Application of LncRNA-32598 in preparation of drug or gene delivery system for inhibiting chondrocyte hypertrophic differentiation

A chondrocyte and hypertrophy technology, which is applied in the application field of preparing drugs or gene delivery systems for inhibiting chondrocyte hypertrophy differentiation, can solve the problems of difficulty in maintaining cell phenotype, repair failure, and easy occurrence of fibrosis.

Active Publication Date: 2020-05-08
ARMY MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In view of this, the purpose of the present invention is to solve the problem that when tissue engineering cartilage is used to repair defects, the seed cells often undergo hypertrophy and differentiation, it is difficult to maintain the cell phenotype, fibrosis is prone to occur, and the problem of repair failure is solved. The lncRNA-LncRNA-32598 related to the hypertrophy process of stem cell-derived chondrocytes, studied its function and molecular mechanism, determined that it can effectively inhibit the hypertrophy and differentiation process of stem cell-derived chondrocytes, and proved that LncRNA-32598 as a marker can Screening of plant extracts that inhibit the hypertrophy process of chondrocytes, the present invention provides the application of long-chain non-coding RNA LncRNA-32598 in the preparation of drugs or gene delivery systems for inhibiting hypertrophy and differentiation of chondrocytes

Method used

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  • Application of LncRNA-32598 in preparation of drug or gene delivery system for inhibiting chondrocyte hypertrophic differentiation
  • Application of LncRNA-32598 in preparation of drug or gene delivery system for inhibiting chondrocyte hypertrophic differentiation
  • Application of LncRNA-32598 in preparation of drug or gene delivery system for inhibiting chondrocyte hypertrophic differentiation

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Experimental program
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Effect test

Embodiment 1

[0041] Example 1, LncRNA-32598 Construction of Tissue Engineering Bone Based on Endochondral Osteogenic System

[0042] 1. Culture of bone marrow MSCs and establishment of MSCs-derived chondrocyte hypertrophic differentiation induction model

[0043] Human bone marrow MSC culture was carried out by the method of lymphocyte separation medium, the buffy coat mononuclear cells after centrifugation were washed twice with DMEM (centrifugation at 1000r / min for 5min), the supernatant was discarded, and the cells were washed with DMEM containing 15% fetal bovine serum -L complete medium resuspended, with 1 × 10 6 / mL cell density inoculated in a culture flask, recorded as the primary generation, placed at 37°C, 5% CO 2 Culture in a constant temperature incubator, change the medium every 3 days, observe the cell growth under an inverted microscope after changing the medium, digest with 0.25% trypsin when the cells are close to confluence, and passage at a ratio of 1:2 to 1:3. The bas...

Embodiment 2

[0048] Example 2, LncRNA-32598 inhibits chondrocyte hypertrophy and cartilage matrix degradation

[0049] In order to detect the regulatory effect of LncRNA-32598 on chondrocyte hypertrophy, cartilage precursor cells (MSCs cells induced for 14 days of chondrogenic differentiation) were infected with lncRNA overexpression and interference lentivirus. It was confirmed by qPCR that LncRNA-32598 interference and overexpression of cartilage precursor cell lines were successfully established ( figure 2 A). At the nucleic acid level, the expression of hypertrophy markers Col10a1 and Runx2 in the interference LncRNA-32598 group was higher than that in the control group, and the expression of Col10a1 and Runx2 in the overexpression LncRNA-32598 group was lower than that in the control group ( figure 2 B, C). WB results showed that the expression of Runx2 and Col10a1 was upregulated in the interference LncRNA-32598 group, and downregulated in the overexpression LncRNA-32598 group ( ...

Embodiment 3

[0051] Example 3, LncRNA-32598 regulates Sox9 expression to inhibit chondrocyte hypertrophy by competitively binding miR-145a-5p and miR-509

[0052] 1. LncRNA-32598 targets miR-145a-5p and miR-509-5p

[0053] LncRNA-32598 interference and overexpression cells were established, and the expression of miR-145a-5p was detected by qPCR. The results are as follows image 3 shown. It showed that the expression of miR-145a-5p in the cells of the interference group was higher than that of the control group, and the expression of miR-145a-5p in the cells of the overexpression group was lower than that of the control group ( image 3 A). The direct targeting relationship between lncRNA-32598 and miR-145a-5p was further verified by a dual-luciferase reporter gene system. The wild-type Lnc 3'-UTR plasmid and mutant Lnc 3'-UTR were transfected into HEK293 cells, and the precursor of microRNA-145 or control were added, respectively. The results showed that the fluorescence intensity det...

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Abstract

The invention discloses an application of LncRNA-32598 in the preparation of a drug or gene delivery system for inhibiting chondrocyte hypertrophic differentiation. A nucleic acid sequence of LncRNA-32598 is as shown in SEQ ID NO.1, and cytology experiments show that LncRNA-32598 can inhibit hypertrophy of chondrocytes, can be used to inhibit a hypertrophic process of MSCs after chondrogenic differentiation and maintain the matrix metabolism balance of chondrocytes, and has good application prospects in tissue engineering cartilage construction and cartilage defect repair, and intervention ofcartilage matrix degeneration in osteoarthritis.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering, and relates to the application of LncRNA-32598 in the preparation of drugs or gene delivery systems for inhibiting chondrocyte hypertrophy and differentiation. Background technique [0002] Human cartilage tissue is composed of chondrocytes and extracellular matrix. Among them, chondrocytes account for only 1% of the total tissue volume, and are responsible for forming cartilage and maintaining the metabolic balance of extracellular matrix in the tissue during development. In the matrix, however, collagen fibrils are the structural framework of the tissue and give the cartilage its tensile strength. Articular cartilage is a kind of hyaline cartilage, the main component of its collagen fibrils is type II collagen, and the minor components are type IX collagen and type XI collagen. A network of collagen fibers encases proteoglycans and glycoproteins, giving cartilage tissue its mechani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/861C12N15/85A61K31/713A61P19/08
CPCC12N15/113C12N15/86C12N15/85A61K31/713A61P19/08C12N2310/141C12N2740/15043C12N2710/10043
Inventor 董世武曹震张珠康菲龚小珊李建美刘蓝懿江虹
Owner ARMY MEDICAL UNIV
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