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Application of immune cells containing a reporter system expressing cd54 and cloned novel cells

A technology of immune cells and reporting systems, applied in the application field of immune cells, can solve problems such as not appearing

Active Publication Date: 2018-11-27
IND TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In line with the trend of animal experiment ethics and reduction, research institutions and related companies in Europe, the United States and Japan have invested considerable resources in the development of alternative animal experiment methods, but currently there is no internationally recognized alternative that can replace animal experiments. Way

Method used

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  • Application of immune cells containing a reporter system expressing cd54 and cloned novel cells
  • Application of immune cells containing a reporter system expressing cd54 and cloned novel cells
  • Application of immune cells containing a reporter system expressing cd54 and cloned novel cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Establishment of ITRI-54M cells

[0047] According to the manufacturer's operating instructions of the LVX-MetLuc (Clontech, PT4422-5) plasmid, the LVX-MetLuc plasmid was treated with BstB I and BamH I restriction enzymes, so that the CD54 promoter sequence (sequence identification number: 1) was transferred to LVX-MetLuc In the MetLuc plasmid, a CD54-promoter mLuc expression system (Fig. 1) is formed, wherein in this expression system, the sequence of the CD54 promoter-mLuc is the sequence of SEQ ID NO: 2. Then the formed CD54-promoter mLuc expression system was used Lenti-X TM Ready-To-Glow TM Secreted Luciferase Reporter System (Clontech, 631746) was transfected into 293FT cells to produce lentivirus (Lentivirus) with CD54-mLuc. THP-1 cells were then infected with this virus. The THP-1 cell line was cultured in THP-1 medium, and its composition was RPMI medium (GIBCO, 31800), containing 10% FBS (GIBCO, 10437), 4.5g / L glucose (Sigma, G7021), 10mM HEPES ( Sigma, H...

Embodiment 2

[0050] Determination of whether the substance to be tested is a skin sensitizer (or an immunomodulatory substance)

[0051] In this experiment, the process of judging whether the substance to be tested is a skin sensitizer is as follows: figure 2 shown. see figure 2 with detailed step-by-step instructions below.

[0052] 1. Toxicity test of the tested substance on ITRI-54M cells

[0053] ITRI-54M cells were cultured in THP-1 medium, which consisted of RPMI medium (GIBCO, 31800), containing 10% fetal bovine serum (GIBCO, 10437), 4.5g / L glucose (Sigma, G7021), 10mM HEPES (Sigma, H4034), 1x penicillin and streptomycin (Biowest, L0022), 1mM sodium pyruvate (sodiumpyruvate) (Biowest, L0642), 0.05mM 2-mercaptoethanol (GIBCO , 21985-023) and 0.5 μg / mL puromycin (puromycin).

[0054] The reference chemical samples listed in Table 1 with known allergenicity or the Chinese herbal extracts listed in Table 2 commonly used in cosmetics were mixed with an appropriate solvent (DMSO or...

Embodiment 3

[0086] Determination of whether the substance to be tested is an anti-inflammatory substance (or an immunomodulatory substance)

[0087] In this experiment, the process of judging whether the substance to be tested is a skin sensitizer is as follows: image 3 shown. see image 3 with detailed step-by-step instructions below.

[0088] 1. Toxicity test of the tested substance on ITRI-54M cells

[0089] ITRI-54M cells were cultured in THP-1 medium, which consisted of RPMI medium (GIBCO, 31800), containing 10% fetal bovine serum (GIBCO, 10437), 4.5g / L glucose (Sigma, G7021), 10mM HEPES (Sigma, H4034), 1x penicillin and streptomycin (Biowest, L0022), 1mM sodium pyruvate (sodiumpyruvate) (Biowest, L0642), 0.05mM 2-mercaptoethanol (GIBCO , 21985-023) and 0.5 μg / mL puromycin (puromycin).

[0090] The test substances listed in Table 6 known to have anti-inflammatory ability were dissolved in an appropriate solvent (DMSO or THP-1 culture solution without 2-ME) to form a stock solut...

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Abstract

The present invention provides a method of confirming an immunoregulation substance, comprising: (a) providing a to-be-measured substance and an immune cell, wherein the immune cell includes a CD54 expression reporting system, and the CD54 expression reporting system comprises a CD54 gene promoter and a reporter gene connected with the CD54 gene promoter and used for reporting expression of the CD54 gene promoter; (b) processing the immune cell by the to-be-measured substance, and (c) measuring the product of the reporter gene produced by the immune cell processed by the to-be-measured substance so as to determine whether the to-be-measured substance has an immune regulation function.

Description

field of invention [0001] The present invention relates to the application of the immune cells containing the reporter system expressing CD54, and particularly relates to the method of applying the immune cells containing the reporter system expressing CD54 to confirm the immunomodulatory substance. Background of the invention [0002] CD54 (Cluster of Difffeentiation 54) (also known as Intercellular Adhesion Molecule 1, ICAM-1) is a protein encoded by the ICAM1 gene in humans. The gene encodes a cell surface glycoprotein normally expressed on endothelial cells and cells of the immune system. CD54 binds to CD11a / CD18 type or CD11b / CD18 type integrins (integrins) , and is also used as a receptor by rhinovirus. It is known that CD54 is a biomarker of immune response, especially allergic response. [0003] The immune response plays an important role in many diseases, such as inflammation, skin sensitivity, etc., so many drugs are developed to regulate the immune system. Howev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/84G01N21/31C12N5/10C12R1/91
Inventor 赖惠敏郭宗铿江彣笙曾湘文
Owner IND TECH RES INST