Implicit Tanshinone For 4 T1 Tumor Cells Based On Platelet Activation And Escape Immune Surveillance Of Nk Cells Intervention Study

Background and Basis:Tumor metastasis is the leading cause of death in tumor patients eventually,The process includes three parts.The first is tumor cells escaping from the primary site, the second is tumor cells running with blood,The last is tumor cells growing in the transfer area.At present,some research have found that platelet plays an important role in tumor metastasis.The detail mechanism of how does platelet play in tumor metastasis process is not clear,but an important one of the maybe mechanism is that the tumor cells activate platelets in the blood,then platelets adhere with tumor cells form something like a "cloak" to help tumor cells escape destruction of the immune system.Most removing of circulating tumor cells in blood is main accomplished by NK cells,so tumor cells activate platelets is the main reason of escaping from NK cell’s immune surveillance. Tumor cells can also stimulate platelet secrete some soluble cell factors, these factors inhibit the killing effect of NK cell and it’s ability to produce interferon (IFN-gamma).In platelet derived active ingredients,TGF-beta inhibit the NK cell activity through reducing immune activity of the receptors on the surface of NK cell natural killer group2,memberD(NKG2D).Theory of Traditional Chinese Medicine believe that emerge of tumor is relative with blood stasis.Some research has found that cryptotanshinone (CPT) has cytotoxic effect on tumor cells,it plays an role on blocking cell proliferation and inducing cell apoptosis,there also has some research find that CPT has the effect on anti-platelet aggregation.The further research about intervention of CPT on the process of tumor cells activating platelet to escape from the NK cell’s immune surveillance,will make contribution to the method of promoting blood circulation and removing blood-stasis on preventing cancer spread to the blood from immune part.Methods:First,take blood from eyeball of healthy female BALB/c mice, centrifuge to get platelets;Second, separate NK cell from the spleen through flow separation technology.Then,put platelets and NK cells and4T1tumor cells together to make a Co-culture system. Watch the adhesion of platelets with tumor cells through microscope and take picture at the same time.Before the intervention of CPT,test the influence of CPT to the natural growth of4T1tumor cells and draw the growth curve,then calculate the IC50to prepare for the following experiment.Make different group of Co-culture system and intervene with CPT which contains4T1tumor cells only(group1),4T1tumor cells with CPT(group2),4T1tumor cells with platelets(group3),4T1tumor cells with platelets and CPT(group4),4T1tumor cells with platelets and NK cells(group5),4T1tumor cells with platelets and NK cells and CPT(group6). Culture these groups for24hours then test the content of TGF-β、IFN-γ in cell supernatant fluid.Results:In the experiment of MTT,we find that CPT has obvious inhibitory effect on cell proliferation of4T1tumor cells and IC50is16.7μmol. From the extraction of platelets and mixed culture with4T1tumor cells to watch the adhesion of them,we found that tumor cells can really activate platelets to adhere around them form something like spherical package.But we also find that there is no obvious difference about the number and state of adhesion when add CPT into the Co-culture system.At the same time,we also find that there forms something like transparent film around the tumor cells.To collect high purity of NK cells through flow separation technology and test the content of TGF-β、IFN-γ in different cell supernatant fluid,we find that average content of TGF-β in group1is791.13pg/ml,group2is551.67pg/ml which is definitely different with group1.group3is2350.04pg/ml which is also definitely different with group1. group4is2120.977pg/ml which is no apparent difference with group2.group5is2367.56pg/ml,group6is2530.38pg/m,there is also no obvious difference between them.When comes to the content of IFN-γ,the result is no significance.Conclusions:As the mixed culture proved platelets can really adhere with tumor cells and wrapped it up.This provide the basis for the hypothesis that tumor cells activating platelet to escape from the NK cell’s immune surveillance.CPT has obvious inhibitory effect on cell proliferation of4T1tumor cells,but the inhibition of platelet aggregation and it’s secretion of TGF-β is not obvious when watch from the adhesion picture and the results of ELISA.Platelets can really secrete a lot of TGF-β and TGF-β can restrain the killing function of NK cell,this experiment have insisted the theory from the angle of cell factors,but the function of secreting IFN-y has not been tested in this experiment.It is proved in this experiment that platelets play an important role in helping tumor cells to escape from the NK cell’s immune surveillance.This point is supported by both pictures and cell factors.And CPT has obvious inhibitory effect on cell proliferation of4T1tumor cells while little effect on platelet aggregation,and next step we will try to use more effective herbs which are more powerful on inhibiting of platelet aggregation.