羟基红花黄色素A对异常增殖人脐静脉内皮细胞的抑制作用及其信号转导机制研究
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摘要
肿瘤的生长是血管依赖性的。研究表明,至少有30种生长因子与肿瘤的血管生成有关,其中最直接的促血管生成因子是血管内皮细胞生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)。如何阻断肿瘤血管的生成已成为目前肿瘤研究领域的一个重要课题。
在新血管的发生过程中,血管内皮细胞增殖是最基本和最重要的环节。由于肿瘤细胞和血管内皮细胞互相依赖介导了肿瘤血管新生。因此,血管内皮细胞与肿瘤细胞的共培养模型是研究体外肿瘤血管新生的重要方法。
中医理论认为“血瘀证”为肿瘤的基本证型,活血化瘀法在抗肿瘤的研究中占有重要地位。红花是传统的活血化瘀类中药,临床上用于治疗多种恶性肿瘤,但其作用机制鲜见相关报道。经基红花黄色素A(HSYA)是红花的主要有效成分,目前对于HSYA抑制肿瘤新生血管形成的作用,国内外尚未见报道。
本研究通过前期实验,首次从十余味活血化瘀中药或中药有效组分中,筛选出HSYA,能显著抑制鸡胚尿囊膜(CAM)新生血管的生成。为进一步探讨HSYA对肿瘤上清液培养环境下血管内皮细胞的作用情况及作用机理,本研究建立了肿瘤细胞上清液刺激下异常增殖人脐静脉内皮细胞(HUVEC)模型,目的是探讨活血化瘀中药红花的有效成分羟基红花黄色素A(HSYA)对肿瘤上清液诱导的人脐静脉内皮细胞增殖、凋亡及血管生成相关因子及受体表达的影响,以及对癌基因、抑癌基因表达的影响并探讨其信号转导机制。实验共分为五个部分:
1、原代培养人脐静脉内皮细胞,传代至3—5代进行实验。以MTT法检测不同浓度肿瘤上清液在不同时间点刺激后内皮细胞的增殖情况。选择最佳最佳浓度、最佳时间点进一步实验。然后以不同浓度羟基红花黄色素A作用于肿瘤上清液刺激后的HUVEC,MTT法检测HSYA对肿瘤上清液刺激后内皮细胞增殖的抑制作用,并从中选择最佳浓度的羟基红花黄色素A。2、设立三个实验组:正常组、肿瘤上清刺激组和加HSYA组。流式细胞术检测各组细胞周期和凋亡情况,并分别以real-time PCR和ELISA法检测凋亡相关细胞因子TNF-α的mRNA和蛋白表达情况。3、以real-time PCR和ELISA法检测与血管生成有关的细胞因子VEGF及其受体KDR,bFGF及其受体bFGFR的mRNA和蛋白表达情况。4、以western blotting法检测HUVEC MAPK通路信号转导分子ras、c-Raf、ERK、p38 MAPK、Akt等的表达。5、以real-time PCR和ELISA法检测癌基因N-ras、c-myc和转录因子NFκB表达情况。
实验结果如下:
1、人脐静脉内皮细胞生长状态良好,以3-5代状态最佳。50%以上的HepG2肿瘤上清液对HUVEC增殖具有较好的刺激效果,且在加药培养24小时后达到最佳效果。终浓度为0.0037g/L的羟基红花黄色素A是抑制肿瘤上清液刺激后HUVEC增殖的最佳浓度。2、HUVEC经肿瘤上清液刺激后G0/G1期细胞比例明显减少(P＜0.05),S期细胞的比例明显增加(P＜0.01),凋亡细胞比例明显减少(P＜0.05);经HSYA作用后,G0/G1期细胞比例增加,S期细胞比例减少,差异不显著。凋亡细胞明显增多(P＜0.05)。同时,凋亡相关细胞因子TNF-α的mRNA和蛋白表达在肿瘤上清液刺激组明显增加(P＜0.05),HSYA组明显减少(P＜0.05)。3、血管生成相关因子表达情况:HUVEC的VEGF及其受体KDR表达经肿瘤上清液刺激后可以明显增加(P＜0.01),经HSYA作用后VEGF表达明显降低(P＜0.05),KDR蛋白表达明显减少(P＜0.01),mRNA表达有减少趋势,差异不显著;bFGF及其受体bFGFR表达经肿瘤上清液刺激后明显增加(P＜0.05),经HSYA作用后有所降低,但差异不显著(P＞0.05)。4、信号转导通路相关因子表达情况:肿瘤上清液刺激组与对照组相比较,Ras、p-raf、p-ERK、p-p38MAPK表达增加;HSYA作用后,以上信号转导分子表达减少。而总raf、总ERK、总p38MAPK和Akt在三组之间变化不明显。4、癌基因和转录因子表达情况:HUVEC经肿瘤上清液刺激后癌基因c-myc、N-ras和转录因子NFκB表达明显增加(P＜0.05);HSYA作用后,c-myc、N-ras、NFκB表达明显减少(P＜0.05)。
分析实验结果,得出如下结论:
1、HSYA可以有效抑制肿瘤上清液刺激后的人脐静脉内皮细胞增殖,促进其凋亡。
2、HSYA可以明显抑制血管生成相关因子VEGF及其受体KDR的表达,并在一定程度上抑制bFGF及其受体的表达。
3、HSYA可以明显抑制异常增殖HUVEC MAPK家族的Ras、Raf、磷酸化ERK、磷酸化p38MAPK表达,对其总蛋白以及Akt表达影响不明显。
4、HSYA可以明显抑制异常增殖HUVEC癌基因c-myc、N-ras和转录因子NFκB的表达。
综上所述,羟基红花黄色素A可以有效抑制肿瘤上清液刺激后的人脐静脉内皮细胞增殖,促进其凋亡。可以明显抑制血管生成相关因子VEGF及其受体KDR的表达,并在一定程度上抑制bFGF及其受体的表达,通过受体酪氨酸激酶信号转导通路,经MAPK家族的Ras→Raf→MEK→ERK途径进行胞内信号转导,最终通过抑制癌基因和转录因子表达实现抑制肿瘤上清液诱导的血管内皮细胞异常增殖的作用,从而抑制肿瘤新生血管的形成。本研究的结果对于进一步阐明红花活血化瘀作用机制,开发HSYA新的药理作用提供了一定的理论和实验依据。
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.
在新血管的发生过程中,血管内皮细胞增殖是最基本和最重要的环节。由于肿瘤细胞和血管内皮细胞互相依赖介导了肿瘤血管新生。因此,血管内皮细胞与肿瘤细胞的共培养模型是研究体外肿瘤血管新生的重要方法。
中医理论认为“血瘀证”为肿瘤的基本证型,活血化瘀法在抗肿瘤的研究中占有重要地位。红花是传统的活血化瘀类中药,临床上用于治疗多种恶性肿瘤,但其作用机制鲜见相关报道。经基红花黄色素A(HSYA)是红花的主要有效成分,目前对于HSYA抑制肿瘤新生血管形成的作用,国内外尚未见报道。
本研究通过前期实验,首次从十余味活血化瘀中药或中药有效组分中,筛选出HSYA,能显著抑制鸡胚尿囊膜(CAM)新生血管的生成。为进一步探讨HSYA对肿瘤上清液培养环境下血管内皮细胞的作用情况及作用机理,本研究建立了肿瘤细胞上清液刺激下异常增殖人脐静脉内皮细胞(HUVEC)模型,目的是探讨活血化瘀中药红花的有效成分羟基红花黄色素A(HSYA)对肿瘤上清液诱导的人脐静脉内皮细胞增殖、凋亡及血管生成相关因子及受体表达的影响,以及对癌基因、抑癌基因表达的影响并探讨其信号转导机制。实验共分为五个部分:
1、原代培养人脐静脉内皮细胞,传代至3—5代进行实验。以MTT法检测不同浓度肿瘤上清液在不同时间点刺激后内皮细胞的增殖情况。选择最佳最佳浓度、最佳时间点进一步实验。然后以不同浓度羟基红花黄色素A作用于肿瘤上清液刺激后的HUVEC,MTT法检测HSYA对肿瘤上清液刺激后内皮细胞增殖的抑制作用,并从中选择最佳浓度的羟基红花黄色素A。2、设立三个实验组:正常组、肿瘤上清刺激组和加HSYA组。流式细胞术检测各组细胞周期和凋亡情况,并分别以real-time PCR和ELISA法检测凋亡相关细胞因子TNF-α的mRNA和蛋白表达情况。3、以real-time PCR和ELISA法检测与血管生成有关的细胞因子VEGF及其受体KDR,bFGF及其受体bFGFR的mRNA和蛋白表达情况。4、以western blotting法检测HUVEC MAPK通路信号转导分子ras、c-Raf、ERK、p38 MAPK、Akt等的表达。5、以real-time PCR和ELISA法检测癌基因N-ras、c-myc和转录因子NFκB表达情况。
实验结果如下:
1、人脐静脉内皮细胞生长状态良好,以3-5代状态最佳。50%以上的HepG2肿瘤上清液对HUVEC增殖具有较好的刺激效果,且在加药培养24小时后达到最佳效果。终浓度为0.0037g/L的羟基红花黄色素A是抑制肿瘤上清液刺激后HUVEC增殖的最佳浓度。2、HUVEC经肿瘤上清液刺激后G0/G1期细胞比例明显减少(P＜0.05),S期细胞的比例明显增加(P＜0.01),凋亡细胞比例明显减少(P＜0.05);经HSYA作用后,G0/G1期细胞比例增加,S期细胞比例减少,差异不显著。凋亡细胞明显增多(P＜0.05)。同时,凋亡相关细胞因子TNF-α的mRNA和蛋白表达在肿瘤上清液刺激组明显增加(P＜0.05),HSYA组明显减少(P＜0.05)。3、血管生成相关因子表达情况:HUVEC的VEGF及其受体KDR表达经肿瘤上清液刺激后可以明显增加(P＜0.01),经HSYA作用后VEGF表达明显降低(P＜0.05),KDR蛋白表达明显减少(P＜0.01),mRNA表达有减少趋势,差异不显著;bFGF及其受体bFGFR表达经肿瘤上清液刺激后明显增加(P＜0.05),经HSYA作用后有所降低,但差异不显著(P＞0.05)。4、信号转导通路相关因子表达情况:肿瘤上清液刺激组与对照组相比较,Ras、p-raf、p-ERK、p-p38MAPK表达增加;HSYA作用后,以上信号转导分子表达减少。而总raf、总ERK、总p38MAPK和Akt在三组之间变化不明显。4、癌基因和转录因子表达情况:HUVEC经肿瘤上清液刺激后癌基因c-myc、N-ras和转录因子NFκB表达明显增加(P＜0.05);HSYA作用后,c-myc、N-ras、NFκB表达明显减少(P＜0.05)。
分析实验结果,得出如下结论:
1、HSYA可以有效抑制肿瘤上清液刺激后的人脐静脉内皮细胞增殖,促进其凋亡。
2、HSYA可以明显抑制血管生成相关因子VEGF及其受体KDR的表达,并在一定程度上抑制bFGF及其受体的表达。
3、HSYA可以明显抑制异常增殖HUVEC MAPK家族的Ras、Raf、磷酸化ERK、磷酸化p38MAPK表达,对其总蛋白以及Akt表达影响不明显。
4、HSYA可以明显抑制异常增殖HUVEC癌基因c-myc、N-ras和转录因子NFκB的表达。
综上所述,羟基红花黄色素A可以有效抑制肿瘤上清液刺激后的人脐静脉内皮细胞增殖,促进其凋亡。可以明显抑制血管生成相关因子VEGF及其受体KDR的表达,并在一定程度上抑制bFGF及其受体的表达,通过受体酪氨酸激酶信号转导通路,经MAPK家族的Ras→Raf→MEK→ERK途径进行胞内信号转导,最终通过抑制癌基因和转录因子表达实现抑制肿瘤上清液诱导的血管内皮细胞异常增殖的作用,从而抑制肿瘤新生血管的形成。本研究的结果对于进一步阐明红花活血化瘀作用机制,开发HSYA新的药理作用提供了一定的理论和实验依据。
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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