| The growth of tumor needs blood vessels supplying nutrition and removing me- tabolite. How to intercept the form of tumor blood vessels has becoming an important topic in the study of tumor.Studies indicated that there are at least 30 growth factors having relation with the growth of tumor blood vessels,among which the most direct vasotropic factors are VEGF and bFGF.In the course of the forming of new vessels,the generation of VEC is the most elemental and important component,so the forming of new vessels can be inhibited by inhibiting the growth of VEC.Because the depending of TC(tumor cell) and VEC each other,which mediates the forming of tumor vessels,the study of the form of extro-tumor vessels needs that VEC should be growth in the circumstance of cultivating TC and VEC together.The theory of Chinese Medicine considers that 'stagnancy of qi and blood stasis'is one of themost important courses lead tumor forming.'blood stasis' is the basic symptom of tumor,for this reason,the method of promoting blood circulation by removing blood stasis has an important position in the study of resisting tumor.Carthamus tinctorius L.is a traditional herb to promote blood circulation and remove blood stasis,which is used to treat many kinds of tumor,but it's mechanism of action is seldom reported.HSYA is the essential component of Carthamus tinctorius L..at present,there is few reports about HSYA.there is no report on the action of inhibiting new vessels forming of HSYA.By preliminary test,we have bolted HSYA from 10 and more herbs or active components of promoting blood circulation and removing blood stasis,which can inhibit the form of CAM new vessels.To approach the action and mechanism of HSYA to HUVEC in the tumor cultured fluid condition,we add the supernatant of TC to HUVEC and construct an abnormal model of VEC generation,the aim is to observe the effect of HSYA on the proliferation,apoptosis of HUVEC and the expression of the angiogenesis correlation factors and their receptors,the expression of oncogenes,transcription factors and the molecules in the signal transduction route,so as to study the mechanism of the inhibition of HSYA on HUVEC.This study includes 5 parts:1.HUVEC was primarily cultured and used in the experiment when passaged to the third to fifth generation.The method of MTT was used to detect the status of proliferation cultured under the conditions of different concentration of HSYA and after different phase of time.We selected the best concentration and time to the further steps of experiment.2.Three experimental groups were constructed:the control group,model group and the HSYA group.Flow cytometry was used to detect the cell cycle and apoptosis in each group. Real-time PCR and ELISA were used to detect the expression of mRNA and proteinum of apoptosis related factor TNF-α.3.Real-time PCR and ELISA were used to detect the expression of mRNA and proteinum of angiogenesis correlation factors and their receptors VEGF,KDR,bFGF and bFGFR.4.Western blotting was used to detect the expression of molecules in the MAPK signal transduction passageways such as Ras,c-Raf,ERK,p38MAPK and Akt.5.Real-time PCR and ELISA were used to detect the expression of mRNA and proteinum of oncogenes N-ras,c-myc and transcription factor NFκB.The results of the experiments were as follows:1.The third to fifth generation of HUVEC have the best condition.50%is the best concentration of tumor cultured liquid.And the best condition of inhibition to the growth of HUVEC is 0.0037g/L HSYA added after 24 hours.2.In the tumor model group,the proporation of HUVEC at G0/G1 stage was lower than the control group(P＜0.05),the proporation of S stage was higher than the control group (P＜0.01),and the apoptosis was inhibited at the same time(P＜0.05).The differences of the proporation of HUVEC at each cell cycle stage between the HSYA group and the model group were not notable.But the proporation of apoptosis was higher than the model(P＜0.05). The expression of TNF-αwas higher in the model group than in the control(P＜0.05),and it is lower in HSYA group than in the model(P＜0.05).3.The expression of VEGF and KDR were enhanced in the model group(P＜0.01),and decreased significantly in the HSYA group(VEGF P＜0.05,KDR P＜0.01).The expression of bFGF and bFGFR were higher in the model group than the control group(bFGF P＜0.01, bFGFR P＜0.05),but the differences between the HSYA group and the model group were not notable(P＞0.05).4.The expression of Ras,raf,p-ERK,p-p38MAPK in the model group wer higher than the control group,and lower than the HSYA group.And the differences of tota-raf,total-ERK, total-p38MAPK and Akt among the three groups were not nota- ble.5.The expression of c-myc,N-ras and NFκB were higher in the model group than in the control group(P＜0.05),and lower than in the HSYA group(P＜0.05).According to the experimental results,Conclusions could be drawn as follows:1.HSYA can effectively inhibit the proliferation of HUVEC,and prmote its apoptosis.2.HSYA can obviously inhibit the expression of angiogenesis correlation factor VEGF and KDR in HUVEC,and it inhibits the expression of bFGF and bFGFR to certain degree.3.HSYA obviously inhibits the expression of molecules in MAPK signal transduction passageway such as Ras,raf,p-ERK,p-p38MAPK.4.HSYA can obviously inhibit the expression of oncogenes N-ras,c-myc and transcription factor NFκB.In Summary,HSYA has the effect of inhibition of the proliferation of HUVEC stimulated with tumor conditioned culture,at the same time it promote apoptosis.The possible mechanism of this is the inhibition of the expression of VEGF and KDR on HUVEC membrane,then through the intra-cellular signal transduction passageway Ras→Raf→MEK→ERK,the expression of oncogenes were inhibited finally.The inhibition of HUVEC brings the result of repression of tumor angiogenesis.The conclusions of this study may provide some bisis to expound the mechanism of Carthamus tinctorius L in treating tumor and may help to exploit the new pharmacological action of HSYA.
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