DC-based therapeutic glioma vaccine and preparation method thereof

Abstract

The invention relates to a DC-based therapeutic glioma vaccine and a preparation method thereof. The preparation method includes the steps: (1) collecting immature DCs; (2) adding in glioma antigen peptides and the immature DCs for co-culture; (3) adding in tumor necrosis factor-alpha and interleukin or lipopolysaccharide for continuous culture for 7-13 days till the DCs are mature and loaded with the glioma antigen peptides. The DC-based therapeutic glioma vaccine is high in immunogenicity and capable of well regulating the body immunity mechanism for gliomas.

Description

technical field

[0001] The invention relates to the fields of bioengineering and biomedicine, in particular to a DC-based therapeutic vaccine for glioma and a preparation method thereof. Background technique

[0002] Glioma is a tumor derived from the neuroepithelium. It is the most common malignant tumor in the brain, accounting for about 40-50% of all intracranial tumors. It has become the mainstay of tumor treatment due to its high incidence, high recurrence rate, and high mortality rate. problem. Traditional treatment methods are mainly surgery and radiotherapy and chemotherapy to improve the survival rate of patients. However, total resection of gliomas located in important functional areas is difficult. Radiation therapy is the routine treatment for almost all types of glioma, but the efficacy evaluation is different. Except for medulloblastoma, which is highly sensitive to radiotherapy, and ependymoma, which is moderately sensitive, other types are not sensitive to ...

Examples

preparation example Construction

[0023] A DC-based glioma therapeutic vaccine provided by the present invention, its preparation method comprises the following steps:

[0024] (1) Obtain immature DC;

[0025] (2) Adding glioma antigen polypeptide to co-culture with immature DC;

[0026] (3) Add tumor necrosis factor-α and interleukin or lipopolysaccharide and continue to culture for 7-13 days, until DC matures and DC is loaded with glioma antigen polypeptide.

[0027] In step (1), immature DCs can be derived from human blood, but because immature DCs only account for 0.5-1.0% of the total number of monocytes in the blood, the number is very small; therefore, in a preferred embodiment of the present invention, induction The method of mononuclear cells to obtain immature DC, the specific steps are:

[0028] (11) isolate monocytes from blood;

[0029] (12) Add in vitro induction medium and cultivate for 72h, and the monocytes obtained in step (11) are amplified;

[0030] (13) Perform a half-quantity exchange...

Embodiment 1

[0048] The preparation method of DC-based glioma therapeutic vaccine comprises the following steps:

[0049] 1) Take 20ml of human peripheral blood, and use Ficon Lymphocyte Separation Medium to separate CD14 + Monocytes, after washing with PBS solution, press 2.5-5x10 7 / 3ml / well was inoculated in a six-well plate, and incubated with RPMI-1640 medium at 37°C, 5% CO 2 placed in an incubator for 90 minutes to collect adherent cells, namely CD14 + monocytes.

[0050] 2) CD14 + Monocytes were inoculated into RPMI-1640 medium containing 800 U / mL granulo-macrophage factor and 1000 U / mL interleukin-4 and incubated at 37°C, CO 2 After induction in an incubator with a concentration of 5% for 3-5 days, half of the medium was changed, and granule-macrophage factor and interleukin-4 were supplemented to maintain the concentration of granule-macrophage factor and interleukin-4 to obtain immature DC;

[0051] 3) Use PBS solution (PH=7.4) to adjust the concentration of immature DC cell...

Embodiment 2

[0054] The difference from Example 1 provided by the present invention is that in this example, the concentration of tumor necrosis factor-α is 10 ng / ml, and the concentration of interleukin-1 is 5 ng / ml.

technical field

[0001] The invention relates to the fields of bioengineering and biomedicine, in particular to a DC-based therapeutic vaccine for glioma and a preparation method thereof. Background technique

[0002] Glioma is a tumor derived from the neuroepithelium. It is the most common malignant tumor in the brain, accounting for about 40-50% of all intracranial tumors. It has become the mainstay of tumor treatment due to its high incidence, high recurrence rate, and high mortality rate. problem. Traditional treatment methods are mainly surgery and radiotherapy and chemotherapy to improve the survival rate of patients. However, total resection of gliomas located in important functional areas is difficult. Radiation therapy is the routine treatment for almost all types of glioma, but the efficacy evaluation is different. Except for medulloblastoma, which is highly sensitive to radiotherapy, and ependymoma, which is moderately sensitive, other types are not sensitive to ...

Examples

preparation example Construction

[0023] A DC-based glioma therapeutic vaccine provided by the present invention, its preparation method comprises the following steps:

[0024] (1) Obtain immature DC;

[0025] (2) Adding glioma antigen polypeptide to co-culture with immature DC;

[0026] (3) Add tumor necrosis factor-α and interleukin or lipopolysaccharide and continue to culture for 7-13 days, until DC matures and DC is loaded with glioma antigen polypeptide.

[0027] In step (1), immature DCs can be derived from human blood, but because immature DCs only account for 0.5-1.0% of the total number of monocytes in the blood, the number is very small; therefore, in a preferred embodiment of the present invention, induction The method of mononuclear cells to obtain immature DC, the specific steps are:

[0028] (11) isolate monocytes from blood;

[0029] (12) Add in vitro induction medium and cultivate for 72h, and the monocytes obtained in step (11) are amplified;

[0030] (13) Perform a half-quantity exchange...

Embodiment 1

[0048] The preparation method of DC-based glioma therapeutic vaccine comprises the following steps:

[0049] 1) Take 20ml of human peripheral blood, and use Ficon Lymphocyte Separation Medium to separate CD14 + Monocytes, after washing with PBS solution, press 2.5-5x10 7 / 3ml / well was inoculated in a six-well plate, and incubated with RPMI-1640 medium at 37°C, 5% CO 2 placed in an incubator for 90 minutes to collect adherent cells, namely CD14 + monocytes.

[0050] 2) CD14 + Monocytes were inoculated into RPMI-1640 medium containing 800 U / mL granulo-macrophage factor and 1000 U / mL interleukin-4 and incubated at 37°C, CO 2 After induction in an incubator with a concentration of 5% for 3-5 days, half of the medium was changed, and granule-macrophage factor and interleukin-4 were supplemented to maintain the concentration of granule-macrophage factor and interleukin-4 to obtain immature DC;

[0051] 3) Use PBS solution (PH=7.4) to adjust the concentration of immature DC cell...

Embodiment 2

[0054] The difference from Example 1 provided by the present invention is that in this example, the concentration of tumor necrosis factor-α is 10 ng / ml, and the concentration of interleukin-1 is 5 ng / ml.

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