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Method for evaluating immunomodulatory function of substance

An immunomodulatory and material technology, applied in biochemical equipment and methods, cells modified by introducing foreign genetic material, blood/immune system cells, etc., can solve cumbersome operations, inability to accurately evaluate the immunomodulatory activity of active substances, and inability to be precise Assess issues such as natural product targeting and regulation of immune cells

Pending Publication Date: 2022-02-25
JIMEI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In vivo animal experiments include in vivo experiments on mice or rats. After long-term feeding of natural products, the secretion of immune-related cytokines in mouse serum is detected to determine the specific regulatory effects of natural products; It is cumbersome, and because cytokines are often not secreted by a single cell, multiple immune cells may produce the same cytokine, so the detection of in vivo animal experiments cannot accurately evaluate the targeting immune cells and regulatory effects of natural products in vivo
The in vitro macrophage model is mostly used to detect the phagocytosis or cytokine secretion of RAW264.7 cells; this cell model only characterizes the phagocytosis and digestion of antigens by some natural immune cells after entering the body, and cannot reflect the phagocytosis of antigens after entering the body , digestion, presentation, T cell differentiation and other immune processes, the in vitro macrophage model has a certain one-sidedness, and cannot accurately evaluate the immunomodulatory activity of active substances

Method used

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  • Method for evaluating immunomodulatory function of substance
  • Method for evaluating immunomodulatory function of substance
  • Method for evaluating immunomodulatory function of substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 In vitro model of T cells to evaluate the effect of asparagus sulfated oligosaccharides on Th1 cell differentiation

[0029] 1) Take the spleen of Rag1 mice, remove the red blood cells, and make the primary natural immune cell suspension, add 1×10 5 Add 2 μg / mL OVA and 300 μg / mL sulfated oligosaccharide of Gracilaria lemaneiformis (GLSO) to the culture system, 37°C, 5% CO 2 cultured in an incubator for 24 hours, wherein no sulfated oligosaccharides from asparagus was used as the control group.

[0030] 2) Collect OT-II mouse spleen single cell suspension, and use magnetic bead sorting method to obtain CD4 + CD62L + Naive T cells, after sorting, 96.3% of the cells expressed CD4 + CD44 + CD62L + Naive T cells (combined with figure 1 ), according to 2×10 5 Add cells / well into the above culture system and continue to culture for 3 days to construct an in vitro model of T cell differentiation, such as figure 2 shown.

[0031] 3) Collect all the cells on t...

Embodiment 2

[0033] Example 2 In vitro model of T cells to evaluate the effect of asparagus sulfated oligosaccharides on Th2 cell differentiation

[0034] 1) Take the spleen of Rag1 mice, remove the red blood cells, and make the primary natural immune cell suspension, add 1×10 5 Add 2 μg / mL OVA and 300 μg / mL sulfated oligosaccharide of Gracilaria lemaneiformis (GLSO) to the culture system, 37°C, 5% CO 2 cultured in an incubator for 24 hours, wherein no sulfated oligosaccharides from asparagus was used as the control group.

[0035] 2) Collect OT-II mouse spleen single cell suspension, and use magnetic bead sorting method to obtain CD4 + CD62L + Naive T cells, after sorting, 96.3% of the cells expressed CD4 + CD44 + CD62L + Naive T cells (combined with figure 1 ), according to 2×10 5 Add cells / well into the culture system and continue to culture for 3 days to construct an in vitro model of T cell differentiation, such as figure 2 shown.

[0036] 3) Collect all the cells on the 4th...

Embodiment 3

[0038] Example 3 In vitro model of T cells to evaluate the effect of asparagus sulfated oligosaccharides on the differentiation of Treg cells

[0039] 1) Take the spleen of Rag1 mice, remove the red blood cells, and make the primary natural immune cell suspension, add 1×10 5 Add 2 μg / mL OVA and 300 μg / mL sulfated oligosaccharide of Gracilaria lemaneiformis (GLSO) to the culture system, 37°C, 5% CO 2 cultured in an incubator for 24 hours, wherein no sulfated oligosaccharides from asparagus was used as the control group.

[0040] 2) Collect OT-II mouse spleen single cell suspension, and use magnetic bead sorting method to obtain CD4 + CD62L + Naive T cells, after sorting, 96.3% of the cells expressed CD4 + CD44 + CD62L + Naive T cells (combined with figure 1 ), according to 2×10 5 Add cells / well into the culture system and continue to culture for 3 days to construct an in vitro model of T cell differentiation, such as figure 2 shown.

[0041] 3) Collect all the cells o...

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Abstract

The invention discloses a method for evaluating the immunomodulatory function of a substance, which comprises the following steps: preparing a primary natural immune cell suspension from the spleen of an acquired immune cell deficient mouse; culturing the immune cells and OVA to obtain a culture system; collecting spleen and lymph node single-cell suspensions of a T cell receptor transgenic mouse, and carrying out magnetic bead sorting to obtain CD4 + CD62L + Naive T cells; adding the CD4 + CD62L + Naive T cells into the culture system, and adding a substance to be evaluated into the culture system at the concentration of 100-500mu g / mL in the culture process; collecting all cells, performing stimulation culture on the cells by using phorbol and ionomycin, performing membrane rupture, matching and fixing, adding a fluorescent antibody, and detecting the proportion of Th1, Th2 or Treg by using a flow cytometer so as to evaluate the immunomodulatory function of the substance. The method can accurately evaluate whether the active substance has the immune regulation function, and is low in cost and short in period.

Description

technical field [0001] The invention relates to the technical field of cell in vitro models, in particular to a method for evaluating the immunoregulatory function of a substance. Background technique [0002] Natural products are rich in active substances such as sugars, proteins, and minerals, and are widely used in food manufacturing and drug research and development. Studies have shown that a variety of natural products have biological functions that regulate the body's immune response, manifested in immunomodulatory activity, anti-inflammation , anti-tumor, anti-allergy, anti-virus and many other aspects. More and more scholars pay attention to the study of natural products regulating the immune response of the body. [0003] At present, the commonly used models for evaluating the immunomodulatory function of active substances are mainly in vivo animal experiments or in vitro macrophage phagocytosis experiments. In vivo animal experiments include in vivo experiments o...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12N5/10C12Q1/02
CPCC12N5/0634C12N5/0636G01N33/505C12N2501/998C12N2510/00C12N2502/11C12N2502/1114C12N2502/99
Inventor 刘庆梅刘光明周钰刘文贤刘晨风
Owner JIMEI UNIV
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