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Mesenchymal stem cell for inhibiting Th17 cell activation as well as preparation method and application thereof

A mesenchymal stem cell and cell technology, applied in the field of gene-modified mesenchymal stem cell preparation

Active Publication Date: 2019-05-28
北京贝来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, anti-mAb antibodies were detected in the serum of some patients (4%) during the use of the above-mentioned antibodies [14], suggesting that resistance to targeted drugs is still a problem that cannot be overcome by monoclonal antibody drugs

Method used

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  • Mesenchymal stem cell for inhibiting Th17 cell activation as well as preparation method and application thereof
  • Mesenchymal stem cell for inhibiting Th17 cell activation as well as preparation method and application thereof
  • Mesenchymal stem cell for inhibiting Th17 cell activation as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Construction of lentiviral gene vector

[0063] We constructed LV-IL23R-ECD-I-EGFP (LV-IL23R for short) and LV-IL23R-ECD:Fc- I-EGFP (abbreviated as LV-IL23R:Fc) fusion protein lentiviral expression plasmid, and LV-EGFP was used as a control virus (LV-null) (Figure 1 is a structure diagram of the lentiviral vector).

[0064] Wherein, the lentiviral transfection system was purchased from OriGene Company. The target gene IL23R-ECD and IL23R-ECD: Fc have an IRES sequence connected to the 3'-end, followed by an enhanced green fluorescent protein (EGFP) gene to form the IL23R-ECD-I-EGFP sequence, IL23R-ECD:Fc-I - EGFP sequence, namely IL23R-ECD and IL23R-ECD:Fc fusion protein, are together downstream of the CMV promoter.

[0065] The IL23R-ECD includes a human interleukin 23 receptor signal peptide and an N-terminal domain, and the amino acid sequence of the human interleukin 23 receptor signal peptide is:

[0066] MNQVTIQWDAVIALYILFSWCHG (SEQ No: 1), the amino ...

Embodiment 2

[0069] Example 2: Establishment of human umbilical cord mesenchymal stem cell seed bank

[0070] Human umbilical cords were obtained from healthy full-term pregnancy cesarean section pregnant woman donors, provided by qualified national second-level hospitals, and pregnant women or their family members signed informed consent and umbilical cord collection registration forms. The extraction, cultivation and establishment of cell bank of umbilical cord mesenchymal stem cells are all completed in our company's GMP production workshop. After the cells are separated, cultured, subcultured and expanded to the second generation (P2), the exogenous microorganisms, viruses, endotoxins, etc. are detected; at the same time, the immune phenotype, differentiation ability and cell biological efficacy of the cells are detected. Qualified cells were used as seed bank cells and stored in a liquid nitrogen tank at -196°C.

Embodiment 3

[0071] Example 3: Genetically Modified Mesenchymal Stem Cells

[0072] The lentiviral plasmid vectors described in Example 1 were mixed with lentiviral pGag / Pol, pRev, pVSV-G and other framework plasmids, introduced into 293T cells through LTX liposomes, packaged to obtain mature lentiviruses, and infected MSCs after harvesting the viruses. After 24 hours, puromycin was added to select successfully infected cells.

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PUM

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Abstract

The invention discloses a mesenchymal stem cell capable of inhibiting Th17 cell activation. The mesenchymal stem cell can specifically express human interleukin 23 acceptor homolog through a gene modifying method, so that combination between IL-23 and an IL-23 acceptor is competitively inhibited. Therefore, the mesenchymal stem cell can be applied to inhibiting correlated signal channels and inflammation reaction of IL-23 mediated inflammation. The invention further discloses the human interleukin 23 acceptor homolog as well as a preparation method and application of the mesenchymal stem cell.

Description

technical field [0001] The present invention relates to a method for preparing gene-modified mesenchymal stem cells, which can inhibit the inflammatory response mediated by Th17 signaling pathway by blocking the IL23 / IL23R conduction pathway, mesenchymal stem cells for treating related autoimmune diseases and the preparation method thereof and The application belongs to the field of biomedicine. Background technique [0002] Human interleukin 23 (hIL-23) is secreted by dendritic cells (DC cells), macrophages or other antigen-presenting cells, and is a heterodimer composed of two subunits of p19 and p40 linked by disulfide bonds Structural inflammatory factor [1]. IL-23 and IL-12 share the p40 subunit, and its coding gene is located on human chromosome 5; while p19 is a unique subunit protein of IL23, and its coding gene is located on human chromosome 12. Research and development found that [2], IL23 mediates many inflammatory responses in the body by binding to its recepto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K14/715C12N15/867A61K35/28A61K38/20A61P19/06A61P29/00A61P19/02A61P11/00A61P1/00A61P37/02
Inventor 刘拥军刘广洋张晨亮王皓
Owner 北京贝来生物科技有限公司
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