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Application of transferrin in preparing immunosuppressant medicines

An immunosuppressant, transferrin technology, applied in the field of biomedicine, can solve the problem of no transferrin activation, and achieve the effects of reducing liver tissue damage, reducing cell apoptosis, and inhibiting inflammatory responses

Pending Publication Date: 2020-05-19
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no record and report that transferrin is used for TLR4 signaling pathway activation in the prior art

Method used

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  • Application of transferrin in preparing immunosuppressant medicines
  • Application of transferrin in preparing immunosuppressant medicines
  • Application of transferrin in preparing immunosuppressant medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Verification of transferrin immune target: BIAcore 2000 (GE, USA) was used to analyze the combination of transferrin and CD14.

[0042] Specific steps are as follows:

[0043] 1. Activate the CM5 chip (BR100012, GE, USA) with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride activation solution, and then use A transferrin solution with a final concentration of 20 μg / ml diluted with sodium acetate solution (10 mM, pH=5) flows through the surface of the activated CM5 chip to covalently couple transferrin (500 reaction value), and the flow rate is 5 μl / ml. min.

[0044] 2. Use the same method and flow rate as step 1) to make 75 μl of blocking solution ethanolamine flow over the surface of the chip to block redundant sites.

[0045] 3. The mobile phase CD14 solution diluted with Tris buffer (20mM, pH 7.4) with different concentrations (125, 250, 500 and 1000nM) flows over the surface of the chip to detect the binding curve of transferrin...

Embodiment 2

[0048] Detection of the inhibitory effect of transferrin on inflammatory factors at the cellular level: detection of transferrin on LPS-induced cellular inflammatory factors.

[0049] The specific operation is as follows:

[0050]Peripheral blood mononuclear cells, neutrophils, dendritic cells derived from mouse bone marrow, mononuclear macrophages and venous endothelial cells were plated on 96-well plates. Then add different concentrations (0.05, 0.5, and 5 μM) of apo-transferrin (apo-transferrin) and holo-transferrin (holo-transferrin) to incubate with the above cells for 10 min, and finally add lipopolysaccharide (LPS, 2 μg / ml ) induced cell inflammation for 8h. Finally, the purchased Elisa kit was used to detect the contents of TNF-α, IL-6, IFN-β and TGF-β in the cell supernatant. The results are: transferrin can significantly inhibit the production of LPS-induced inflammatory cytokines ( Figure 2-A Indicates that transferrin inhibits the production of inflammatory fac...

Embodiment 3

[0052] Detection of the inhibitory effect of transferrin on inflammation in animals: overexpression of transferrin (PLP-Tf), knockdown (RNR-Tf) and tail vein injection of transferrin on the plasma inflammatory factors induced by lipopolysaccharide LPS, liver Effects of tissue damage.

[0053] First construct mouse transferrin overexpression (PLP-Tf) and knockdown (RNR-Tf) lentivirus and retroviral vectors and infect mouse models, the specific operation is: synthetic mouse transferrin coding region gene (GenBank: BC092046.1) and cloned into the pLVX-Puro (Clontech, USA) lentiviral overexpression vector. Design and synthesis of transferrin knockdown interference RNA (shRNA, nucleotide sequence shown in SEQ ID NO.1, specifically: 5'-AAGAATCTGAAGCAGGAAGACTTCAAGAGAGTCTTCCTGCTTCAGA TTCTTCTTTTTT-3'(forward oligo),) sequence and cloned into RNAi- ReadypSIREN-RetroQ (Clontech, USA) retroviral knockdown vector. The transferrin overexpression plasmid together with the viral packaging p...

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Abstract

The invention provides application of transferrin in preparing immunosuppressant medicines and belongs to the technical field of biomedicines. Transferrin is capable of targeting at a co-receptor monocyte surface antigen CD14 of a TLR4 (toll-like receptor 4), then recognition of lipopolysaccharide (LPS) and CD14 is inhibited, LPS induced TLR4 signal path activation is also inhibited, LPS induced inflammatory factors are inhibited, LPS induced plasma inflammatory factors TNF-alpha, IL-6, IL-1beta and IFN-beta are inhibited, and LPS induced liver tissue damage and cell apoptosis can be alleviated. The transferrin has a very remarkable effect in inhibiting inflammation induced by bacterium products. The transferrin is capable of inhibiting inflammation reactions induced by bacterium productsand in addition, has the potential of preparing immunosuppressant medicines.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application of transferrin in the preparation of immunosuppressant drugs. Background technique [0002] Toll-like receptors (Toll-like receptors, TLRs) can recognize various disease-associated molecular patterns (PAMPs), and are an important barrier for the body to resist infection. Microbes infect and stimulate the TLRs family to cause pro-inflammatory responses and then mediate host immune defense, but excessive inflammation is very harmful to the body, and the consequences are also very serious. TLR4 is an important member of the TLRs family that recognizes bacteria. It mainly recognizes lipopolysaccharide (LPS) of Gram-negative bacteria to activate the NF-κB signaling pathway and then activate the host immune response. In order to prevent excessive inflammation and avoid damage to the body caused by inflammation, the TLR4 signaling pathway is also strictly negatively...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/40A61P37/06A61P29/00
CPCA61K38/40A61P37/06A61P29/00
Inventor 赖仞唐小芃方鸣谦
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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