Experimental Study On The Change Of The Function Of NK Cells After Interleukin-12Gene Transduced Intratumorally In Treatment Of HCC-hu-PBL-NOD/SCID Mice

Objective: Through studing the receptor activity and the expression of the inhibitoryreceptors on the surface of NK cells of primary hepatocellular carcinoma and its adjacenttissues，we analyzed the relationship of these two receptor contents in the genesis anddevelopment of primary liver cancer and its clinical value；And to investigate thetherapeutic effect and mechanism of the change of NK cell receptor after the treatment ofhuman interleukin12gene in the NOD/SCID murine models.Methods:1.The number and activity of the NK cells as well as the expression of theinhibitory receptors on the surface of those cells which were derived from52cases ofprimary hepatocellular carcinoma and its adjacent tissues were detected with the methodof flow cytometry and immunohistochemical staining. The results were analyzedstatistically with the consideration of clinical relative factors.2.To establish theHCC-hu-PBL-NOD/SCID model, NOD/SCID mice were injected with HepG2cellssubcutaneously, and then with human PBL;The mice were randomized into4groups aftertumorigenesis including IL-12gene of lung cancer cell line9597/IL-12group, IL-12group、empty vactor group and control group.On day1,4,7the mice in the first groupwere injected intratumorally with irradiated lethally9597/IL-12cells, the mice in thesecend group with IL-12the mice in the third group with irradiated lethally9597/plasmid cells, and the fourth group with PBS;3.Observe the changes in tumorvolume of mice, as well as the body weight and general conditions.3.Detect the level ofINF-r and IL-12in murine peripheral blood and tumors with ELISA;4. Detect thechange of NK cell receptor after the treatment of human interleukin12gene withimmunohistochemistry.Results:1. In the tissue of primary hepacellular carcinoma,the number of NK cells islower than that in the adjacent tissues obviously (P<0.01); the expression of activatingreceptos, NKG2D and NKP44, is also lower than that in the adjacent tissues obviously(P<0.05); the expression of inhibitory receptors, CD158b and CD159a, is significantlyhigher than that in the adjacent tissues (P<0.05).A negative correlation was foundbetween the expression of NKG2D, NKP30and NKP44and the clinical stage of the livercancer--higher in patients with early and middle stages(P<0.05).The content of theinhibitory receptors of NK cells,CD158b and CD159a, in tissues from partients withadvanced cancer stage, would be higher(P<0.05).That’s also correlated with the level ofAFP and the HBSAg infection.There is no significant statistical difference between theexpression of NK receptors and the distant metastasis, tumor differentiation as well as thetumor size (P>0.05).2.Tumors developed in all the NOD/SCID mice after injected withHepG2cells subcutaneously and then with human PBL intraperitoneal. the overall success rate of100%, four groups of mice tumor volume and body weight in mice, Therewas no difference in the body weight between the treatment group and the other groupsduring the experimental period;3. The level of IFN-γ in the gene group and IL-12groupwhich were higher than the other two groups;4. Detect the NK cell receptor of tumortissue and spleen with Immunohistochemical assay:the activation receptors of NKG2D,NKP44, NKP30expression were increased, there were statistically significant after thetreatment of interleukin12gene and interleukin12;the expression of NKP46wassignificantly elevated, but that was not statistically significant after the treatments;Simultaneous,the inhibitory receptors of CD158b, CD159a (NKG2A) expression wereinhibited, there were statistically significant after the treatment of interleukin12gene andinterleukin12.Conclusion:1. The decrease of NK cell number and activating receptors and theincreased expression of the inhibitory receptors would be relevant to the incidence ofprimary hepacellular carcinoma.2.IL-12gene and IL-12intratumor injection caneffectively inhibit HCC-hu-PBL-NOD/SCID mouse model of tumor growth; The level ofIFN-γ in the gene group and IL-12group which were higher than the other twogroups;3.The surface activation receptors of NKcells in the intratumoral of mice withliver cancer are up-regulmion by interleukin12gene and IL-12，while The inhibitoryreceptors are down regulation.The change of the balance between activation receptorsand inhibitory receptors is one of the Important factors to which is to improve bodyimmunesurveillance function．Provide new ideas and theoretical basis for immunotherapyof liver cancer.