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Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell

A dendritic cell and specific technology, applied in the field of medicine and biology, can solve the problems of difficult virus removal, low clinical cure rate, high recurrence rate, etc., and achieve the effect of improving specific cellular immunity

Inactive Publication Date: 2016-03-02
黄月华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing treatment drugs are difficult to clear the virus, the clinical cure rate is extremely low, and the recurrence rate after drug withdrawal is high, requiring lifelong treatment

Method used

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  • Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell
  • Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell
  • Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Obtaining antigen-loaded monocyte-derived dendritic cells

[0033] Collect 10ml of peripheral blood from patients with chronic hepatitis B, separate peripheral blood mononuclear cells (PBMC) according to conventional methods, resuspend in 4ml of AIM-V culture medium, and then place them at 37°C, 50% (volume concentration) CO 2 Incubate for 2 hours in an incubator.

[0034] After 2 hours, take it out, shake it gently, and remove the suspended non-adherent cells. Add 4ml of AIM-V medium to the adherent cells, which contains IL-4 (20ng / ml) and GM-CSF (100ng / ml ).

[0035] After 3 days, the adherent cells were supplemented with 2ml of AIM-V culture medium, which contained IL-4 (20ng / ml) and GM-CSF (100ng / ml), and was still placed at 37°C and 50% (volume concentration) CO 2 cultured in an incubator.

[0036] Day 6: Adherent cells: Add 10 μg of hepatitis B vaccine stock solution (Shenzhen Kangtai Company), culture for 24 hours, and obtain functional DC loaded wi...

Embodiment 2

[0038] Example 2: T cell expansion

[0039] The suspended cells (i.e. T cells) obtained in Example 1 were transferred to the AIM-VT75 culture flask containing 4ml, and after the AIM-V medium was washed 3 times (washing liquid volume 2ml / time), 1ug / ml OKT3, 1ug / ml OKT3, 1ug / ml anti-CD28, placed at 37°C, 50% (volume concentration) CO 2 cultured in an incubator.

[0040]Day 2: T cell culture medium was supplemented with 5-10ml of AIM-V culture medium, which contained IL-2 (300IU / ml) and 1.5ml of 20% (mass volume concentration) albumin.

[0041] Day 4: Supplement 5-10ml of AIM‐V culture medium, which contains IL‐2 (300IU / ml) and 1.5ml of 20% (mass volume concentration) albumin )CO 2 Cultured in an incubator until day 8 to obtain proliferating T cells.

Embodiment 3

[0042] Example 3: HBV-specific T cells

[0043] Day 8: The "antigen-loaded functional DC" obtained in Example 1 and the proliferating T cells obtained in Example 2 were mixed and co-cultured.

[0044] Day 10: Supplement 5-10ml of AIM‐V culture medium, which contains IL2 (300IU / ml) and 1.5ml of 20% albumin.

[0045] Day 12: Supplement 5-10ml of AIM‐V culture medium, which contains IL2 (300IU / ml) and 1.5ml of 20% albumin.

[0046] Day 14: Obtain HBV-specific T cells.

[0047] figure 2 : HBV-sensitized DC co-cultured with T cells can produce more CD8+ cytotoxic T cells.

[0048] Such as figure 2 As shown, the following methods were used to induce PBMCs from chronic HBV-infected patients and healthy people with hepatitis B protective antibodies: method 1: adding hepatitis B vaccine directly to PBMC; method 2: adding hepatitis B vaccine and interleukin 4 to PBMC; method 3 : Co-culture of HBV-sensitized DC and T cells. The results showed that methods 2 and 3 produced higher ...

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Abstract

The invention provides a construction method of a hepatitis vaccine sensitization dendritic cell inducing specific T cell. The method comprises the following steps that (1) an antigen load mononuclear cell derivative dendritic cell is obtained; (2) the T cell is amplified; (3) the specific hepatitis virus T cell is obtained. The method can efficiently present viral antigen.

Description

technical field [0001] The invention belongs to the field of medicine and biology, and in particular relates to a method for constructing specific T cells induced by dendritic cells sensitized by hepatitis vaccine. Background technique [0002] Chronic HBV (hepatitis B virus) infection is still the primary factor in the progression of liver cancer and end-stage liver disease. The existing therapeutic drugs are difficult to clear the virus, the clinical cure rate is extremely low, and the recurrence rate after drug withdrawal is high, requiring life-long treatment. The core theory of chronic HBV infection is that the insufficiency of specific T cells leads to the body's immune tolerance to the virus. [0003] Dendritic cells (DC) are the key promoters of T cell activation, the strongest antigen-presenting cells in the body, and are responsible for the initiation of T cell maturation, activation, and proliferation. However, the lack of DC number or function in patients with c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P31/14A61P31/20
Inventor 黄月华
Owner 黄月华
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