Application of vitamin D acceptor and ligand thereof to improvement of antitumor capacity of immunocyte
A technology of immune cells and vitamins, applied in the direction of blood/immune system cells, receptors/cell surface antigens/cell surface determinants, anti-tumor drugs, etc., can solve the problems of expensive and side effects, achieve good safety performance, and promote secretion , well tolerated effect
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Embodiment 1
[0068] Example 1: The ligand structural formula of the vitamin D receptor is as follows:
[0069]
[0070] The above compounds (vitamin D 3 (VD 3 ), 25(OH)D 3 , 1α,25(OH) 2 D. 3 ) as an activator of the vitamin D receptor. This compound is commercially available from Selleck Corporation.
Embodiment 2
[0071] Example 2: Isolation and culture of CD8T and CD4T cells
[0072] CD8T and CD4T cells are derived from peripheral blood mononuclear cells (PBMCs), and PBMCs are derived from the peripheral blood of healthy people. Will 1×10 7 For every 50 μL of PBMCs cells (according to the instructions, every 50 μL of sorting magnetic beads liquid, add 1×10 7 1 PBMCs) sorting magnetic beads CD8 and CD4 (both magnetic beads were purchased from BD Company), and incubated at 4°C for 25 min. Add 950 μL of PBS to resuspend in the flow tube, put it into the magnetic stand for 5 minutes, remove the supernatant, and repeat this step 2 times. Count and plate, add the sorted CD8T or CD4T cells to the 48 wells of the pre-coated plate (αCD3, 5 μg / mL, Biolegend) in advance, add 1 μg / mL (final concentration) αCD28 (Biolegend) to stimulate for 48 h, two Add rhIL-2 (final concentration: 40 ng / mL) to cells available one day later (γδT / CD3>90% detected by flow cytometry).
Embodiment 3
[0073] Embodiment 3: recombinant vitamin D receptor gene cloning
[0074] (1) Overexpression of VDR gene
[0075] The vitamin D receptor (vitamin D receptor, VDR) gene was cloned from CD8 T cells (obtained in Example 2). RNA (Trizol) in CD8 T cells was extracted, cDNA library was obtained by reverse transcription (Takara), and primers were designed to clone the full-length fragment of VDR (upstream primer sequence: 5'-ATGGAGGCAATGGCGGC-3', downstream primer sequence: 5' -TCAGGAGATCTCATTGCCAAACA-3'), double digested with XbaI and EcoRI (NEB), and cloned into the plasmid pTSB-CMV-MCS-SBP-3Flag-copGFP-F2A-PuroR (TranSheepBio) to obtain the plasmid pTSB-CMV-MCS -SBP-3Flag-copGFP-F2A-PuroR-VDR, and then transform it into Escherichia coli DH5α (Takara), after BGI sequencing and NCBI database comparison, the cloned gene is indeed the vitamin D receptor gene, The sequence is as follows (SEQ ID NO.1):
[0076] atggaggcaatggcggccagcacttccctgcctgaccctggagactttgaccggaacgtgccccggatctgtg...
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