The Primary Study On The Effect And Mechanism Of Anti-proliferation In Human Lung Adenocarcinoma By Tilianin

Uygur medicine Dracocephalum moldavica(Dracocephalum moldavica L.)is one of distinctive medicinal herb in XinJiang region.The medicine whose raw material is Dracocephalum moldavica has been listed,which has been proved by researches that it directly has the function of inhibiting platelet activation and stabilizing platelet.The mechanism of stabilizing the platelet is related to some active ingredients such as total flavonoid Dracocephalum and tilianin.Recently,studies have shown that the active ingredient in DML Dracocephalum total flavonoids can downgrade P-selectin,which cause anti-platelet aggregation,inhibition of thrombosis and anti-endothelial cell activation,which inhibit their proliferation.Modern oncology research shows that tumor angiogenesis and thus mediated tumorigenesis,invasion and metastasis are closed linked with the activation of endothelial cells and platelet.The tumor-induced activation of endothelial cells and platelet form a vicious circle with platelet and endothelial growth factor-induced tumor growth.It is possible to break the circle by the regulation of platelet and endothelial cell activation and aggregation,maintain normal intravascular environment and inhibit tumor angiogenesis invasion and metastasis.As a natural medicinal plant,DML can not only reduce P-selectin,but also has many other pharmacological mechanisms such as anti-inflammatory,anti-oxidation and inhibition of calcium influx.What's more,it can inhibit platelet aggregation from multiple links,so that play a more stable role to improve its advantages of anti-platelet aggregation,antithrombosis and anti-endothelial cell activation,which inhibit proliferation.So can the active monomer tilianin inhibit the activation of endothelial cells and platelets through this mechanism,and develop appropriate anti-tumor effect further?Experimenting on vitro cells and tumor-bearing nude mice,through molecular biology and pathology way,the study researched the function of TFD's active monomer tilianin to affect platelet and inhibit tumor angiogenesis by adjusting P-selectin,thus the possibility for cancer prevention.And it also describes the future of DML medicinal applications in the new field of anti-tumor.1.Cytotoxicity of tilianinUsing CCK-8 assay to evaluate the toxicity of tilianin and endothelial cell stimulating factor TNF-? endothelial cell EA.hy926 at each concentration and investigate the vitro cytotoxicity of tilianin and TNF-?.And then researches lay out the concentration in later vitro experiments: TNF-? at a final concentration of culture system 25 ng / ml for activating endothelial cells;TIL at a final concentration of 40 ?g / ml culture system for intervention by TNF-? to activate endothelial cells.2.The impact of tilianin on endothelial cell adhesion molecule expressionUsing the Western Blot to detect tilianin's interventing activated endothelial cells Pselectin,VEGF and VCAM-1 protein expression,in order to confirm which endothelial cell adhesion molecule is regulated by tilianin.It was found that endothelial cell expression of VCAM-1 protein induced by TNF-? is most significantly suppressed by tilianin;overexpression of P-selectin also has a slight inhibitory effect,while the expression of VEGF in the stimulated TNF-? is slightly reduced.3.The impact of tilianin on endothelial cell adhesion molecule gene regulationUsing Real-time PCR assay and setting GAPDH as reference gene,mRNA expression of VCAM-1,P-selectin and VEGF are semi-quantitativly meatured.The results confirmed that VCAM-1 and P-selectin mRNA expression has the same trends with it in protein level,and tilianin has an inhibiting effect on expression of TNF-? stimulation increased VCAM-1 and P-selectin.Changes in VEGF protein level is different from the testing,after TNF-? stimulating mRNA upregulation causing by intervention of tilianin which suppressd its expression.4.Research on tilianin affecting lung cancer growth in nude miceFirstly,through tumor tissue block transplantation,human lung adenocarcinoma xenograft model in nude mice can be established.And then researchers compared inhibition of tilianin and positive control recombinant on human endostatin hormone to tumor growth in nude mice in early time.It can be observed that tumor in nude mice grow more slowly in tilianin group in the experiment,and tumor inhibition rate is higher then it in positive control group,but the results are not statistically significant.Secondly,researchers sliced tumor tissue,did immunohistochemical staining to it,determinated P-selectin,VCAM-1 and VEGF levels and used CD34 staining levels analysis to calculate the gross tumor microvessel density(MVD).After detecting effect of tilianin on tumor microenvironment and angiogenesis in nude mice,the result has comfirmed that the expression of P-selectin and VCAM-1 in tumor tissues of tumor bearing nude mice was decreased and the expression of VEGF was not changed,which shows that the inhibition of tumor growth in nude mice and tumor angiogenesis of tilianin are related to P-selectin and VCAM-1 those two cell adhesion molecules.