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Reprogramming induction scheme for direct transformation of subchondral bone cells to articular cartilage cells

A technology of articular cartilage and subchondral bone, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., to achieve the effect of improving induction efficiency and transformation reliability and reducing dependence

Pending Publication Date: 2021-06-01
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical applications, there are still many problems such as in vitro operations such as cell acquisition, culture and storage, and precise regulation of cell-directed differentiation to avoid ossification and calcification, and technical bottlenecks need to be broken through

Method used

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  • Reprogramming induction scheme for direct transformation of subchondral bone cells to articular cartilage cells
  • Reprogramming induction scheme for direct transformation of subchondral bone cells to articular cartilage cells
  • Reprogramming induction scheme for direct transformation of subchondral bone cells to articular cartilage cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Experimental materials

[0034] Subchondral bone osteoblasts and 293T cells of mouse knee joint

[0035] Lentiviral packaging plasmid psPAX2, envelope plasmid pMD2.G and shuttle plasmid pSIN4-CMV

[0036] 2. Experimental method

[0037] 1.a. Construction of lentiviral vectors carrying reprogramming factor gene fragments

[0038] The Sox9, Sox5 and Plagl1 reprogramming factor genes amplified from cDNA were constructed on the pSIN4 lentiviral vector backbone, the resulting plasmid was purified, and the titer was obtained by the second-generation lentiviral packaging system at 1–3×10 8 TU / ml of lentiviral particles.

[0039] Table 1 Primer Sequence

[0040]

[0041]

[0042] 1.b. Induction of reprogramming factors based on osteoblasts from subchondral bone of mouse knee joint

[0043] Osteoblasts in subchondral bone at 5000 / cm 2 24 hours after inoculation, the reprogramming factor lentivirus was transduced by Sox9, Sox5, Sox6, Sox8, Zcchc5 or Plagl1 single f...

Embodiment 2

[0053] 1. Experimental materials

[0054] Subchondral bone osteoblasts and 293T cells of mouse knee joint

[0055] Lentiviral packaging plasmid psPAX2, envelope plasmid pMD2.G and shuttle plasmid pSIN4-CMV

[0056] 2. Experimental method

[0057] 2.a. Construction of lentiviral vectors carrying reprogramming factor gene fragments

[0058] The Sox9, Sox5, Sox8 and Zcchc5 reprogramming factor genes amplified from cDNA were constructed on the pSIN4 lentiviral vector backbone, the obtained plasmid was purified, and the titer was obtained by the second-generation lentiviral packaging system at 1–3×10 8 TU / ml of lentiviral particles.

[0059] Table 3 Primer Sequence

[0060]

[0061] 2.b. Induction of reprogramming factors based on osteoblasts from subchondral bone of mouse knee joint

[0062] Osteoblasts in subchondral bone at 5000 / cm 2 24 hours after inoculation, the reprogramming factor lentivirus was added to the culture medium containing 8 μg / ml polybrene under the con...

Embodiment 3

[0071] 1. Experimental materials

[0072] Subchondral bone osteoblasts and 293T cells of mouse knee joint

[0073] Lentiviral packaging plasmid psPAX2, envelope plasmid pMD2.G and shuttle plasmid pSIN4-CMV

[0074] 2. Experimental method

[0075] 3.a. Construction of lentiviral vectors carrying reprogramming factor gene fragments

[0076] The Sox9, Sox6 and Zcchc5 reprogramming factor genes amplified from cDNA were constructed on the pSIN4 lentiviral vector backbone, the resulting plasmid was purified, and the titer was obtained by the second-generation lentiviral packaging system at 1–3×10 8 TU / ml of lentiviral particles.

[0077] Table 5 Primer Sequence

[0078]

[0079] 3.b. Induction of reprogramming factors based on osteoblasts from subchondral bone of mouse knee joint

[0080]Osteoblasts in subchondral bone at 5000 / cm 2 24 hours after inoculation, the reprogramming factor lentivirus was added to the culture medium containing 8 μg / ml polybrene with a combination ...

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Abstract

The invention belongs to the technical field of biology, discloses an induction scheme for direct transformation of subchondral bone osteoblasts to functional articular cartilage cells through reprogramming, and is suitable for regeneration and repair treatment of severe articular cartilage defects. According to the induction scheme, Sox9, Sox5 and Plagl1 or Sox8 and Zcchc5 genes are introduced into subchondral bone cells, and the cartilage-like cells are obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to cell and molecular biology, eukaryotic cell reprogramming, cell transformation and differentiation, tissue regeneration and repair, and also relates to the application of the technical scheme of cell reprogramming in the treatment of articular cartilage damage. Background technique [0002] Articular cartilage has a fine layered structure and contains chondrocytes with different functions. There is a lack of blood vessels, nerves and lymphatic vessels in the middle and deep structures, and the number of chondrocytes is scarce. Although the matrix mainly contains components such as collagen, proteoglycan and non-collagen, which play a buffering and lubricating role, it lacks self-renewal ability (Bhosale and Richardson2008). If joint activities cause high-intensity impact and excessive wear, the cartilage will be severely damaged, and the cartilage tissue will not be able to repair its...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K38/17A61P19/04
CPCC12N15/86C12N5/0655A61K38/17A61P19/04C12N2740/15043C12N2510/00A61K2300/00
Inventor 竺青李美含
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI