Cancer cell/DC fusion tumor vaccine for expressing Endoglin and preparation method for cancer cell/DC fusion tumor vaccine
A technology of tumor cells and fusion cells, which is applied in the field of cancer cell/DC fusion tumor vaccines expressing tumor blood vessel marker molecules and its preparation, and can solve the problems of unsatisfactory therapeutic effects of fusion vaccines
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Embodiment 1
[0092] Embodiment 1, the preparation of fusion cell
[0093] The method for preparing fusion cells, including S1) and S2):
[0094] S1) introducing Endoglin coding gene into tumor cells to obtain recombinant cells;
[0095] S2) Fusing the recombinant cells with dendritic cells to obtain the fused cells.
[0096] The specific method is as follows:
[0097] 1. Preparation of recombinant cells
[0098] Replace the DNA fragment between the BamHI and XbaI recognition sites on the pLVX-Puro vector with the DNA molecule shown in SEQ ID No.2 in the sequence table, keep the other sequences of pLVX-Puro unchanged, and obtain the recombinant vector pLVX-Puro / Eng , the recombinant vector pLVX-Puro / Eng expresses the Endoglin shown in SEQ ID No.1. The recombinant vector pLVX-Puro / Eng was introduced into HepG2 cells to obtain recombinant cells containing Endoglin coding sequence and EGFP coding sequence, and the recombinant cells were named HepG2(Eng+).
[0099] Wherein, the 1st-1683rd ...
Embodiment 2
[0117] Example 2, DC / HepG2 (Eng+) in vitro induces the generation of T lymphocytes that secrete IFN-γ
[0118] The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:
[0119] The T lymphocytes secreting IFN-γ induced by DC / HepG2 (Eng+) were obtained according to the following method, and the IFN-γ secreted by T lymphocytes was detected by enzyme linked immunospot assay (ELISPOT). ×Washing buffer, biotin-labeled antibody, and enzyme-labeled avidin are all Human IFN-gamma precoated ELISPOT kit (Human IFN-gamma precoated ELISPOT kit is a product of Daktronix Biotechnology Co., Ltd., the catalog number is DKW22-1000-048) The reagents in the specific steps are as follows:
[0120] 1) Take out the well plate in the kit, add 200 μL of incomplete RIPM 1640 medium to each well, let stand at room temperature for 5-10 minutes, and pour off the liquid.
[0121] 2) Add DC / HepG2 (Eng+) 3×10 of Example 1 suspended in incomplete RIPM 164...
Embodiment 3
[0134]Example 3. Therapeutic effect of T lymphocytes secreting IFN-γ induced by fusion cells DC / HepG2 (Eng+) on tumors
[0135] The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:
[0136] The T lymphocytes secreting IFN-γ induced by DC / HepG2 (Eng+) of Example 2, the T lymphocytes secreting IFN-γ induced by DC / HepG2, and the IFN-γ secreted induced by DC / HepG2 (pLVX-Puro) T lymphocytes induced by DC, T lymphocytes secreting IFN-γ induced by DC, T lymphocytes induced by HepG2 (Eng+), T lymphocytes induced by HepG2 and T lymphocytes induced by HepG2 (pLVX-Puro) were suspended in PBS , respectively, the cell content is 10 5 cells / μL of DC / HepG2(Eng+)-induced IFN-γ-secreting T lymphocyte suspension, DC / HepG2-induced IFN-γ-secreting T-lymphocyte suspension, DC / HepG2(pLVX-Puro)-induced secretion IFN-γ T lymphocyte suspension, DC induced IFN-γ secreting T lymphocyte suspension, HepG2(Eng+) induced T lymphocyte suspension, HepG...
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