Synergistic drug compound for treating tumor and preparation method thereof
A drug complex, tumor technology, applied in antitumor drugs, drug combinations, pharmaceutical formulations, etc., can solve problems such as increasing the sensitivity of tumor cells, and achieve the effect of enhancing the therapeutic effect
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Embodiment 1
[0036] Embodiment 1: Preparation of human recombinant arginase
[0037] The coding sequence of encoding human arginase was obtained by using the prior art PCR method. The coding sequence of the gene is 969bp in length, with enzyme cutting sites XhoI and EcoRI. Empty plasmid vector, connect the target gene to the vector and transform the ampicillin resistance plate, pick several positive transformants, take one of them to amplify and extract the plasmid, and carry out double digestion identification with XhoI and EcoRI endonuclease; extract the recombinant plasmid, The recombinant vector was digested with BglII to linearize it, and transformed into Pichia pastoris by electroporation; positive transformants were picked and screened on RDB, MM and MD plates to obtain clones with the phenotype his+muts, which were further carried out at the shaker level Induce expression, detect the expression of arginase on the shaker level by SDS-PAGE protein electrophoresis, thereby screening r...
Embodiment 2
[0039] Example 2: Formulating autophagy inhibitor drugs
[0040] (1) Preparation of chloroquine: Dissolve an appropriate amount of chloroquine in pure water to make a 10mmol / L storage solution, filter and sterilize with a 0.1 μm filter, store at 4°C, and dilute 500-1000 times for in vitro experiments to inhibit cell autophagy ;
[0041] (2) Preparation of ammonium chloride: Dissolve an appropriate amount of ammonium chloride in water to prepare a 0.4 mol / L storage solution, filter and sterilize with a 0.1 μm filter, and store at 4°C. In vitro experiments were diluted 50-80 times to inhibit cell autophagy;
[0042] (3) Preparation of hydroxychloroquine: Dissolve an appropriate amount of hydroxychloroquine in pure water to form a 10mmol / L stock solution, filter and sterilize with a 0.1 μm filter, store at 4°C, and dilute 500-1000 times in the in vitro laboratory for inhibition autophagy;
[0043] (4) Preparation of 3-MA: Take an appropriate amount of 3-MA dry powder to prepar...
Embodiment 3
[0047] Example 3: Human recombinant arginase induces autophagy in triple-negative breast cancer MDA-MB-231 cells
[0048] MDA-MB-231 cells were treated with 1U / ml arginase for 24h, then paraffin-embedded, sectioned, and stained, and the submicroscopic structure of the cells was observed under a transmission electron microscope. The results were as follows: figure 1 As shown in A, there are a large number of typical double-membrane structure autophagosomes in the cells of the administration group, but not found in the control group; treated with 1IU / ml arginase for 24h, and then Cyto-ID and Hoechst triple negative In breast cancer MDA-MB-231 cells, the green fluorescent spots were observed under the laser confocal microscope, and the results were as follows figure 1 B shows that there are a large number of fluorescent spots in the cells of the administration group, while the control group has fewer;
[0049] The collected triple-negative breast cancer MDA-MB-231 cells were was...
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