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Immune toxicity evaluation method for new medicine development

A technology of immunotoxicity and drugs, applied in the field of biomedicine, can solve the problems of no immunotoxicity prediction and safety evaluation

Active Publication Date: 2015-12-30
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In applied research, human peripheral blood mononuclear cells are widely used in the safety and immunogenicity of vaccination; but so far, there is no use of human peripheral blood mononuclear cell systems for immunotoxicity prediction and safety in new drug development. Evaluation of performance and as an effective surrogate for predicting potential immunotoxicity in new drug development

Method used

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  • Immune toxicity evaluation method for new medicine development
  • Immune toxicity evaluation method for new medicine development
  • Immune toxicity evaluation method for new medicine development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] [Example 1] Separation and preparation of peripheral blood mononuclear cells

[0036] Human peripheral blood mononuclear cells were separated from plasma samples by density gradient centrifugation using Ficoll-Paque (ρ=1.077 g / ml) purchased from GE. The isolated peripheral blood mononuclear cells were treated with RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum and 0.5% blue chain double antibody ( , ThermoFisher) resuspended. About 3-4×10 per 100ml of plasma can be separated 7 Peripheral blood mononuclear cells, the viability of which was detected by trypan blue staining was greater than 95%, and the isolated cells were immediately used for subsequent experimental research.

[0037] 1.1: Exploration of the conditions for the isolation of human peripheral blood mononuclear cells

[0038] The key points for separating human peripheral blood are as follows: 1. The dilution degree of plasma; 2. The ratio of plasma to lymphocyte separation medium; T...

Embodiment 2

[0041] [Example 2] T cell proliferation test

[0042] The proliferation of T cells was detected by CCK-8 method and CFSE staining method respectively. In the detection of CCK-8, human peripheral blood mononuclear cells treated with four test samples (Phytohemagglutinin, PHA, PBS, pHSA, OsrHSA) respectively were seeded in 96-well plates, 100 per well A microliter of medium contains approximately 2 x 10 5 Personal peripheral blood mononuclear cells, the inoculated cells are cultured in a carbon dioxide incubator. After reaching each detection time point (24h, 48h, 72h), add 10 microliters of CCK-8 reagent to each well, and continue to incubate at 37°C for 4h, and then use a microplate reader to detect the absorbance at 450nm. In the CFSE assay, peripheral blood mononuclear cells were stained with 3 μM CFSE at 37°C for 5 minutes and then seeded into 24-well plates. Each well contained approximately 4×10 5 Peripheral blood mononuclear cells, also after reaching each detection t...

Embodiment 3

[0046] [Example 3] Detection of T cell subsets

[0047] The detection of T cell subtypes was all determined by flow cytometry. Peripheral blood mononuclear cells were seeded in 12-well plates after being treated as shown in Table 1, and each well contained about 5×10 5 Peripheral blood mononuclear cells, after reaching each detection time point, the cells were rinsed with PBS and stained with CD3-PE, CD4-FITC and CD8-APC for 30 minutes, and the cells were detected by BD flow cytometry after staining. The ratio of CD3+ / CD4+ and CD3+ / CD8+ cells was detected after lymphocyte circle gate.

[0048] The results showed that the T cells treated with plant-derived recombinant human serum albumin had similar subset ratios to the T cells treated with human serum albumin, as shown in Table 2.

[0049] Table 2. The proportion of subgroups of T cells under different treatments and at different time points

[0050]

[0051]

[0052] a.*p<0.05; **p<0.01. The significant difference in...

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Abstract

The invention provides immune toxicity evaluation application of human peripheral blood monocytes for new medicine development. The method for new medicine toxicology evaluation through human peripheral blood monocytes comprises the steps of 1, preparing the human peripheral blood monocytes; 2, using control medicine and new medicine to be measured for processing the human peripheral blood monocytes obtained in the step 1; 3, using a CCK-8 method and a CFSE dyeing method for detecting proliferation of T cells in the human peripheral blood monocytes obtained in the step 2; 4, using flow cytometry for determining T-cell subsets in the human peripheral blood monocytes obtained in the step 2; 5, using a CBA reagent box and a BD flow cytometer for detecting cell factors of the human peripheral blood monocytes obtained in the step 2.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an evaluation method of immunotoxicity for new drug development. Background technique [0002] Plant-derived drugs are a class of drugs developed based on plant platforms. They have developed rapidly in the past two decades. As of today, there are at least 108 types of drugs based on plant cells, and more than 30 of them have entered the clinical evaluation stage. Nine species have been approved to enter the market. A variety of plants are used to develop drugs today, including rice, tobacco, corn, soybeans, potatoes, barley, carrots, and safflowers. Various pharmaceutical preparations are produced from these plants, including antibodies, cytokines, Vaccines and enzyme preparations, etc. At present, rice seeds have successfully expressed a variety of recombinant medicinal proteins, including cholera toxin B subunit protein, human transferrin, human serum albumin, human α-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
CPCC12Q1/02
Inventor 杨代常付凯
Owner WUHAN UNIV
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