| Programmed cell death ligand-1(PD-L1/B7-H1)is a transmembrane protein that is highly expressed in many tumor cells.It can combine with PD-1 protein on effector T cells,conduct immunosuppressive signals,inhibit the activity of immune effector T cells,and facilitate the growth and escape of tumor cells.It has been found that PD-L1 immune checkpoint inhibitor can specifically combine with PD-L1,block PD-1/PD-L1 pathway,and relieve the inhibition of T cell activity,thus increasing the anti-tumor immunity of the body(improving the tumor killing ability of T cells)and achieving the anti-tumor effect.However,clinical studies have found that immune checkpoint inhibitors also have some clinical limitations.PD-L1 expression in cells is regulated by some internal mechanisms,such as FOXO,NF-?B,HIF-1,STAT3,SRF,STAT1 and other signal pathways.Constitutive carcinogens in tumor cells can promote overexpression of PD-L1 through these signaling pathways.PURPOSE:Due to some limitations of immune checkpoint inhibitors in clinical application,this topic intends to start with the signal pathway that regulates PD-L1 expression and try to reduce the expression level of PD-L1 on the surface of tumor cell membrane.The whole subject is divided into the following six parts:1)Using each signal pathway inhibitor to detect the modulation of PD-L1 expression by each signal pathway;2)Construction of a screening system for signaling pathway reporter genes;3)Screening the traditional Chinese medicine monomer drugs by using the constructed reporter gene system;4)Using Flow cytometry to verify the down-regulation effect of the screened traditional Chinese medicine monomer on tumor cell PD-L1;5)Carrying out a cell killing experiment on the verified traditional Chinese medicine monomer;6)Carrying out an in vivo experiment on the traditional Chinese medicine monomer determined after detection.METHODS and RESULTS:1)Flow cytometry was used to detect the regulatory effects of six signaling pathway inhibitors on the expression of PD-L1 on the cell surface of tumor cell MDA-MB-231.The results showed that the inhibitors of signaling pathways STAT3,STAT1 and FOXO had a down-regulation effect on the expression of PD-L1 on the cell surface of tumor cell MDA-MB-231.2)Construction and verification of luciferase reporter gene system for stable expression of STAT3,STAT1 and ISRE(STAT1/2)transcription factors:construction of corresponding recombinant gene expression vectors using basic operations such as enzyme digestion and ligation of molecular clones;Stable expressed monoclonal cell lines Raw264.7-STAT3 and A549-STAT1;were constructed by plasmid transfection.Raw264.7-STAT3 cell line showed concentration gradient dependence on IL-6 stimulation,indicating that the cell line stably expressing STAT3 luciferase regulated by STAT3 transcription factor was successfully constructed and can be used to screen drugs related to STAT3 activation regulation.However,the stimulation of IFNy by A549-STAT1 and A549-ISRE cell lines has not changed,so there is no cell line stably expressing STAT1 and ISRE luciferase.3)The constructed Raw264.7-STAT3 cell line is used to screen the traditional Chinese medicine monomer compound,and the traditional Chinese medicine monomer compound which can inhibit the activation of STAT3 is screened based on Nluc/OD value.Results more than ten kinds of traditional Chinese medicine monomers that can down-regulate STAT3 signaling pathway were screened out.4)The screened traditional Chinese medicine monomer is applied to MDA-MB-231 cells and A549 cells,the expression amount of tumor cells PD-L1 and HLA is taken as an index,Metformin is taken as a positive control,and flow cytometry is used to screen the traditional Chinese medicine monomer which can down-regulate tumor cells PD-L1 but has no down-regulation or no obvious down-regulation effect on HLA.Results Nine kinds of traditional Chinese medicine monomers including Y028,Y162,Q002,L011,X045,E011,B7,A9,Metformin were screened out.5)The nine kinds of traditional Chinese medicine monomers are verified by cell killing experiments.After human peripheral blood lymphocytes(PBMC)are activated by stimulation factors CD3 and CD28,IL-2 is added to stimulate them into cytotoxic T lymphocytes,and the activity of T cells is verified by flow.The MDA-MB-231-Fluc cells were co-cultured with CTL cells,and Chinese medicine monomers were added.After overnight culture,the activity rates of MDA-MB-231 cells in the experimental group and the corresponding blank group were calculated.The results showed that traditional Chinese medicine monomers Y028,E011,A9,B7 and Metformin could enhance the tumor killing effect of T cells.6)NU/NU nude mice were tested for the tumor-killing effect of Chinese medicine monomer in vivo:MDA-MB-231-Flue cells were inoculated into the skin of the fourth breast pad of mice,and then the stimulated T cells were reinfused to BALB/c nunu mice via tail vein,and T cells were reinfused once every three days.At the same time,the screened Chinese medicine monomer was injected intraperitoneally every day,and whether the screened Chinese medicine monomer enhanced the tumor-killing effect of T cells was detected by small animal living imaging system.CONCLUSIONS:1)This experiment proves that STAT1 and STAT3 signal pathways are correlated with PD-L1 expression.2)The STAT3 transcription factor reporter gene system with stable expression has been successfully constructed,and more than ten kinds of traditional Chinese medicine monomer compounds capable of regulating the expression of the transcription factor have been screened out through the system.3)Traditional Chinese medicine monomers X045,E011,B7,A9,L011,Y162,Y028,Q002,etc.can down-regulate the expression of PD-L1 in tumor cells MDA-MB-231.4)Traditional Chinese medicine monomers Y028,E011,A9,B7,Metformin,etc.can promote the tumor killing effect of T cells on tumor cells.5)Chinese medicine monomers B7 and Metformin can play a promoting role in CTL reinfusion in tumor inhibition of tail vein of nude mice. |
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