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Genetically modified stem cell for cartilage repair treatment and application thereof

A cartilage repair and gene modification technology, applied in the field of genetic engineering, can solve problems such as limited understanding of factors regulating its activity, effectiveness and safety limitations of clinical application of mesenchymal stem cells, etc., to achieve good synergistic effects, relieve reduction/avoidance Swelling, the effect of suppressing the activity of immune cells

Pending Publication Date: 2021-12-10
BEIJING JIYUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although there is great demand and support for stem cells to treat clinical diseases such as bone and knee joint repair, due to the limited understanding of the factors that regulate the activity of mesenchymal stem cells and the lack of understanding of mesenchymal stem cells and their ecological microbiome The understanding of the complex interaction between the components of the environment, the effectiveness and safety of the clinical application of mesenchymal stem cells is limited, so there is still a lot of room for exploration in the related research of mesenchymal stem cells repairing bone and knee joints

Method used

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  • Genetically modified stem cell for cartilage repair treatment and application thereof
  • Genetically modified stem cell for cartilage repair treatment and application thereof
  • Genetically modified stem cell for cartilage repair treatment and application thereof

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Experimental program
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Embodiment approach

[0058] According to a preferred embodiment of the present invention, the genetically modified stem cells further include an expression vector, and the nucleic acid encoding the anti-inflammatory factor and the nucleic acid encoding the cartilage repair factor are contained in the same or different expression vectors, preferably contained in the same expression vector. in the carrier.

[0059] In a further preferred embodiment, the nucleic acid encoding the anti-inflammatory factor and the nucleic acid encoding the cartilage repair factor are connected through a medium-strength promoter.

[0060] In a preferred embodiment of the present invention, the nucleic acid encoding the anti-inflammatory factor is linked to the nucleic acid encoding the cartilage repair factor, for example, through a medium-strength promoter.

[0061] More preferably, a medium-strength promoter such as EF1a is linked to an enhancer, the enhancer is linked to the 5' end of the nucleic acid encoding a cart...

Embodiment 1

[0121] Synthetic human XbaI-IL10-T2A-IL37-EF1α-5'LTR-FGF18-Sall

[0122] The IL10 coding nucleotide (the nucleotide sequence is shown in SEQIDNO:1) and the IL37 coding nucleotide (the nucleotide sequence is shown in SEQIDNO:2) are connected with T2A (the nucleotide sequence is shown in SEQIDNO:3) , FGF18 coding nucleotide (nucleotide sequence is shown in SEQIDNO:4) before connecting 5'LTR sequence (sequence shown in SEQIDNO:5) and EF1α promoter (sequence shown in SEQIDNO:7), IL37 and EF1α start The two ends were connected with restriction sites XbaI and SaII, and human XbaI-IL10-T2A-IL37-EF1α-5'LTR-FGF18-Sall was synthesized, named as Sequence103718, whose sequence is shown in SEQ ID NO:6.

Embodiment 2

[0123] Example 2 Construction of pCDH-103718 plasmid

[0124] The pCDH-CMV plasmid (Addgene) was digested with XbaI and SaII, and a fragment of about 6195 bp was recovered from the product gel as a vector.

[0125]Sequence103718 was digested with XbaI and SaII, and a fragment of about 2487bp was recovered from the product gel.

[0126] Take the pCDH-CMV plasmid digestion product vector and the Sequence103718 digestion product fragment, connect with T4 ligase overnight at 4°C, transform DH5α competent cells, take 100 μL of bacterial liquid and spread it on the LB plate containing ampicillin resistance, Cultivate overnight at 37°C, pick a single clone for colony PCR, send the positive clone to the sample for sequencing, save the clone with the correct sequencing result and extract the plasmid, named pCDH-103718, its map is as follows figure 1 shown.

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Abstract

The invention discloses a genetically modified stem cell for cartilage repair treatment as well as a preparation method and application of the genetically modified stem cell, and further provides a pharmaceutical composition containing the stem cell. The stem cells comprise nucleic acid for coding an anti-inflammatory factor and nucleic acid for coding a cartilage repair factor, by simultaneously modifying the mesenchymal stem cells by adopting the anti-inflammatory factor and a factor for promoting cartilage differentiation, the mesenchymal stem cells can be effectively promoted to be differentiated into cartilage, and by comprehensively acting on various immune cells through combination of various factors, and the activity of various immune cells is inhibited, so that the local inflammatory environment problem of cartilage repair of parts such as bones and knees is solved, and the swelling condition of a clinical patient is reduced or decreased when the mesenchymal stem cells are applied to bone and knee repair.

Description

technical field [0001] The invention relates to genetic engineering, in particular to the technical field of stem cell therapy, in particular to genetically modified stem cells for cartilage repair. Background technique [0002] Stem cells are a type of cells with self-renewal and differentiation potential, and are an important means for the research or treatment of tumors, cardiovascular diseases and other malignant diseases. Stem cell therapy has great research and application value in the three major fields of life sciences, new drug testing and disease research. It has been widely used in the fields of medical regenerative cell replacement therapy and drug screening, and has become the focus of world attention and research. [0003] The indications involved in stem cell drugs are mainly knee osteoarthritis and diabetic foot. Stem cell clinical treatment and research diseases involve all major systems of the body, including: acute myocardial infarction, cerebral palsy in...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/24C12N15/12A61K35/28A61K48/00A61K38/20A61K38/18A61K38/17A61P19/02A61P19/04A61P19/08A61P29/00
CPCC12N5/0668C12N15/86C07K14/5403C07K14/5406C07K14/5409C07K14/5412C07K14/5428C07K14/5437C07K14/54C07K14/50C07K14/71A61K35/28A61K48/005A61K38/202A61K38/2026A61K38/2033A61K38/204A61K38/2066A61K38/2086A61K38/20A61K38/1825A61K38/179A61P19/02A61P19/04A61P19/08A61P29/00C12N2740/15043C12N2800/107C12N2510/00A61K2300/00
Inventor 薛冰华于婷婷张振利
Owner BEIJING JIYUAN BIOTECH
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