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Mesenchymal stem cells with enhanced antimicrobial function as well as application of mesenchymal stem cells in sepsis

A technology of stem cells and antibacterial peptides, applied in the field of construction and its application in sepsis, can solve the problems of difficulty in achieving antibacterial, anti-endotoxin and anti-inflammatory effects, weak functions, etc., and achieve enhanced neutralization of endotoxin. ability, enhance antibacterial effect

Inactive Publication Date: 2018-03-23
重庆赛托斯创生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above-mentioned problem that mesenchymal stem cells self-secret the antibacterial peptide LL-37 function is not strong, and it is difficult to achieve good antibacterial, anti-endotoxin and anti-inflammatory effects. Tandem with the human bactericidal / permeability enhancing protein active fragment gene (rBPI21) and clone into a virus vector, the virus infects and modifies the mesenchymal stem cells, so that the mesenchymal stem cells can efficiently secrete and express while possessing the ability of tissue regeneration and repair Broad-spectrum antibacterial fusion protein obtains high-efficiency and broad-spectrum antibacterial ability

Method used

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  • Mesenchymal stem cells with enhanced antimicrobial function as well as application of mesenchymal stem cells in sepsis
  • Mesenchymal stem cells with enhanced antimicrobial function as well as application of mesenchymal stem cells in sepsis
  • Mesenchymal stem cells with enhanced antimicrobial function as well as application of mesenchymal stem cells in sepsis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Structure of expression vectors of pLVX-rBPI21-LL37 and pLVX-LL37-rBPI21

[0039] According to the amino acid sequence of the antibacterial fusion protein and the preferred codon of Chinese hamster, the fusion protein gene was designed and synthesized, and the synthetic gene was cloned into the pLVX-Tight-Puro vector to construct the fusion protein expression vector. Finally, two forms of fusion protein gene expression vectors, BPI21-LL37(BL) or LL37-BPI21(LB), were obtained. The difference between the two expression vectors is that the order of fusion is different, and the signal peptide of each protein located at the N' end guides the exocrine expression of the entire fusion protein. The schematic diagram of the construction of the vector is as follows figure 1 as shown, figure 1 A is the schematic diagram of the vector structure of pLVX-rBPI21-LL-37, figure 1 B is the structural representation of pLVX-LL-37-rBPI21 vector.

Embodiment 2

[0040] Example 2 Western Blot detection of rBPI21 / LL-37 antibacterial fusion protein expression

[0041] (1) Packaging of lentivirus

[0042] The cultured 293FT cells were digested with trypsin, the growth medium was discarded, and the cells were resuspended in the lentiviral packaging medium to make a cell suspension. The cells were counted and the concentration of the cell suspension was calculated. Take 1×10 5 / cm 2 Cells were seeded into 10 cm culture dishes at a density of . According to the liposome transfection method, add 3 μg lentiviral vector (pCDH), 3 μg pLP1, 3 μg pLP2, 3 μg pLP / VSVG to 1.5 ml serum-free DMEM basic medium, and shake gently to mix. Add 36 μl lipofectamine transfection reagent to 1.5 ml serum-free. Slowly add the liposomes to the plasmid mixture, mix gently and let stand at room temperature for 20 minutes until the DNA / liposome complexes are formed. Add all the DNA / liposome complexes to the 293FT cells, make up the medium to 10ml, gently pipette...

Embodiment 3

[0055] Example 3 Detection of stemness maintenance of BL-hUC-MSCs and LB-hUC-MSCs

[0056] Stemness maintenance plays a very important role in the function of mesenchymal stem cells. After lentivirus infection of the 4th passage umbilical cord-derived mesenchymal stem cells, flow cytometry was used to identify cell surface molecular markers and detect BL-hUC-MSCs And the change of stemness of LB-hUC-MSCs.

[0057] The result is as image 3 As shown, the results of flow cytometry showed that after lentivirus infection, the positive rates of CD29, CD44, CD90, and CD105 and the negative rates of CD34, CD45 on the surface of hUC-MSCs were in line with the standards recommended by the International Society for Cell Therapy (ISCT) in 2006, which indicated that Lentivirus infection had no significant effect on the stemness maintenance of mesenchymal stem cells.

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Abstract

The invention belongs to the technical field of stem cell treatment, and mainly relates to construction of mesenchymal stem cells modified by antibacterial fusion protein, and application of the mesenchymal stem cells in sepsis. Aiming at the problems that the mesenchymal stem cells are weaker in antimicrobial peptide LL-37 secretion function and cannot easily achieve good antibacterial, anti-endotoxin and anti-inflammatory effects, a recombinant carrier is constructed, a human LL-37 antimicrobial peptide gene and a human bactericidal / permeability enhanced protein active fragment gene rBPI21 are connected in series and are cloned into a virus vector, and the mesenchymal stem cells are infected with a virus and are modified by the virus, so that the mesenchymal stem cells can efficiently secrete and express the broad-spectrum antimicrobial fusion protein and further obtain high efficiency broad-spectrum antibacterial ability while having the ability of regenerating and repairing tissues. The stem cell treatment is effectively combined with gene therapy, so that a new choice is provided for the treatment of the sepsis.

Description

technical field [0001] The invention belongs to the technical field of stem cell therapy, and mainly relates to the construction of an antibacterial fusion protein-modified mesenchymal stem cell and its application in sepsis. Background technique [0002] Sepsis is a syndrome in which the body's immune response to infection is dysregulated, leading to life-threatening organ dysfunction. Further development can lead to shock, acute lung injury / acute respiratory distress syndrome, acute kidney injury, disseminated intravascular coagulation, etc. It is characterized by rapid deterioration and high mortality, and is the main cause of late death in patients with severe infection and severe burns. It is mainly manifested in that the body's immune system is overactivated under stressful conditions such as infection or trauma, and inflammatory cytokines such as IL-1, TNF-α, and IL-6 are rapidly produced in large quantities, causing septic shock and multiple organ dysfunction. failu...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867C07K19/00A61K38/17A61P31/04
CPCA61K38/00C07K14/4723C07K2319/00C12N5/0665C12N15/86C12N2740/15043C12N2800/22
Inventor 徐祥李战宋昱庆袁培淞
Owner 重庆赛托斯创生物科技发展有限公司
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