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In vitro culture method for promoting maturation of dendritic cells and special culture medium for the same

A dendritic cell and culture medium technology, applied in the biological field, can solve the problems of poor curative effect of HBeAg-positive chronic hepatitis B and affect the therapeutic effect of HBVDC vaccine, and achieve less stimulating factors and strong DC maturation ability. Effect

Inactive Publication Date: 2013-01-02
PEKING UNIV FIRST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the HBV DC vaccine has a certain effect, especially for HBeAg-negative chronic hepatitis B, it is less effective for HBeAg-positive chronic hepatitis B. Therefore, more in-depth research is needed on these issues
In addition, there are still some issues to be considered in the clinical application of HBV DC vaccine, such as the source of DC, the optimal antigen loading scheme, in vitro culture time, maturation promotion scheme, the number of immune cells, the interval time of immunization, the frequency of immunization and the way of immunization etc. These links may affect the therapeutic effect of HBV DC vaccine

Method used

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  • In vitro culture method for promoting maturation of dendritic cells and special culture medium for the same
  • In vitro culture method for promoting maturation of dendritic cells and special culture medium for the same
  • In vitro culture method for promoting maturation of dendritic cells and special culture medium for the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Exploration and phenotype detection of conditions for promoting dendritic cell maturation

[0042] 1. Peripheral blood mononuclear cell isolation (PBMC)

[0043] (1) Divide the concentrated white blood cells isolated from healthy people into 10mL centrifuge tubes, about 4mL in each tube, add PH 7.2 phosphate buffer solution to dilute in equal times and mix well;

[0044] (2) Slowly add the diluted and concentrated leukocytes to a 10mL centrifuge tube prefilled with 4-5mL lymphocyte separation medium (density 1.077g / mL), keeping the interface between the two clear;

[0045] (3) Centrifuge at room temperature (25°C), 1600rpm, 35min;

[0046] (4) Carefully absorb the middle buffy coat (peripheral blood mononuclear cells), add it to a centrifuge tube prefilled with 4-5mL PBS and mix gently;

[0047] (5) Centrifuge at room temperature, 1500rpm, 10min;

[0048] (6) Discard the supernatant, add PBS and mix gently, centrifuge at room temperature, 1000rpm, 10min; ...

Embodiment 2

[0095] Example 2. Functional identification of mature dendritic cells in different maturation-promoting schemes

[0096] 1. Separation of peripheral blood mononuclear cells (PBMC): the same as the method of 1 in Example 1;

[0097] 2. Sorting of CD14 positive monocytes: the same method as in Example 1, 2; obtain CD14+ monocytes and remove PBMCs of CD14+ monocytes;

[0098] 3. GM-CSF / IL-4 induction to obtain immature DC: the same method as in Example 1 and 3; obtain imDC;

[0099] 4. From immature DC (imDC) to mature DC (DC).

[0100] After aspirating and discarding most of the imDC culture medium, perform the following two groups of treatments:

[0101] Control group: add imDC to standard medium to promote maturation (37° C., in a 5% carbon dioxide incubator) for 2 days to obtain mature DC1 in the control group.

[0102] The standard medium is prepared as follows: IL-1β, IL-6, TNF-α, PGE2, penicillin, streptomycin and serum-free AIM-V medium are mixed to obtain medium, IL-1...

Embodiment 3

[0159] Example 3, Study on the Function of Dendritic Cells Sensitized by HBV Antigen Peptides

[0160] Peripheral blood concentrated leukocytes of HBV chronically infected patients were collected from the blood of isolated HBV chronically infected patients (provided by the 302 Hospital of the People’s Liberation Army, and patients were informed); the clinical data are as follows in Table 5:

[0161] Table 5 Clinical data of patients with chronic HBV infection

[0162]

[0163] 1. Separation of peripheral blood mononuclear cells (PBMC): obtain the concentrated leukocytes from the peripheral blood of 1 case of HBV chronic infection in vitro, and the method is the same as in 1 of Example 1;

[0164] 2. Sorting of CD14 positive monocytes: same as in 2 of Example 1 to obtain CD14+ monocytes.

[0165] 3. Detection of immature DC loaded with HBV multiple antigen peptides

[0166] After sorting CD14+ monocytes, plant CD14+ monocytes in a 12-well plate, 1 million per well, add med...

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Abstract

The invention discloses an in vitro culture method for promoting maturation of dendritic cells and special culture medium for the method. The invention provides a maturation promoting culture medium prepared from in vitro immature dendritic cells. The method includes mixing R848 and PGE2 with serum-free AIM-V culture medium to obtain a culture medium, the concentration of R848 in the maturation promoting culture medium being (1-10) mu g / mL, the concentration of PGE2 in the maturation promoting culture medium being 1 mu g / mL. The inventive experiment shows that the inventive maturation promoting culture medium is superior to the control standard culture medium. The inventive maturation promoting culture medium has few stimulating factors, strong DC maturation promoting capacity, and high survival rate of DC after maturation promotion.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an in vitro culture method and a special culture medium for promoting the maturation of dendritic cells. Background technique [0002] At present, there are about 400 million HBV chronically infected people in the world, mainly in developing countries, and more than 200,000 and 300,000 HBV chronically infected people die of liver cirrhosis and hepatocellular carcinoma (HCC) respectively every year. Antiviral therapy is the key to the treatment of chronic hepatitis B (CHB). At present, the main antiviral drugs include interferon (including common interferon and pegylated interferon) and nucleoside (acid) analogs such as lamivudine, Adefovir dipivoxil, entecavir and telbivudine, etc., but the efficacy is not satisfactory. The interferon response ratio is about 30%, but the response to nucleoside (acid) analog therapy is not durable, the recurrence rate is high after drug withdrawal, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784C12N5/0786A61K39/00A61P1/16A61P31/20
Inventor 王贵强吴学杰刘永哲任媛媛李世红
Owner PEKING UNIV FIRST HOSPITAL
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