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Modified dendritic cell and vaccine containing the same

A dendritic cell and vaccine technology, which is applied in the fields of biotechnology and medicine, and achieves the effects of simple preparation process and good active immunotherapy effect

Active Publication Date: 2014-11-05
广东省医学医疗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of SYK gene in tumor vaccines has not been reported.

Method used

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  • Modified dendritic cell and vaccine containing the same
  • Modified dendritic cell and vaccine containing the same
  • Modified dendritic cell and vaccine containing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A modified dendritic cell comprising the SYK gene transfected thereinto using a viral vector. Wherein, the preparation method of the modified dendritic cells is as follows:

[0041] 1. Amplification of SYK gene fragment

[0042] The PCR amplification template is a plasmid (pCMV6-XL5) carrying the full-length cDNA of the SYK gene (NCBI RefSeq: NM 003177.3), which was purchased from OriGen Biological Company. The CDS sequence (SEQ ID NO: 1) of the SYK gene is about 1.9kb.

[0043] The primer sequences used for PCR amplification are:

[0044] P1: 5'-ATGGCCAGCAGCGGCATGGCTG-3' (SEQ ID NO: 2)

[0045] P2: 5'-TTAGTTCACCACGTCATAGTAG-3' (SEQ ID NO: 3)

[0046] Where P1 is the forward primer and P2 is the reverse primer.

[0047] The PCR reaction system for amplifying the SYK gene is as follows:

[0048]

[0049]

[0050] PCR reaction conditions such as Figure 6 shown.

[0051] 2. Purification of SYK gene amplified fragment

[0052] Agarose gel electrophoresis is ...

Embodiment 2

[0106] Determination of DC vaccine and related parameters

[0107] 1. Detection of DC cell maturity

[0108] The LV-DC cells obtained in Step 8 of Example 1 can be directly used as a DC cell vaccine. The expression of CD80, CD83, and CD86 molecules on the surface of LV-DC cells obtained in Example 1 was detected by flow cytometry, and the detection results are shown in Table 2 and figure 1 , 2 , 3.

[0109] 2. Detection of tumor killing rate of LV-DC-CIK cells in vitro

[0110] Will 2×10 5 The LV-DC cell obtained in step 8 in Example 1 was mixed with 2×10 6 CIK cells obtained in Step 8 were co-cultured (LV-DC:CIK=1:10), supplemented with 0.5% (V / V) autologous plasma, 1000U / ml IL-2, 100ng / ml GM-CSF every two days Alys-505 medium, 5-7 days later to obtain effector cells LV-DC-CIK cells.

[0111] Take CFSE-labeled RB cells as target cells, and adjust the cell density to 1×10 5 / ml and 1×10 6 / ml.

[0112] Take a flow tube, add 800 μl and 200 μl of effector target cells ...

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Abstract

The invention relates to the fields of biotechnology and medicine, and provides a modified dendritic cell. Viral vectors are used to transfect an SYK gene into the modified dendritic cells. The invention also provides a vaccine with the modified dendritic cell as an active component. The vaccine is used for prevention and active immunotherapy of tumor. According to the invention, maturation of the DC cells transfected with the SYK gene, percentage and tumor killing rate of CD3 + CD56 + T cells in the obtained LV-DC-CIK cells, and IFN-gamma secretion rate in a cell culture supernatant are all significantly higher than those in a control group.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular, the invention relates to a modified dendritic cell and a vaccine comprising the dendritic cell. Background technique [0002] Dendritic cell (DC) is currently known as the antigen-presenting cell with the strongest antigen-presenting ability, which has attracted close attention in current tumor immunotherapy. DCs can ingest and process antigens, and express high levels of MHC molecules, co-stimulatory molecules, cell adhesion molecules ICAM-1, ICAM-3, and lymphocyte function associated antigen-3 (1ymphocyte function associated antigen LFA-3), etc. The presented molecules secrete a high level of Thl-type cytokine IL-12, which effectively presents antigens to T lymphocytes and initiates the patient's own specific tumor-killing immune response. However, a large number of experiments have shown that DCs in tumor patients are in an "incompetent state", and their ability to pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/12C12N15/867A61K39/00A61K48/00A61P35/00
Inventor 刘韬陈雪梅
Owner 广东省医学医疗有限公司
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