Method of inducing antitumor immunity and its application in preparing medicine

An anti-tumor immunity and cell induction technology, applied in the field of biomedicine, to prevent and inhibit the growth of myeloma, improve the immune system, and improve the survival rate

Inactive Publication Date: 2007-01-03
南京大学生物制药工程研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, fixed primary umbilical vein endothelial cells have been used to induce anti-tumor immunity [41，42] , but there is no method for inducing anti-tumor immunity using live primary umbilical vein endothelial cells and tumor vascular endothelial cell membrane surface protein extracts

Method used

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  • Method of inducing antitumor immunity and its application in preparing medicine
  • Method of inducing antitumor immunity and its application in preparing medicine
  • Method of inducing antitumor immunity and its application in preparing medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Low-dose live myeloma FO cells induce anti-tumor immunity

[0084] 1. Vaccine preparation:

[0085] 1.1 Dendritic cells derived from splenocytes: The erythrocytes in the mouse spleen were lysed with 8.3 g / ml ammonium chloride solution dissolved in 0.01M Tris-HCl. Unlysed spleen cells were cultured in serum-free RPMI1640 containing 2 mM glutamine, 2 mg / ml sodium bicarbonate, 25 mM HEPEs, 100 U / ml penicillin and 100 μg / ml streptomycin. at 5% CO 2 After incubation for 1 hour in the incubator, non-adherent cells were washed away. Adherent spleen cells continued to be treated with 10% fetal bovine serum, 2mM glutamine, 2mg / ml sodium bicarbonate, 25mM HEPEs, 10ng / ml IL-4 (PeproTech, UK), 10ng / ml GM-CSF (PeproTech, UK), 100U / ml penicillin and 100μg / ml streptomycin medium, and differentiated into dendritic cells. After 4 days in culture, adherent and loosely adherent dendritic cells were harvested by gentle pipetting, and the cell surface exhibited typical dendri...

Embodiment 2

[0105] Example 2: Myeloma FO cells transfected with GM-CSF induce anti-tumor immunity

[0106] 1. Vaccine preparation:

[0107] 1.1 Transfection of recombinant GM-CSF adenovirus into myeloma FO cells: Incubate cells with recombinant GM-CSF adenovirus suspension diluted 10 times with serum-free RPMI-1640. After culturing at 37°C for 1 hour, calf serum and serum-free RPMI-1640 were added to adjust the serum concentration to 10%, and incubated in an incubator for 24 hours.

[0108] 1.2 Determination of transfection efficiency and GM-CSF secretion: collect transfected and non-transfected myeloma FO cells, fix with 2% (v / v) paraformaldehyde for 15 minutes, and then wash with 0.5% (v / v) paraformaldehyde ) Triton X-100 and 0.5% (w / v) BSA in PBS to permeate the cell membrane. Anti-mouse GM-CSF antibody (0.5 μg / 10 6 cells) were stained for 1 hour at room temperature and observed under a fluorescence microscope. Depend on Figure 4 It can be seen that more than 90% of the myeloma F...

Embodiment 3

[0116] Example 3: Live primary umbilical vein endothelial cells induce anti-tumor immunity

[0117] 1. Vaccine preparation:

[0118] 1.1 Preparation of primary human vascular endothelial cells: We prepared according to the method of separating vascular endothelial cells by Jaffe EA et al. [43] . Fresh human umbilical vein vessels were digested with type II collagenase, and the obtained human umbilical vein endothelial cells were cultured in 1% gelatin-coated culture dishes with IMDM medium containing 10% FBS.

[0119] 2. Evaluation of the anti-tumor effect of the vaccine:

[0120] 2.1 Live cell preventive immune anti-tumor model:

[0121] Mouse myeloma FO model: Primary human umbilical vein endothelial cells were harvested and resuspended in serum-free RPMI1640 medium. Female BALB / c mice were treated with 10 6 Immunization with primary human umbilical vein endothelial cells. Vaccine once a week for a total of four times. Inoculate subcutaneously 10 weeks after the last ...

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Abstract

The present invention discloses method of inducing antitumor immunity and its application in preparing medicine. The method is to induce antitumor immunity with live myeloma FO cell, freeze dried powder of GM-CSF transfected myeloma FO cell, primary human umbilical vein vessel endothelial cell, or the extract of tumor vessel endothelial cell membrane surface protein. The method may be used in preparing antitumor medicine and has simple operation and high antitumor immunity inducing effect.

Description

technical field [0001] The invention belongs to the field of biomedicine, relates to a method for inducing anti-tumor immunity, and also relates to the application of the method in pharmacy. Background technique [0002] With the development of gene transfer technology [1，2] , the presence of oncogenes has been discovered [3] , which clarifies the relationship that tumors originate from normal cells but are different from normal cells. Tumor development involves many cascades of events, such as immortal proliferation, sustained angiogenesis, and tissue invasion [4] . Tumor cells often express tumor-specific or tumor-associated antigens, which constitute targets for attack by the host immune system. Antibodies can attack tumors through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Immune cells, such as natural killer cells and cytotoxic T cells, can lyse tumor cells through cell-to-cell interactions. At the same time, cytokines sec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/12A61K35/28A61K9/19A61P35/00C12N5/10C12N15/861A61K35/44
Inventor 刘建宁谭向阳张菁
Owner 南京大学生物制药工程研究中心
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