A polypeptide specifically targeting triple-negative breast cancer stem cells and its application
A triple-negative breast cancer, application technology, applied in the direction of non-active ingredient medical preparations, active ingredient-containing medical preparations, peptides, etc., can solve the problem of incomplete and hindered triple-negative breast cancer stem cell targeted drug research and development, Lack of specific targets and other issues
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Embodiment 1
[0042] 1. Culture and identification of triple-negative breast cancer stem cells
[0043] A large number of triple-negative breast cancer cells with stem cell characteristics were cultivated by a serum-free low-adsorption suspension culture method, and their stem cell characteristics were verified for subsequent screening of targeted polypeptides.
[0044] The specific operation steps are as follows:
[0045] (1) Use serum-free low-adsorption suspension to culture triple-negative breast cancer stem cells. The medium components used include DMEM / F12 medium, epidermal growth factor, basic fibroblast growth factor, insulin, and B27.
[0046] (2) qRT-PCR verification in triple-negative breast cancer stem cells SOX2 , OCT4 , NANOG Wait for the expression level of the biomarker to verify whether it has stem cell characteristics.
[0047] figure 1 It is a stem cell-related gene in triple-negative breast cancer stem cells (CSC) and triple-negative breast cancer cell line MDA-MB...
Embodiment 2
[0071] In this example, flow cytometry and surface plasmon resonance (SPR) were used to analyze the three polypeptides screened in Example 1 (the polypeptides of SEQ ID No.1~SEQ ID No.3 are represented by polypeptides 1~3 respectively) Affinity and binding constants with triple-negative breast cancer stem cells, the specific steps are as follows:
[0072] a. After the three polypeptides were labeled with quantum dots, they were incubated with triple-negative breast cancer stem cells, and the fluorescence intensity was analyzed by flow cytometry to determine the affinity of the three polypeptides with triple-negative breast cancer stem cells.
[0073] b. Extract the membrane protein of triple-negative breast cancer stem cells.
[0074] c. Using EDC / NHS to couple triple-negative breast cancer stem cell membrane proteins to the SPR chip.
[0075] d. The targeting polypeptides were formulated into different concentrations, and flowed through the surface of the SPR chip in sequenc...
Embodiment 3
[0079] Fluorescent probe targeting triple-negative breast cancer stem cells
[0080] A fluorescent probe specifically targeting triple-negative breast cancer stem cells, comprising the polypeptide whose amino acid sequence is shown in SEQ ID No.1 in Example 1 and the fluorescent group FITC coupled to the peptide chain.
[0081] To detect the targeting specificity of the fluorescent probe, the specific steps are as follows:
[0082] 1) Synthesis of peptides linked to FITC fluorescent groups;
[0083] 2) Inoculate triple-negative breast cancer stem cells and MDA-MB-231 cells as a control group in a 24-well plate, and wait for the cells to grow to an appropriate density;
[0084] 3) Aspirate the medium, wash with PBS three times, each time for 3 minutes, add 500 μL of 4% paraformaldehyde solution to each well, fix at room temperature for 20 minutes, absorb the fixative, wash with PBS solution three times, each time for 3 minutes;
[0085] 4) Dilute the peptide at 1:500, add it ...
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