| The first part is theoretical research:The first chapter studies the theory of blood stasis: it focuses on the formation of blood stasis,the characteristics of pathogenesis and the treatment of blood stasis theory has been systemically arranged.The main causes of blood stasis include exogenous six evils,pestilent pathogens,improper diet,bad mood,trauma,phlegm and stagnant fluid,Qi-Blood and Yin-Yang less,seven aspects.Blood stasis is easy to block Qi machine,affect blood vessel operation,affect the formation of new blood,and cause a wide range of complex pathogenetic characteristics.In modern medicine,Blood stasis is closely related to microcirculation disorder,blood hypercoagulability,changes in vasoactive factors,changes in blood rheology,changes in platelet function,vascular endothelial damage and so on.Activating blood circulation to remove blood stasis is the basic treatment of blood stasis.The second chapter is theoretical study of lung cancer: focus on the causes,pathogenesis,treatment and treatment of lung cancer.The understanding of lung cancer in traditional Chinese medicine is a gradual and in-depth process.The primary etiology of lung cancer is mainly the invasion of exogenous pathogens,the internal injury of seven emotions and improper diet,and the secondary causes are phlegm and blood stasis.The pathogenesis of lung cancer is complicated,which is mainly composed of deficiency of qi,accumulation of phlegm and dampness,stagnation of qi and blood stasis and blood stasis and toxin.The TCM treatment of lung cancer mainly includes supplementing qi and nourishing yin,removing blood stasis and detoxifying toxin,invigorating spleen and resolving phlegm and removing blood stasis and detoxification.Nourishing yin and clearing away heat,removing blood stasis and detoxification,regulating qi and activating blood circulation,removing blood stasis and detoxification.The treatment of lung cancer should be considered comprehensively,according to the specific clinical condition,according to syndrome differentiation and treatment,all kinds of prescriptions should be selected as appropriate.The third chapter is the TCM treatment of hypercoagulable state of lung cancer:stagnation in the meridian or viscera tissue in the slow movement of the state of blood,that is,blood stasis,is very close to the state of hypercoagulability in modern medicine;hypercoagulable state of lung cancer from the blood stasis treatment of rich connotations,effective.The fourth chapter summarized the theoretical study on Anti-lung Cancer effect of Salvia miltiorrhiza: Salvia miltiorrhiza can cause stasis accumulation,vexation,recovery of qi,and achieve the function of promoting blood circulation and removing blood stasis and detoxification.The anti-lung cancer effects of various effective components of Salvia miltiorrhiza include promoting tumor cell apoptosis,inhibiting tumor cell proliferation,and inhibiting tumor invasion and migration.Inhibition of tumor angiogenesis and improvement of hypercoagulability,chemoradiotherapy,synergism and reduction of toxicity and so on.The second part is the experimental research:Objective Diterpenoid Tanshinone(DT)was used as experimental drug to investigate the molecular mechanism of its regulation of coagulation-related factors,improving the hypercoagulability of lung cancer and inhibiting the invasion and metastasis of lung cancer PC9 cells in vivo and in vitro.Methods In vivo:(1)PC9 lung cancer transplanted tumor model was established in nude mice.The effect of DT on the growth of lung cancer transplanted tumor was investigated by comparing the tumor volume of nude mice with transplanted tumor;(2)Detection of APTTT PTT and study of the effect of DT on coagulation function in nude mice transplanted with lung cancer;(3)Determination of plasma coagulation factor by ELISA;(4)PI3K-Akt related marker protein p-AKT and Tissue factor were analyzed by Western Blot experiment.In vitro:(1)Effects of DT on proliferation of PC9 cells the effect of DT on the mobility of PC9 cells was investigated by CCK-8;(2)Study on the effect of DT on the motor ability of PC9 cells by cell scratch assay;(3)Effects of DT on migration and invasion of PC9 cells by Transwell assay;(4)Effect of DT on m RNA expression of PSGL-1 and TF genes in MUC1 by q PCR experiment;(5)PI3K-Akt related marker protein p-AKT、AKT were analyzed by Western Blot experiment.ResultsIn vivo:(1)The trend of tumor volume in model control group and DT intervention group was approximately the same.The tumor volume of DT intervention group was lower than that of model control group,which indicated that DT intervention group had better effect than model control group.After repeated measurement of ANOVA,there was significant difference in tumor volume among different groups(P<0.05),and there was interaction between groups at different time points and among groups(P<0.05).(2)The results of coagulation test showed that the APTT time of transplanted tumor nude mice in the model control group was significantly shorter than that in the normal group(P<0.05),suggesting that the plasma APTT time of the nude mice treated with DT was longer than that of the model control group.The DT high dose group had statistical significance(P<0.05).The PT time of nude mice in the model control group was significantly shorter than that in the normal group(P<0.05).The PT time of transplanted tumor nude mice in each group was longer than that in the model control group.The significance of calculation was(P<0.05).(3)The results of Elisa showed that the contents of PAI-1 and TXB2 in the model control group were significantly higher than those in the control group(P<0.05)and the contents of 6-Keto-PGF 1α in the model group were significantly lower than those in the control group(P<0.05),and the DT was higher and higher in the model group than in the model group.The contents of PAI-1 and TXB2 in nude mice in low dose group were significantly lower than those in control group(P<0.05)and 6-Keto-PGF 1α in middle dose group were significantly higher than those in control group(P<0.05).(4)The results of Western blot showed that the expression of TF and p-AKT protein was significantly decreased in each dose of DT compared with that in the model groupe.The expression of p-AKT protein was significantly down-regulated in each dose of DT than that in the model group(P<0.05).In vitro:(1)The results of CCK8 showed that the inhibitory rate of DT with 10μg/ml on PC9 cell proliferation was 52.89±4.34%.The inhibitory rate of each concentration group was significantly different from that of the control group.(P<0.05)(2)Cell scratch assay showed that PC9 cells had strong migration and motor ability.After treatment with DT with no cytotoxic concentration of 2,4μg/ml for 24 h,the cell scratch distance was significantly larger than that of control group.(3)Transwell experiment showed that compared with the normal control group,the number of PC9 cells passing through the polycarbonate membrane decreased significantly after 24 hours of treatment with DT.(P<0.05)(4)The results of q PCR showed that the m RNA expression levels of PSGL-1 and TF in PC9 cells treated with DT at different concentrations for 24 hours were significantly down-regulated(P<0.05).(5)Western Blot experiments show that the protein levels of AKT and p-AKT in PC9 cells were significantly down-regulated by different concentrations of DT for 24 hours.(P<0.05).Conclusion DT could inhibit the growth of transplanted tumor of lung cancer in nude mice,improve the hypercoagulability of blood in nude mice,inhibit the proliferation and migration of PC9 cells in vitro,and improve the hypercoagulability of lung cancer by regulating the coagulation related factor PAI-1,TXB2,6-Keto-PGF1αTF,MUC1,PSGL-1.It may inhibit the development of lung cancer by inhibiting the activation of PI3K-AKT signaling pathway. |
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