Blood Medicine, Yiqihuoxue Drugs On Tumor Growth And Metastasis Of The Process Of Regulatory T Cell-mediated Immune Escape The Impact Of Research,

The role that blood activating drugs play in tumor therapy is always in dispute. The clinical experience showed that we can enhance the effect of blood activating drugs on improving the high blood coagulation state of tumor-bearing body and reverse(or decrease) the possible trend of promoting tumor metastasis by reasonable compatibility.The earlier studies of my mentor have observed influence of different types of blood activating drugs compatibility with qi-deficiency drug(Radix Astragali) in different doses on tumor metastasis.The results showed that high dosage qi-deficiency drug compatibility with blood activating drugs could improve the tumor inhibiting rate and reverse the trend of promoting tumor metastasis of simple blood activating drugs.The results of mechanism study showed that the supplementing qi and activating blood circulation drugs could down-regulate the expression of CD4~+CD25~+ Treg.The study needs further research.The study was carried out on the base of the earlier studies,and was supported by NSFC.We carried out a small sample clinical research on the difference of the expression of CD4~+CD25~+Foxp3~+ Treg in peripheral blood between patients with NSCLC and healthy volunteers and its relationship with main features and TCM syndrome of lung cancer.After the difference comparison studying on the inhibiting rate of lung metastasis in Lewis lung cancer model in mice,we chose a pair of representative drugs(Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ)) from three types of blood activating drugs(Salvia Miltiorrhiza Bunge,Caesalpinina sappan,Hirudo) and three types of supplementing qi and activating blood circulation drugs(blood activating drugs compatibility with Radix Astragali).In animal experiments we observed the expressions of CD4~+CD25~+Treg and its related activated molecules,regulatory molecules,effector molecules,main genes in Jagged1-Notch signal transduction pathway and cell surface molecules of DC intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ),using the main techniques of MACS,FCM,real-time PCR, fluorescence microscope,ELISA and CBA flexset.In vitro experiments we observed the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum, using the main techniques of MTT and transwell.We also established the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG to study the T cell differentiation intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+ Radix Astragali(SMHQ) medicated serum.The study explored the molecular mechanisms and key links of the different effects of blood activating drugs and supplementing qi and activating blood circulation drugs on tumor growth and metastasis from the angle of tumor immune escape.We have obtained high academic valuable data.1 The primary research conclusions1.1 Detection of CD4~+CD25~+Foxp3~+ regulatory T cells in peripheral blood of patients with NSCLC and its clinical significanceBy detecting CD4~+CD25~+Foxp3~+ regulatory T cells in peripheral blood of 30 patients with NSCLC and 12 healthy volunteers and analyzing their clinical information,we found that:The population of CD4~+CD25~+Foxp3~+ Treg in peripheral blood from patients with NSCLC was higher in comparison with that in healthy volunteers,which conformed the documental reports.The proportion of CD4~+CD25~+Foxp3~+ Treg in the patients with stageⅣlung cancer was higher than those in stageⅠ-Ⅲ.The proportion of CD4~+CD25~+Foxp3~+ Treg in the patients with tumor was higher than those in the patients without tumor.Treg expression was not obviously correlated with different genders,ages,pathological types,KPS and level of tumor markers.The increase of the proportion of CD4~+CD25~+Foxp3~+ Treg in the patients most closely correlated with Qi deficiency syndrome and blood stasis syndrome.These results had provided data basis for animal and vitro experiments on the relationship between CD4~+CD25~+Foxp3~+Treg and lung cancer.1.2 The effects on Lewis lung cancer growth and metastasis intervened by different types of blood activating drugs and supplementing qi and activating blood circulation drugs By studying on the inhibiting rate of lung metastasis in Lewis lung cancer model in mice,we observed the intervention effect of three types of blood activating drugs (Salvia Miltiorrhiza Bunge,Caesalpinina sappan,Hirudo) and three types of supplementing qi and activating blood circulation drugs(blood activating drugs compatibility with Radix Astragali).We found that:①Salvia Miltiorrhiza Bunge had showed the trend of promoting tumor metastasis while Salvia Miltiorrhiza Bunge+Radix Astragali could not reverse it.Caesalpinina sappan,Caesalpinina sappan+Radix Astragali,Hirudo,Hirudo+Radix Astragali had showed good curative effect on decrease tumor metastasis,which suggested that different types of blood activating drugs and supplementing qi and activating blood circulation drugs had different effects.②Supplementing qi and activating blood circulation drugs had better effect on decrease tumor growth and metastasis than blood activating drugs.So we chose a pair of representative drugs(Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ)) for further research.1.3 Continuous observation on the expression of CD4~+CD25~+Foxp3~+ regulatory T cells in spleen of tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugsBy Continuous observing on the body weight,tumor weight,expression of CD4~+CD25~+Foxp3~+ regulatory T cells in spleen of tumor-beating mice intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) and their survival time,we found that:①Traditional Chinese medicine had showed a more obvious tumor inhibiting effect in the early stage.②Caesalpinina sappan(SM) had showed a better tumor inhibiting effect in the early stage,while the advantages of Caesalpinina sappan+Radix Astragali(SMHQ) was in the inhibiting rate of lung metastasis and survival time.③The expression of CD4~+CD25~+Foxp3~+ Treg in spleen of tumor-beating mice in control group was increased followed with the tumor progression.④Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) had different effect on the changing tendency of the expression of CD4~+CD25~+Foxp3~+ Treg.The decreased of CD4~+CD25~+Foxp3~+ Treg(20d) was obvious in SMHQ group.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by Treg down-regulation and this effect was better than that of blood activating drugs.1.4 The effects on the levels of cytokines in CD4~+ cell culture supernatant in spleen of tumor-bearing mice and the expression of main genes in Jagged1-Notch signal transduction pathway intervened by blood activating drugs and supplementing qi and activating blood circulation drugsBy detecting the levels of cytokines in CD4~+ cell culture supernatant in spleen of tumor-bearing mice and the expression of main genes in Jagged1-Notch signal transduction pathway intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ),we found that:①The levels of IL-2,IFN-γin CD4~+ cell culture supernatant in spleen of tumor-bearing mice in SMHQ group was obviously higher than that in control group,while the levels of TGF-β1 was lower than that in control group.②Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) had no significant effect on the expression of Notch1,Jagged1 mRNA.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by regulating the levels of cytokines in tumor-bearing mice and this effect was better than that of blood activating drugs.1.5 The effects on the expressions of genes related with immune escape in tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugsBy real-time RT-PCR,we detected the expressions of Foxp3 and CTLA-4 mRNA in tumor tissues and in CD4~+CD25~+cells selected from spleen cells of tumor-bearing mice by magnetic activated cell sorting system.We discovered that:The expressions of Foxp3 and CTLA-4 mRNA in tumor tissues and the expression of Foxp3 mRNA in CD4~+CD25~+cells selected from spleen cells of tumor-bearing mice in SMHQ group were lower than that in control group.These results showed that the supplementing qi and activating blood circulation drugs could improve the tumor-bearing mouse immune tolerance condition by down-regulating the expressions of genes related with immune escape and this effect was showed in both lymphoid tissue and tumor micro-environment.1.6 The effects on the expressions of phenotypes of DC in spleen of tumor-bearing mice intervened by blood activating drugs and supplementing qi and activating blood circulation drugsBy flow cytometry,we detected the expressions of phenotypes of DC in spleen of tumor-bearing mice.We discovered that:①There was a significantly decrease of the expressions ofphenotypes of DC in tumor-bearing bodies.②Radix Astragali(SMHQ) could increase the expressions of mature phenotypes of DC in spleen of tumor-bearing mice and this effect was better than that of blood activating drugs.These results had provided data basis for vitro experiments on the relationship between the mature degree of DC and differentiation of T cells.1.7 The effects on the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serumBy detecting the effects on the ability of cell proliferation,movement,invasion in PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum,we found that:①Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) had no significant effect on the ability of cell proliferation in PG.②Caesalpinina sappan+ Radix Astragali(SMHQ) medicated serum had significant inhibitory effect on the ability of cell movement,invasion in PG.These results showed that traditional Chinese medicine could inhibit tumor growth and metastasis in many different targets and many different ways.Directly killing the tumor cells was only one side of these effects.1.8 The effects on the expressions of phenotypes of DC in vitro intervened by culture supernatant of PGIn this study,PBMC was isolated from human peripheral blood,differentiation into different mature degrees of DC after induction in vitro.Then culture supernatant of PG was added into the culture system to simulate tumor microenvironment.By flow cytometry,we detected the expressions of phenotypes of DC in the two culture systems.We discovered that:The culture supernatant of PG could decrease the mature degree of DC in vitro.These results had provided premise conditions and experimental method for further study.1.9 The effects on the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) medicated serumIn this study,we established the mixed culture system of DCs,CD4~+T cells and culture supernatant of PG.By detecting the expression of CD4~+CD25~+Foxp3~+Treg, the level of IL-12 and the expression of Jagged1,Notch1 mRNA in the mixed culture system,we found that:①In the mixed culture system intervened by culture supernatant of PG,the expression of CD4~+CD25~+Foxp3~+Treg and Jagged1,Notch1 mRNA was higher compared with the control group while the level of IL-12 was decreased.②Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum had no significant effect on the differentiation of T cells and the level of IL-12 in the mixed culture system.③Caesalpinina sappan+Radix Astragali(SMHQ) medicated serum had inhibitory effect on CD4~+CD25-T cells differentiating into CD4~+CD25~+T cells.④Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum could increase the level of IL-12 in the mixed culture system.⑤Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali (SMHQ) medicated serum had no significant effect on the expression of Notch1, Jagged1 mRNA in the mixed culture system.These results showed that the CD4~+CD25T cells differentiating into CD4~+CD25~+T cells in the mixed culture system intervened by culture supematant of PG related to many factors,including inhibitory cytokines secreted by tumor cells,the mature degree of DC and stimulating amount of tumor antigen.In this research,patients with NSCLC,Lewis lung cancer-bearing mice and PG cells were taken as the research objects.We discussed on the relationship between CD4~+CD25~+Foxp3~+Treg and the tumor growth and metastasis from three different levels of clinical experiments,animal experiments and in vitro experiments.The expressions of CD4~+CD25~+Treg and its related activated molecules,regulatory molecules,effector molecules,main genes in Jaggedl-Notch signal transduction pathway and cell surface molecules of DC intervened by Caesalpinina sappan(SM) and Caesalpinina sappan+Radix Astragali(SMHQ) were observed in both tumor tissue and T cells.The study explored the molecular mechanisms and key links of the different effects of blood activating drugs and supplementing qi and activating blood circulation drugs on tumor growth and metastasis from the levels of whole body,cell mechanism and molecular mechanism.The results had provided a scientific basis for more reasonable and effective clinical application of blood activating drugs and supplementing qi and activating blood circulation drugs.