27 or analog thereof to in vitro restraining of differentiation of mesenchymal stem cells to hypertrophic chondrocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Primary isolation and purification of human bone marrow mesenchymal stem cells:

[0054] Add 5ml of bone marrow fluid from subjects diagnosed as having no blood disease provided by the Hematology Department of Jilin University First Hospital into a centrifuge tube, add 20ml of PBS buffer solution (pH value 7.4) into the centrifuge tube, centrifuge at 1500rpm for 5min, and centrifuge at room temperature 2 times. Aspirate the supernatant and remove the anticoagulant. After centrifugation, add 10ml of culture medium (DME / F12 containing 10% FBS, 10ng / ml bFGF), and then put it into a 10mm culture dish for adherent cells. 2 , 37 ° C incubator 48h after replacement of culture medium. After changing the medium twice, wash away the red blood cells with PBS buffer, and observe the bone marrow mesenchymal stem cells under a microscope (such as figure 1 -A). When the cells were confluent to 90%, they were subcultured, and when they were subcultured to P4, 2×10 5 Each well was ...

Embodiment 2

[0056] Chondrocyte induction and qRT-PCR identification

[0057] The 4th generation bone marrow mesenchymal stem cells prepared in Example 1 were mixed with 5×10 5 cells / ml density suspended in 0.5ml chondrocyte induction medium (high sugar DMEM medium, 1% ITS, 10ng / ml TGF-β3, 100nM dexamethasone, 40mg / ml proline, 25mg / ml ascorbic acid) and transferred to 15ml centrifuge Centrifuge the cells at 1500rpm for high-density cell pellet induction, replace the induction solution every 2 days, fix the cell mass with 4% paraformaldehyde at 3 weeks, and perform HE, Alcian blue staining and safranin O after embedding in paraffin dyeing. The formed cartilage balls were collected on the 7th day, 14th day, and 21d day of cartilage induction.

[0058] For the qRT-PCR identification of miR-27b-3P, the cartilage balls were collected on the 7th day and 14th day of cartilage induction, and the total RNA was extracted to identify the expression of miR-27b-3P.

[0059]The total RNA was extracte...

Embodiment 3

[0069] Bone marrow mesenchymal stem cells transfected with lentivirus carrying miR-27b-3P and identified by qRT-PCR

[0070] Divide the above by 2×10 5 Bone marrow mesenchymal stem cells seeded per well in a six-well plate were cultured for 24 hours, and when the confluence reached about 70%, they were transfected with a lentivirus carrying miR-27b-3P (Jikai Gene, Shanghai). Add the lentivirus (MOI index is 50) into the normal culture medium, divide into miR-27b-3P group, Scramble group and NC group, mix them and place them at 37°C, 5% CO 2 Incubate overnight in the incubator. After 24 hours, the culture medium containing lentivirus was replaced with normal culture medium. After 3 days of transfection, the fluorescent expression was observed under an inverted fluorescent microscope, and detected by fluorescent quantitative PCR (Gene Copoeia, China).

[0071] qRT-PCR detection kit for miRNAs (Gene Copoeia, USA) was used to detect target genes.

[0072] reaction system

[0...

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Abstract

The invention provides an application of miR-27 or an analog thereof to in vitro restraining of differentiation of mesenchymal stem cells to hypertrophic chondrocyte, and belongs to the technical field of medicines for inducing differentiation from the mesenchymal stem cells to chondrocyte and cartilage injury, an application of miR-27 to restraining of the mesenchymal stem cells to the hypertrophic chondrocyte, an application of the miR-27 to promotion of differentiation of the hypertrophic chondrocyte to the chondrocyte, and an application of miR-27 to preparation of medicines for treating cartilage injury. When the articular cartilage is damaged, the miR-27 or the analog thereof is used for restraining the differentiation of the mesenchymal stem cells to hypertrophic chondrocyte, and the chondrocyte apoptosis and the subsequent ossification process are prevented, so that the miR-27 can maintain and stabilize normal physiological function of the chondrocyte, and articular cartilage injury is promoted to regenerate and repair.

Description

technical field [0001] The invention belongs to the technical field of cartilage damage drugs, and in particular relates to the application of miR-27 and its analogues in the preparation of drugs for inhibiting the differentiation of bone marrow mesenchymal stem cells into hypertrophic chondrocytes. Background technique [0002] Bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells) are typical adult stem cells with multiple differentiation potential. Numerous research results have shown that bone marrow mesenchymal stem cells can be effectively differentiated into chondrocytes under in vitro induction conditions and in the microenvironment of cartilage damage in vivo, and a certain amount of bone marrow can be extracted from the patient's bone such as the ilium through bone marrow puncture. Bone marrow mesenchymal stem cells are isolated and expanded for the repair of autogenous cartilage damage without the risk of immune rejection, and are considered by th...

Claims

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Application Information

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IPC IPC(8): A61K31/715A61K48/00A61P19/00
Inventor 池光范李玉林吕爽徐金影陈林
Owner JILIN UNIV
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