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Polygene transfection tumor cell strain and fusion vaccine thereof, as well as preparation methods

A technology of tumor cell lines and transfected cells, which is applied in the field of multi-gene transfection of tumor cell lines and their fusion tumor vaccines and preparation, can solve the problem of inability to effectively remove tumors, and achieve the effect of enhancing the immune effect against pancreatic cancer

Inactive Publication Date: 2014-10-15
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the regulation of danger signals, Th1 cell immune response is activated to eliminate tumors; while under the action of inhibitory signals, Th2 responses are activated, which cannot effectively eliminate tumors

Method used

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  • Polygene transfection tumor cell strain and fusion vaccine thereof, as well as preparation methods
  • Polygene transfection tumor cell strain and fusion vaccine thereof, as well as preparation methods
  • Polygene transfection tumor cell strain and fusion vaccine thereof, as well as preparation methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Foxp3 overexpression, A20miRNA plasmid construction and determination

[0054] (1) Foxp3 overexpression plasmid construction

[0055] For the target gene Rat Foxp3NCBI search sequence NM_001108250.1, use the Invitrogen design software to design the amplification sequence, use PacI / AscI to digest the NM_001108250.1 target sequence, according to the enzyme digestion system (10×buffer5uL, 12GS0498-1 (Rat Foxp3 gene Sequence) 10uL, PacI, AscI each 1uL, add double distilled water to 23uL, 37°C), digest for 2 hours, use PacI / AscI to pLenti6.3-MCS-IRES2-DsRed Monomer (the target vector for overexpressing Foxp3 gene, US Invitrogen), according to the enzyme digestion system (10×buffer 5uL, pLenti6.3-MCS-IRES2-DsRed Monomer 2uL, PacI, AscI 1uL, add distilled water to 41uL, 37°C), enzyme digestion for 2 hours, electrophoresis, and recovery Digested target fragment. Use T4DNA ligase to connect the recovered fragments to the carrier, according to the ligation system (Lig...

Embodiment 2

[0062] Example 2 Foxp3 overexpression, A20miRAN plasmid lentivirus packaging and activity determination

[0063] (1) Lentiviral packaging

[0064] ① Take 293T cells in good condition and in the logarithmic growth phase. 6 The number of cells was seeded in a culture dish at 37°C, 5% CO 2 ②Remove the culture medium before transfection the next day, and replace with 5mL Opti-MEM culture medium; ③Take 9ug Packaging Mix and 3ug lentiviral expression plasmid (overexpression Foxp3 plasmid and A20miRNA plasmid) and add 1.5mL In Opti-MEM (preheated at 37°C), mix gently; ④ Take 36uL lipofectamine2000 and add it to 1.5mL Opti-MEM, mix gently, and let stand at room temperature for 5min; ⑤ Gently mix the plasmid solution and lipofectamine2000 dilution, set 20min at room temperature; ⑥Carefully add 3mL of plasmid-liposome complex to the cell culture dish, mix gently, 37°C, 5% CO 2 After incubating in the incubator for 6 hours, replace the complete culture solution DMEM+10% FBS; ⑦After 48...

Embodiment 3

[0072] Example 3 Cell Recovery, Culture and Freezing

[0073] (1) Recovery of cells

[0074] ①Take out the frozen cell line (DSL6A / C1 cells) from the liquid nitrogen, quickly put it in a water bath at 37°C for about 1 minute, and take it out immediately after it is completely dissolved. During the incubation process, shake the cryopreservation tube fully to make it thaw quickly; ② Transfer the cryopreserved cells to a graduated centrifuge tube filled with 9 mL of DMEM medium culture medium containing 10% FBS, centrifuge at 800 rpm for 5 minutes , remove the supernatant and wash again with base culture; ③ Discard the medium, add 5 mL of DMEM medium containing 10% FBS, and gently blow and mix; ④ Transfer the cell suspension to the culture bottle, and screw the cap Loose 1 / 4 turn, 37°C 5% CO 2 Cultivate in a constant temperature incubator; ⑤ Change the medium once after 24 hours. When the cells grow to 80-90% confluent, perform passage or inoculation according to the above meth...

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Abstract

The invention discloses a polygene transfection tumor cell strain and a fusion vaccine thereof, as well as preparation methods. The cell strain comprises an Foxp3 gene and an A20 gene, and adopts the pancreatic cancer tumor cell line DSL6A / C1 of a rat as a transfection cell. The preparation methods of thepolygene transfection tumor cell strain and the fusion vaccine comprise the following steps: modifying the fusion vaccine of DSL6A / C1 cell and a dendritic cell of Treg by building over-expression Foxp3 and A20miRNA, so as to obtain a novel tumor vaccine which not only has a DC special function but also can express tumor antigen; adopting the fusion vaccine of the tumor cell and the dendritic cell which are modified through co-transfection of Foxp3-A20 to further enhance the anti-pancreatic cancer immunity effect. The fusion vaccine has not only a complete tumor antigen, but also the characteristic of an antigen presenting cell, and can effectively present an tumor antigen to a T lymphocyte, thereby stimulating an organism to generate a specific anti-tumor immunity response, being a new direction of tumor immunological therapy, providing a new method for curing tumor patients, and having a potential application value.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a polygene-transfected tumor cell line and its fusion tumor vaccine and a preparation method. Background technique [0002] In the process of tumor immunity, the immune function of T cells plays a key role. In particular, it is closely related to the immunosuppressive effect of regulatory T cells (regulatory T cells, Treg), so eliminating Treg in patients is expected to improve the immune function of patients. However, recent studies have found that the level of Tregs in cancer patients will quickly recover after Tregs are eliminated and easily cause autoimmune diseases. Therefore, the tumor treatment strategy targeting Tregs should be changed from "depletion" to "inhibition". Inhibit the number and function of Treg in vivo. Currently, Foxp3 is currently recognized as the most sensitive marker of Treg cells. Foxp3 is an autoantigen expressed in the thymus, and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K39/00A61K48/00A61P35/00
Inventor 周国雄黄莉莉
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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