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A kind of dendritic cell vaccine and its preparation method and application

A dendritic cell and vaccine technology, applied in the field of medical bioengineering, can solve problems such as limited expression, and achieve the effect of less toxic side effects and high safety

Active Publication Date: 2018-08-10
杭州朔溪生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Given their high expression and high activity in oncogenic cells and cancer stem cells but limited expression in normal tissues, Oct4 and Sox2 are potential targets for immune intervention

Method used

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  • A kind of dendritic cell vaccine and its preparation method and application
  • A kind of dendritic cell vaccine and its preparation method and application
  • A kind of dendritic cell vaccine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Integration of Oct4 and Sox2 genes at the AAVS1 site

[0032] Tumor cells exist in heterogeneous populations. Therefore, in order to obtain a consistent effect of genetic engineering of liver cancer cells, the present invention obtains HepG2 of a homologous population for the first time through single cell cloning. Single cells were seeded in 96-well plates, and the holes were enlarged. When its parental cell line HepG2, SC was selected for further application, the clone showed similar morphology and growth rate.

[0033] In order to produce HepG2 CSC-like cells, the present invention uses baculovirus-mediated zinc finger nuclease technology to introduce gene cassettes containing Oct4 and Sox2 genes (the specific gene sequences of Oct4 and Sox are described in the attached gene sequence table) into HepG2 SC Cellular AAVS1 locus. Two different promoters, CMV and EF1A, were tested for the expression of these two genes. Liver cancer cells HepG2 and BV-ZFN wer...

Embodiment 2

[0035] Example 2: Preparation of liver cancer cells with expression of cancer stem cell markers

[0036] Routine cells were cultured in DMEM medium (Invitrogen Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin / streptomycin (Invitrogen), in a humidified incubator at 37°C and 5% carbon dioxide. in progress. The sphere culture technique is commonly used to aggregate, maintain and expand the number of CSCs from cultured adherent cells. The above-mentioned transgenic liver cancer cells were cultured in spheres to detect whether up-regulating the gene expression of Oct4 and Sox2 can increase the number of CSCs, and the criteria for defining CSCs are the expression profiles and expression levels of marker genes.

[0037] First, human hepatoma HepG2 cells were isolated for single cell cloning. Cells are seeded individually in culture medium in a 96-well culture plate. Each clone was then propagated individually. Human hepatoma cell s...

Embodiment 3

[0046] Example 3: DC vaccine against tumor stem cell markers

[0047] Dendritic cells are derived from human peripheral blood mononuclear cells (PBMC). PBMCs are Ficoll GradientFicoll-Paque TM Technology (GE Healthcare Life Sciences) isolated from fresh buffy coat. Frozen / thawed hepatocellular carcinoma stem cell-producing lysates and human peripheral mononuclear cell-derived dendritic cells and lysates used in the present invention. To study the priming capacity of the resulting dendritic cells, T cells were obtained from easysep TM Peripheral blood mononuclear cells were isolated from human naive CD8+ T cell enrichment kit (Stem Cell Isolation Technology). The immature T cells were cultured in the Cellgro medium specially for culturing T cells, adding 5% human AB serum (US Life Technologies), IL-2 (50IU / ml), 20ng / ml IL-7 (for ) The ratio of primed naive T cells to mature dendritic cells is 1:2.5, 1:5 or 1:10. Grow and expand on the cell culture medium described above. ...

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Abstract

The invention relates to the technical field of medical biology engineering, in particular to a dendritic cell vaccine and a preparation method and application of the dendritic cell vaccine. Disclosed is a method for preparing a dendritic cell (DC) cancer vaccine for persons with liver cancer stem cells. The method comprises the following steps of inserting Oct4 and Sox2 genes in an AAVSl locus of a hepatoma cell line genome by using a zinc finger nuclease technology; enriching similar liver cancer stem cells in corrected liver cancer cells by using a tumor stem cell spheroid forming method; and separating prepared dendritic cells by stimulating autologous blood through similar liver cancer stem cell freeze thawing and cracking liquid to obtain antigen presentation DC cells loaded with liver cancer stem cell antigens. Established liver cancer stem cell strains can express molecular markers of the liver cancer stem cells at a high level, expression of a plurality of liver cancer stem cell markers such as CDl3, CD90, EpCAM and the like can be improved obviously, and the specificity is high; the used antigen presentation dendritic cells source from the autologous blood, are high in safety and have little toxic and side effects; and after being tried out, the prepared DC cell cancer vaccine can obviously stimulate CTL (cytotoxic T lymphocyte ) reaction of the specificity cell toxic effect of the tumor stem cells.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to a dendritic cell vaccine and its preparation method and application. Background technique [0002] Hepatocellular carcinoma (HCC), a primary liver malignancy, is the third most common cause of cancer-related death worldwide. The vast majority of hepatocellular carcinomas are secondary to viral infection (hepatitis B or C) or cirrhosis (alcoholism is the most common cause of cirrhosis). Surgery offers the best chance of cure, but only 10-20% of liver cancers can be completely eradicated by surgery. Many patients who cannot be operated on for liver cancer (ie, HCC) or who develop metastatic recurrence after surgery will receive chemotherapy / radiation therapy. Unfortunately, HCC is a tumor that is highly resistant to conventional chemotherapeutic agents, and radiation therapy is rarely effective. Therefore, liver cancer brings a great burden to individuals, familie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/00A61P35/00A61P1/16C12N5/10C12N5/0784
Inventor 卓朗王晖王柯
Owner 杭州朔溪生物医药有限公司
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