Application of pharmaceutical composition of metformin and anti-PD-1 antibody in preparation of liver cancer drugs
A technology of metformin and a composition is applied in the application field of preparing liver cancer drugs, which can solve the problems of unfavorable promotion and application, lack of tumor markers, and inability to satisfy both accuracy and practicability.
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Embodiment 1
[0032] Embodiment 1 A kind of detection method of PSPH protein molecule in liver cancer tissue
[0033] 1. Sample testing
[0034] (1) Select a paraffin section of liver cancer that includes the tumor area, and ensure that there is no large area of necrosis;
[0035] (2) Obtain a 4 μm paraffin section, bake it at 60°C for 2 hours, take it out, and cool it slightly;
[0036] (3) Dewaxing with xylene twice at room temperature, 10 minutes each time;
[0037] (4) Wash away the xylene in 100% ethanol, and then pass through 95% alcohol, 80% alcohol, and 70% alcohol successively, each time for 5 minutes;
[0038] (5) Wash in double distilled water for 5 minutes;
[0039] (6) with 0.3% H 2 o 2 Block endogenous peroxidase activity, room temperature for 10 minutes;
[0040] (7) Wash in double distilled water 4 times, 5 minutes each time;
[0041] (8) Antigen retrieval: 10mM sodium citrate buffer (pH6.0), microwave on high heat for 5 minutes, medium and low heat for 25 minutes; ...
Embodiment 2
[0056] Example 2 Up-regulated expression of PSPH in tumor nests of most liver cancer patients
[0057] 1. Experimental method
[0058] The method of Example 1 was used to detect the expression of PSPH in the paraffin sections of the cancer nest tissues of fresh liver cancer patients.
[0059] 2. Experimental results
[0060] The result is as figure 1 As shown, the results showed that the expression of PSPH (phosphoserine phosphatase) was up-regulated in the tumor nests of most liver cancer patients.
Embodiment 3
[0061] Example 3 In vivo experiments in mice found that the PSPH positive group did not respond to anti-PD-1 antibody treatment
[0062] 1. Experimental method
[0063] 1. The method for establishing the subcutaneous tumorigenesis model of liver cancer in mice is as follows:
[0064] Through the lentiviral transfection system, the expression of PSPH in Hepa1-6 cells was knocked out, and the Hepa1-6 cell line with stable and low expression of PSPH was obtained. The pLKO.1-TCR Cloning Vector plasmid vector was used, and the sh fragment filling region and the shPSPH sequence of the vector were respectively digested by Age1 and EcoR1, and then the shPSPH and the vector were connected to construct a complete knockout plasmid. Hepa1-6 cell line can be stably knocked out of PSPH protein, and the specific sequence of shPSPH is PSPH-shF-CCGGAGGCTGAAGTTCTACTTTTAATCTCGAGATTAAAGTAGAACTTCAGCCTTTTTTG- (the nucleotide sequence is shown in SEQ OD NO: 1), PSPH-shR-AATTCAAAAAAGGCTGAAGTTCTACTTT...
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