人参、三七组方促血管生成的信号转导机制及相关临床文献评价
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摘要
血管生成在缺血性疾病发病机制和治疗中作用的研究已成为目前医学研究的热点课题。益气活血中药防治冠心病疗效肯定,其改善心肌缺血与促进缺血心肌血管生成的相关性,已为大量临床和实验研究所证实,提示促血管生成可能是益气活血药治疗冠心病的重要机制之一。本研究通过探讨益气活血药促血管生成作用的分子机制,揭示其干预血管生成的作用环节和分子靶标,阐明益气活血药祛瘀生新的科学内涵,旨在为中医药防治缺血性疾病提供新的实验依据。
本论文分为三部分:文献综述、临床文献系统评价和基础研究。
文献综述部分,分别从中医药治疗性血管生成的研究进展以及血管生成在冠心病防治中的研究进展两个方面进行了综述。
临床文献评价部分,对1994年～2007年在科技期刊上公开发表的益气活血中药治疗不稳定心绞痛的临床文献进行系统评价。
目的:对益气活血中药治疗不稳定心绞痛临床研究进行系统评价。方法:①以[(中文标题:不稳定心绞痛)AND(全部检索条目:中医or中药or中西医结合)AND(全部检索条目:益气and活血)]的检索式检索中国生物医学文献光盘数据库,年限为1994年1月至2007年12月。②由2位研究者按照文献纳入、排除标准独立进行文献筛选,意见不同者通过讨论解决。③获取文献的相关研究特征资料,并加以评价。④对合格的随机对照试验,进行定量的Meta分析。结果:①共检索到15个文献。②经两次筛选,剔除不符合要求以及重复报道的文献,最终共有6篇文献入选评价。③对6篇文献进行Meta分析程序,总病例数仅385例,研究的质量普遍不高。Meta分析显示,加用益气活血中药进行干预对不稳定心绞痛患者心绞痛症状的改善、心电图的改善、血脂的改善较单纯西医基础治疗显著提高。结论:益气活血中药干预不稳定心绞痛在改善心绞痛的症状、心电图表现以及改善血脂方面有一定的疗效。但由于纳入研究的病例数较少和质量不高,还有待进一步研究。
基础研究部分,包括以下八个实验研究。
实验一HUVEC生长曲线测定
目的:明确不同细胞浓度、不同时间点HUVEC的生长情况,为后续实验选用合适的细胞浓度及最佳的给药时间做准备。
方法:将6个浓度的细胞接种于96孔板中,每隔24h比色测定,连续5天,重复一次。
结果:接种细胞后第2天,各细胞密度的生长均进入指数生长期。第3、4天,4×10~4个/ml,6×10~4个/ml,8×10~4个/ml 3个浓度的细胞进入平台期;第5天,1×10~4个/ml,2×10~4个/ml,3×10~4个/ml这3个浓度的细胞还继续增殖,而4×10~4个/ml的细胞己到饱和的密度;6×10~4个/ml,8×10~4个/ml两个浓度的细胞第2天后逐渐进入饱和状态。
结论:选用2×10~4个/ml～3×10~4个/ml这个区间的细胞数接种;细胞接种36h后给药作为后续实验的基础。
实验二人参、三七不同配比和时间点对HUVEC增殖的影响
目的:探讨人参、三七组方促HUVEC增殖的最佳剂量、两药比例和最佳作用时间。
方法:将人参和三七分别按照1:1,2:1,1:2三种配比制备成0.25mg/ml、0.5mg/ml、1mg/ml、2mg/ml、4mg/ml 15个不同浓度的给药组和1个空白组,分别在给药24 h和48 h后比色测定。
结果:三种配比的不同浓度作用于HUVEC 24 h后,只有人参、三七之比为2:1时,出现一定的量效关系,且4 mg/ml对细胞的增殖作用最强,增值率达37.3%;作用于HUVEC 48 h后,三个配比均出现一定的量效关系,人参:三七=1:1,浓度4mg/ml促细胞的增殖作用最强,增值率达26.12%。
结论:人参、三七为2:1,浓度4mg/ml,药物作用时间为24 h时,促HUVEC增殖作用最明显。
实验三人参、三七组方促HUVEC增殖及分泌VEGF的研究
目的:探讨人参、三七组方对脐静脉内皮细胞增殖及分泌VEGF的影响。
方法:细胞分为对照组、bFGF阳性对照组、人参三七大剂量组、人参三七中剂量、人参三七小剂量组,给药24 h比色测定。用双抗体夹心ELISA法,检测上清中VEGF的含量。
结果:人参、三七组方能够促进HUVEC增殖和分泌VEGF,其作用和bFGF相当,与空白组有显著性差异。
结论:人参、三七组方能够促进HUVEC增殖和分泌VEGF。
实验四人参、三七组方对HUVEC VEGFR-2表达的影响
目的:探讨人参、三七组方对HUVEC VEGFR-2表达的影响。
方法:采用免疫组织化学的方法,观察人参、三七组方干预后,HUVECVEGFR-2的表达。
结果:人参、三七组方大剂量组促进了HUVEC VEGFR-2的表达。其作用和bFGF相当,和空白组相比有明显增加,且组间差异显著。
结论:人参、三七组方可能通过促进VEGFR-2的表达而促进HUVEC的增殖。
实验五人参、三七组方对血管生成信号蛋白及其mRNA的影响
目的:探讨人参、三七组方对HUVEC的血管生成信号蛋白及mRNA的影响
方法:人参、三七组方干预后,运用Western-blotting检测方法,分析该组方对HUVEC的VEGFR-2、Ras、MAPK表达的影响;运用realtime-PCR对以上三种蛋白的mRNA表达进行分析。
结果:人参、三七组方可不同程度的促进血管生成信号蛋白VEGFR-2、Ras、MAPK的表达,但是这三种蛋白的mRNA表达却有不同程度的降低。
结论:人参、三七组方可促进血管生成信号蛋白VEGFR-2、Ras、MAPK的表达,这可能是中药促进内皮增殖,产生促血管生成的基础。这三种蛋白mRNA表达的下降可能与其扩增时间相关。
实验六SU5416对下游信号蛋白的影响以及中药的干预作用
目的:探讨中药在血管生成信号通路上的作用靶点。
方法:用IC50的SU5416对HUVEC VEGFR-2进行阻断,运用Western-blotting检测方法,观察人参、三七组方对下游信号蛋白Ras、MAPK表达的影响。
结果:IC50的SU5416对HUVEC VEGFR-2阻断后,人参、三七组方干预后,可以增加下游信号蛋白Ras、MAPK的表达。
结论:人参、三七组方可直接作用在血管生成信号通路上Ras蛋白上,促进其表达,但还不能说明中药可直接作用在MAPK这一上。
实验七FPP对下游信号蛋白MAPK的影响以及中药的干预作用
目的:探讨人参、三七组方在血管生成信号通路上的作用靶点。
方法:用IC50的FPP对HUVEC Ras进行阻断,运用Western-blotting检测方法,观察人参、三七组方对下游信号蛋白MAPK的表达。
结果:IC50的FPP对HUVEC Ras阻断后,人参、三七组方干预后可以增加下游信号蛋白MAPK的表达。
结论:人参、三七组方可直接作用在血管生成信号通路上的MAPK蛋白上。
实验八三维培养脐静脉内皮细胞形成血管样结构的研究
目的:比较二维与三维细胞培养模型之间的差异,模拟血管生成的真实环境。
方法:分别采用24孔培养板和Transwell小室进行二维和三维细胞培养,通过倒置相差显微镜比较二维与三维细胞培养之间的差异,并利用激光共聚焦显微镜观察HUVEC在胶原中血管样结构的形成。
结果:在二维细胞培养中,HUVEC出现经典的铺路石样改变;在三维细胞培养中,出现从零星的不完整的网状逐渐演变成管腔样结构。
结论:利用Transwell小室成功构建HUVEC的三维培养体系,真实地模拟了在体血管新生的过程,从而可以更加准确和真实地评价血管生成作用。
小结
利用Transwell小室成功构建HUVEC的三维培养体系。
人参三七组方可以促进HUVEC增殖,并可促进HUVEC分泌VEGF。
人参三七组方可以直接作用于VEGFR-2,促进其表达。
人参三七组方可以直接作用于Ras蛋白,而不是因为上游VEGFR-2的表达增加而引发下游信号的增强。
人参三七组方可以直接作用于MAPK蛋白,而不是因为上游Ras蛋白表达增加而引发下游信号的增强。
以上研究,部分阐明了益气活血中药促血管生成作用的分子机制,并初步揭示了干预血管生成的作用环节和分子靶标。
The mechanisms and treatment of Angiogenesis in ischemic disease have become a hot research topic now.The Chinese medicine of Tonifying Qi and Activating Blood has a certain efficacy in treating coronary heart disease(CHD).It can improve myocardial ischemic and angiogenesis of myocardial ischemia,and it has been confirmed by lots of clinical and experimental researches,suggesting that promoting angiogenesis may be one of important mechanism of Chinese medicine in the treatment of CHD.
Therefore,in-depthly probe the angiogenesis molecular mechanism of Tonifying Qi and Activating Blood herb;reveal the links and molecular target;set out the scientific connotation of function of Tonifying Qi and Activating Blood Chinese medicine and provides a new experimental evidence for prevention and treatment of ischemic disease,are important tasks.
The research includes three parts:The literature review,The meta analysis and The basic research
Literature review:Traditional Chinese Medicine in Therapeutic Angiogenesis and Therapeutic Angiogenesis for CHD were reviewed.
Clinical study:Chinese Medicine of Tonifying Qi and Activating Blood for Unstable Angina:A Meta Analysis
Objective:To evaluate the effect of Chinese Medicine of Tonifying Qi and Activating Blood on unstable angina pectoris.Methods:①Search items were[Title: unstable angina]AND[All field:(Chinese medicine) OR(Chinese herb) OR (Integrated Traditional and Western Medicine)]AND[All field:(Tonifying Qi) OR (Activating Blood)],the search was applied to the CBM database(1994.1～2007.12).②Literature bolting was performed by 2 reviewers independently according to the standard of in and out,different opinions should be solved through negotiation.③The relation data of literature were extracted and evaluated.④A meta analysis was performed based on qualified literatures.Result:①15 literatures were found out.②Through twice blotting,there were 6 clinical study literatures meet the evaluate procedure.③6 literatures came into meta analysis procedures,the total number of cases only 385 cases,but these studies are not high quality generally.Meta analysis showed that compared with the single conventional basic treatment,to the patients with unstable angina,the total effective of plus the intervention of Chinese medicine of Tonifying Qi and Activating Blood was significantly improved than the control group on the symptoms of angina,the electrocardiogram and blood lipid.Conclusion: Chinese medicine of Tonifying Qi and Activating Blood improve the symptoms of unstable angina,electrocardiogram and the lipid.Due to the small number of researches and the quality is not high,the further study is need.
Basic research:including the following eight experiments.
The 1~(st) Experiment:Drawing the growth curve of HUVEC
Objective:To understand the growth condition in different concentrations and at different time points and to choose the appropriate concentration and the best time for the following experiments.
Methods:Six concentration of cells were inoculate in the 96 well,detect colorimetfic assay every 24 h,for five consecutive days,and repeated it once.
Results:After inoculated,the cells were in incubation period in first day.In the second day,the growth of the cell got into the logarithmic growth period.In the 3rd and 4th day,the cells of 4×10~4/ml,6×10~4/ml and 8×10~4/ml concentration got into the platform period;while the 1×10~4/ml,2×10~4/ml and 3×10~4/ml concentration also showed the trend of proliferation;however,growth rates were less than the first two days.In the 5th day,1×10~4/ml,2×10~4/ml and 3×10~4/ml concentration also continued proliferating,while the 4×10~4/ml cell proliferating that has saturated the density.From the second day,the 6×10~4/ml and 8×10~4/ml cell gradually got into saturation.
Conclusion:In the follow-up experiments,vaccinated the of 2×10~4/ml～3×10~4/ ml;and after 36 h,the cells were supplied.
The 2~(nd) Experiment:A study on the effect of proliferation of HUVEC with ginseng and Sanqi recipe of different ratio,concentration and time point
Objective:To explore the best dose,ratio and the best time point of the ginseng and Sanqi recipe promoting the proliferation of HUVEC.
Method:Prepared the ginseng and Sanqi recipe into 5 different groups: 0.25mg/ml,0.5mg/ml,1mg/ml,2mg/ml,and 4mg/ml,respectively,in accordance with the ratio of 1:1,2:1,1:2,and with a control group.After 24h and 48h,test the colorimetric assay.
Results:After 24h,only 2:1 ratio appeared a certain dose-effect relationship,and 4mg/ml showed the most cell proliferation,and the rate is as high as 37.3%.And 48h later,three different ratio of concentrations all showed a certain proportion of the dose-effect relationship,amang them,the 1:1 ratio and the concentration of 4 mg/ml showed the strongest proliferation,the rate of 26.12%.
Conclusion:The the most obvious proliferation showed in 2:1 ratio,while the concentration is 4 mg/ml,after the 24 h promoting HUVEC.
The 3~(rd) Experiment:The study of ginseng and Sanqi recipe promoting HUVEC proliferation and secretion of VEGF
Objective:To explore the effects of ginseng and Sanqi recipe on the umbilical vein endothelial cells proliferation and secretion of VEGF.
Method:The cells were divided into 5 groups:The control group,bFGF positive control group,ginseng and Sanqi high-dose group,ginseng and Sanqi moderate- dose group,ginseng and Sanqi low-dose group.After 24h,colorimetric assay.Then the supernatant collected each group,and detected the VEGF with double-antibody sandwich ELISA.
Results:Ginseng and Sanqi Group promoted the proliferation of HUVEC and secretion of VEGF,the affect is same as bFGF,and there were significant differences to the control group.
Conclusion:The ginseng and Sanqi recipe can promote the HUVEC proliferation and secretion of VEGF.
The 4~(th) Experiment:The effect of the ginseng and Sanqi recipe on the expression of HUVEC VEGFR-2.
Objective:To explore the effect of the ginseng and Sanqi recipe on the expression of HUVEC VEGFR-2.
Method:To observe the HUVEC VEGFR-2 expression by the immunohisto-chemistry methods after ginseng and Sanqi recipe intervention.
Results:The ginseng and Sanqi recipe high-dose group promote the HUVEC VEGFR-2 expression,same as bFGF,and were more than those of the control group, there were significant differences to the control group.
Conclusion:The ginseng and Sanqi recipe can promote the expression of VEGFR-2,and then cause HUVEC proliferation.
The 5~(th) Experiment:The effect of ginseng and Sanqi recipe on the relative signal protein and its mRNA of angiogenesis.
Objective:To explore the effect of ginseng and Sanqi recipe on the signal protein and its mRNA of angiogenesis.
Methods:To analysis the VEGFR-2,Ras,MAPK of HUVEC,With the Western-blotting protein detection methods;and analysis the mRNA expression of the three proteins with realtime-PCR,after ginseng and Sanqi recipe intervention.
Results:The ginseng and sanqi recipe can promote the expression of signaling protein of angiogenesis(VEGFR-2,Ras,MAPK) in different levels,however,the their mRNA expression have different degrees of reduction.
Conclusion:The ginseng and sanqi recipe can promote the expression of angiogenesis signaling protein,VEGFR-2,Ras and MAPK.These may be the foundation of Chinese medicine promoting endothelial proliferation and angiogenesis. The decline of the three mRNA expression may be related to its amplification time.
The 6~(th) Experiment:The effect of SU5416 on the downstream signal protein and the role of the ginseng and Sanqi recipe
Objective:To study the act targets of the ginseng and Sanqi recipe to the angiogenesis signaling pathway.
Methods:To observe the expression of the downstream signaling protein Ras,MAPK, with the HUVEC VEGFR-2 blocked by SU5416 of IC50 of Western-blotting detection method,after ginseng and Sanqi recipe intervention.
Results:The intervention of the ginseng and Sanqi recipe can increase the downstream signaling protein Ras,MAPK expression,when the HUVEC VEGFR-2 was blocked by SU5416 of IC50.
Conclusion:The ginseng and Sanqi recipe has a direct role in angiogenesis in the Ras protein signaling pathway,to promote its expression,but it can not explain the direct role of Chinese medicine on MAPK.
The 7~(th) Experiment:The effect of FPP on the downstream signal protein and the role of the ginseng and Sanqi recipe
Objective:To study the role targets of the ginseng and Sanqi recipe on angiogenesis signaling pathway,after ginseng and Sanqi recipe intervention.
Methods:To observe the expression of the downstream signaling protein MAPK, with the HUVEC Ras blocked by FPP of IC50 of Western-blotting detection method.
Results:The intervention of the ginseng and Sanqi recipe can increase the downstream signaling protein,MAPK expression,when the HUVEC Ras was blocked by FPP of IC50.
Conclusion:The ginseng and Sanqi group has a direct role on the MAPK protein of angiogenesis signaling pathway.
The 8~(th) Experiment:The research of umbilical vein endothelial cells forming vessel-like structure in three-dimensional culture.
Objective:To compare the differences of the two-dimensional model and three-dimensional model of cell culture,and analog the real environment of angiogenesis.
Method:A 24 well culture board and Transwell Inserts were adopted for two-dimensional and three-dimensional cell culture respectively,and the inverted phase contrast microscope was adopted for compare the difference of cell culture between two and three-dimensional,and the laser scanning confocal microscopy (LSCM) was adopted to observate the formation of HUVEC in the vascular liked structure of collagen.
Results:In two-dimensional cell culture,a classical paving stones and the lumen-like appearance of HUVEC were observed;in three-dimensional cell culture, the structure of sporadic incomplete mesh evolving into the lumen-like gradually was observed.
Conclusion:With the Transwell Inserts,the three-dimensional culturing system of HUVEC was successfully constructed,and the process of angiogenesis was simulated,and thus we can accurately assess the function of angiogenesis.
Summary
With the Transwell Inserts,we successfully constructed the three-dimensional culture system of HUVEC,and observated the differences of two-dimensional and three-dimensional cell culture,with inverted phase contrast microscope and LSCM.
With the immunohistochemistry,ELISA,Western-blotting and realtime-PCR method,we found:
1 The ginseng and Sanqi recipe can promote HUVEC proliferation.
2 The ginseng and Sanqi recipe can promote HUVEC to secrete VEGF.
3 The ginseng and Sanqi recipe can directly role in VEGFR-2,to promote its expression.
4 The ginseng and Sanqi recipe can direct role in the Ras protein,and not because the upper VEGFR-2 expression increased the expression of downstream signal.
5 The ginseng and Sanqi recipe can directly role on MAPK protein,but not because the upper Ras expression increased the expression of downstream signal.
By the above studies,we clarified that the molecular mechanism of Chinese Medicine of Tonifying Qi and Activating Blood can promote angiogenesis,and revealed the intervention role of angiogenesis and molecular targets.
本论文分为三部分:文献综述、临床文献系统评价和基础研究。
文献综述部分,分别从中医药治疗性血管生成的研究进展以及血管生成在冠心病防治中的研究进展两个方面进行了综述。
临床文献评价部分,对1994年～2007年在科技期刊上公开发表的益气活血中药治疗不稳定心绞痛的临床文献进行系统评价。
目的:对益气活血中药治疗不稳定心绞痛临床研究进行系统评价。方法:①以[(中文标题:不稳定心绞痛)AND(全部检索条目:中医or中药or中西医结合)AND(全部检索条目:益气and活血)]的检索式检索中国生物医学文献光盘数据库,年限为1994年1月至2007年12月。②由2位研究者按照文献纳入、排除标准独立进行文献筛选,意见不同者通过讨论解决。③获取文献的相关研究特征资料,并加以评价。④对合格的随机对照试验,进行定量的Meta分析。结果:①共检索到15个文献。②经两次筛选,剔除不符合要求以及重复报道的文献,最终共有6篇文献入选评价。③对6篇文献进行Meta分析程序,总病例数仅385例,研究的质量普遍不高。Meta分析显示,加用益气活血中药进行干预对不稳定心绞痛患者心绞痛症状的改善、心电图的改善、血脂的改善较单纯西医基础治疗显著提高。结论:益气活血中药干预不稳定心绞痛在改善心绞痛的症状、心电图表现以及改善血脂方面有一定的疗效。但由于纳入研究的病例数较少和质量不高,还有待进一步研究。
基础研究部分,包括以下八个实验研究。
实验一HUVEC生长曲线测定
目的:明确不同细胞浓度、不同时间点HUVEC的生长情况,为后续实验选用合适的细胞浓度及最佳的给药时间做准备。
方法:将6个浓度的细胞接种于96孔板中,每隔24h比色测定,连续5天,重复一次。
结果:接种细胞后第2天,各细胞密度的生长均进入指数生长期。第3、4天,4×10~4个/ml,6×10~4个/ml,8×10~4个/ml 3个浓度的细胞进入平台期;第5天,1×10~4个/ml,2×10~4个/ml,3×10~4个/ml这3个浓度的细胞还继续增殖,而4×10~4个/ml的细胞己到饱和的密度;6×10~4个/ml,8×10~4个/ml两个浓度的细胞第2天后逐渐进入饱和状态。
结论:选用2×10~4个/ml～3×10~4个/ml这个区间的细胞数接种;细胞接种36h后给药作为后续实验的基础。
实验二人参、三七不同配比和时间点对HUVEC增殖的影响
目的:探讨人参、三七组方促HUVEC增殖的最佳剂量、两药比例和最佳作用时间。
方法:将人参和三七分别按照1:1,2:1,1:2三种配比制备成0.25mg/ml、0.5mg/ml、1mg/ml、2mg/ml、4mg/ml 15个不同浓度的给药组和1个空白组,分别在给药24 h和48 h后比色测定。
结果:三种配比的不同浓度作用于HUVEC 24 h后,只有人参、三七之比为2:1时,出现一定的量效关系,且4 mg/ml对细胞的增殖作用最强,增值率达37.3%;作用于HUVEC 48 h后,三个配比均出现一定的量效关系,人参:三七=1:1,浓度4mg/ml促细胞的增殖作用最强,增值率达26.12%。
结论:人参、三七为2:1,浓度4mg/ml,药物作用时间为24 h时,促HUVEC增殖作用最明显。
实验三人参、三七组方促HUVEC增殖及分泌VEGF的研究
目的:探讨人参、三七组方对脐静脉内皮细胞增殖及分泌VEGF的影响。
方法:细胞分为对照组、bFGF阳性对照组、人参三七大剂量组、人参三七中剂量、人参三七小剂量组,给药24 h比色测定。用双抗体夹心ELISA法,检测上清中VEGF的含量。
结果:人参、三七组方能够促进HUVEC增殖和分泌VEGF,其作用和bFGF相当,与空白组有显著性差异。
结论:人参、三七组方能够促进HUVEC增殖和分泌VEGF。
实验四人参、三七组方对HUVEC VEGFR-2表达的影响
目的:探讨人参、三七组方对HUVEC VEGFR-2表达的影响。
方法:采用免疫组织化学的方法,观察人参、三七组方干预后,HUVECVEGFR-2的表达。
结果:人参、三七组方大剂量组促进了HUVEC VEGFR-2的表达。其作用和bFGF相当,和空白组相比有明显增加,且组间差异显著。
结论:人参、三七组方可能通过促进VEGFR-2的表达而促进HUVEC的增殖。
实验五人参、三七组方对血管生成信号蛋白及其mRNA的影响
目的:探讨人参、三七组方对HUVEC的血管生成信号蛋白及mRNA的影响
方法:人参、三七组方干预后,运用Western-blotting检测方法,分析该组方对HUVEC的VEGFR-2、Ras、MAPK表达的影响;运用realtime-PCR对以上三种蛋白的mRNA表达进行分析。
结果:人参、三七组方可不同程度的促进血管生成信号蛋白VEGFR-2、Ras、MAPK的表达,但是这三种蛋白的mRNA表达却有不同程度的降低。
结论:人参、三七组方可促进血管生成信号蛋白VEGFR-2、Ras、MAPK的表达,这可能是中药促进内皮增殖,产生促血管生成的基础。这三种蛋白mRNA表达的下降可能与其扩增时间相关。
实验六SU5416对下游信号蛋白的影响以及中药的干预作用
目的:探讨中药在血管生成信号通路上的作用靶点。
方法:用IC50的SU5416对HUVEC VEGFR-2进行阻断,运用Western-blotting检测方法,观察人参、三七组方对下游信号蛋白Ras、MAPK表达的影响。
结果:IC50的SU5416对HUVEC VEGFR-2阻断后,人参、三七组方干预后,可以增加下游信号蛋白Ras、MAPK的表达。
结论:人参、三七组方可直接作用在血管生成信号通路上Ras蛋白上,促进其表达,但还不能说明中药可直接作用在MAPK这一上。
实验七FPP对下游信号蛋白MAPK的影响以及中药的干预作用
目的:探讨人参、三七组方在血管生成信号通路上的作用靶点。
方法:用IC50的FPP对HUVEC Ras进行阻断,运用Western-blotting检测方法,观察人参、三七组方对下游信号蛋白MAPK的表达。
结果:IC50的FPP对HUVEC Ras阻断后,人参、三七组方干预后可以增加下游信号蛋白MAPK的表达。
结论:人参、三七组方可直接作用在血管生成信号通路上的MAPK蛋白上。
实验八三维培养脐静脉内皮细胞形成血管样结构的研究
目的:比较二维与三维细胞培养模型之间的差异,模拟血管生成的真实环境。
方法:分别采用24孔培养板和Transwell小室进行二维和三维细胞培养,通过倒置相差显微镜比较二维与三维细胞培养之间的差异,并利用激光共聚焦显微镜观察HUVEC在胶原中血管样结构的形成。
结果:在二维细胞培养中,HUVEC出现经典的铺路石样改变;在三维细胞培养中,出现从零星的不完整的网状逐渐演变成管腔样结构。
结论:利用Transwell小室成功构建HUVEC的三维培养体系,真实地模拟了在体血管新生的过程,从而可以更加准确和真实地评价血管生成作用。
小结
利用Transwell小室成功构建HUVEC的三维培养体系。
人参三七组方可以促进HUVEC增殖,并可促进HUVEC分泌VEGF。
人参三七组方可以直接作用于VEGFR-2,促进其表达。
人参三七组方可以直接作用于Ras蛋白,而不是因为上游VEGFR-2的表达增加而引发下游信号的增强。
人参三七组方可以直接作用于MAPK蛋白,而不是因为上游Ras蛋白表达增加而引发下游信号的增强。
以上研究,部分阐明了益气活血中药促血管生成作用的分子机制,并初步揭示了干预血管生成的作用环节和分子靶标。
The mechanisms and treatment of Angiogenesis in ischemic disease have become a hot research topic now.The Chinese medicine of Tonifying Qi and Activating Blood has a certain efficacy in treating coronary heart disease(CHD).It can improve myocardial ischemic and angiogenesis of myocardial ischemia,and it has been confirmed by lots of clinical and experimental researches,suggesting that promoting angiogenesis may be one of important mechanism of Chinese medicine in the treatment of CHD.
Therefore,in-depthly probe the angiogenesis molecular mechanism of Tonifying Qi and Activating Blood herb;reveal the links and molecular target;set out the scientific connotation of function of Tonifying Qi and Activating Blood Chinese medicine and provides a new experimental evidence for prevention and treatment of ischemic disease,are important tasks.
The research includes three parts:The literature review,The meta analysis and The basic research
Literature review:Traditional Chinese Medicine in Therapeutic Angiogenesis and Therapeutic Angiogenesis for CHD were reviewed.
Clinical study:Chinese Medicine of Tonifying Qi and Activating Blood for Unstable Angina:A Meta Analysis
Objective:To evaluate the effect of Chinese Medicine of Tonifying Qi and Activating Blood on unstable angina pectoris.Methods:①Search items were[Title: unstable angina]AND[All field:(Chinese medicine) OR(Chinese herb) OR (Integrated Traditional and Western Medicine)]AND[All field:(Tonifying Qi) OR (Activating Blood)],the search was applied to the CBM database(1994.1～2007.12).②Literature bolting was performed by 2 reviewers independently according to the standard of in and out,different opinions should be solved through negotiation.③The relation data of literature were extracted and evaluated.④A meta analysis was performed based on qualified literatures.Result:①15 literatures were found out.②Through twice blotting,there were 6 clinical study literatures meet the evaluate procedure.③6 literatures came into meta analysis procedures,the total number of cases only 385 cases,but these studies are not high quality generally.Meta analysis showed that compared with the single conventional basic treatment,to the patients with unstable angina,the total effective of plus the intervention of Chinese medicine of Tonifying Qi and Activating Blood was significantly improved than the control group on the symptoms of angina,the electrocardiogram and blood lipid.Conclusion: Chinese medicine of Tonifying Qi and Activating Blood improve the symptoms of unstable angina,electrocardiogram and the lipid.Due to the small number of researches and the quality is not high,the further study is need.
Basic research:including the following eight experiments.
The 1~(st) Experiment:Drawing the growth curve of HUVEC
Objective:To understand the growth condition in different concentrations and at different time points and to choose the appropriate concentration and the best time for the following experiments.
Methods:Six concentration of cells were inoculate in the 96 well,detect colorimetfic assay every 24 h,for five consecutive days,and repeated it once.
Results:After inoculated,the cells were in incubation period in first day.In the second day,the growth of the cell got into the logarithmic growth period.In the 3rd and 4th day,the cells of 4×10~4/ml,6×10~4/ml and 8×10~4/ml concentration got into the platform period;while the 1×10~4/ml,2×10~4/ml and 3×10~4/ml concentration also showed the trend of proliferation;however,growth rates were less than the first two days.In the 5th day,1×10~4/ml,2×10~4/ml and 3×10~4/ml concentration also continued proliferating,while the 4×10~4/ml cell proliferating that has saturated the density.From the second day,the 6×10~4/ml and 8×10~4/ml cell gradually got into saturation.
Conclusion:In the follow-up experiments,vaccinated the of 2×10~4/ml～3×10~4/ ml;and after 36 h,the cells were supplied.
The 2~(nd) Experiment:A study on the effect of proliferation of HUVEC with ginseng and Sanqi recipe of different ratio,concentration and time point
Objective:To explore the best dose,ratio and the best time point of the ginseng and Sanqi recipe promoting the proliferation of HUVEC.
Method:Prepared the ginseng and Sanqi recipe into 5 different groups: 0.25mg/ml,0.5mg/ml,1mg/ml,2mg/ml,and 4mg/ml,respectively,in accordance with the ratio of 1:1,2:1,1:2,and with a control group.After 24h and 48h,test the colorimetric assay.
Results:After 24h,only 2:1 ratio appeared a certain dose-effect relationship,and 4mg/ml showed the most cell proliferation,and the rate is as high as 37.3%.And 48h later,three different ratio of concentrations all showed a certain proportion of the dose-effect relationship,amang them,the 1:1 ratio and the concentration of 4 mg/ml showed the strongest proliferation,the rate of 26.12%.
Conclusion:The the most obvious proliferation showed in 2:1 ratio,while the concentration is 4 mg/ml,after the 24 h promoting HUVEC.
The 3~(rd) Experiment:The study of ginseng and Sanqi recipe promoting HUVEC proliferation and secretion of VEGF
Objective:To explore the effects of ginseng and Sanqi recipe on the umbilical vein endothelial cells proliferation and secretion of VEGF.
Method:The cells were divided into 5 groups:The control group,bFGF positive control group,ginseng and Sanqi high-dose group,ginseng and Sanqi moderate- dose group,ginseng and Sanqi low-dose group.After 24h,colorimetric assay.Then the supernatant collected each group,and detected the VEGF with double-antibody sandwich ELISA.
Results:Ginseng and Sanqi Group promoted the proliferation of HUVEC and secretion of VEGF,the affect is same as bFGF,and there were significant differences to the control group.
Conclusion:The ginseng and Sanqi recipe can promote the HUVEC proliferation and secretion of VEGF.
The 4~(th) Experiment:The effect of the ginseng and Sanqi recipe on the expression of HUVEC VEGFR-2.
Objective:To explore the effect of the ginseng and Sanqi recipe on the expression of HUVEC VEGFR-2.
Method:To observe the HUVEC VEGFR-2 expression by the immunohisto-chemistry methods after ginseng and Sanqi recipe intervention.
Results:The ginseng and Sanqi recipe high-dose group promote the HUVEC VEGFR-2 expression,same as bFGF,and were more than those of the control group, there were significant differences to the control group.
Conclusion:The ginseng and Sanqi recipe can promote the expression of VEGFR-2,and then cause HUVEC proliferation.
The 5~(th) Experiment:The effect of ginseng and Sanqi recipe on the relative signal protein and its mRNA of angiogenesis.
Objective:To explore the effect of ginseng and Sanqi recipe on the signal protein and its mRNA of angiogenesis.
Methods:To analysis the VEGFR-2,Ras,MAPK of HUVEC,With the Western-blotting protein detection methods;and analysis the mRNA expression of the three proteins with realtime-PCR,after ginseng and Sanqi recipe intervention.
Results:The ginseng and sanqi recipe can promote the expression of signaling protein of angiogenesis(VEGFR-2,Ras,MAPK) in different levels,however,the their mRNA expression have different degrees of reduction.
Conclusion:The ginseng and sanqi recipe can promote the expression of angiogenesis signaling protein,VEGFR-2,Ras and MAPK.These may be the foundation of Chinese medicine promoting endothelial proliferation and angiogenesis. The decline of the three mRNA expression may be related to its amplification time.
The 6~(th) Experiment:The effect of SU5416 on the downstream signal protein and the role of the ginseng and Sanqi recipe
Objective:To study the act targets of the ginseng and Sanqi recipe to the angiogenesis signaling pathway.
Methods:To observe the expression of the downstream signaling protein Ras,MAPK, with the HUVEC VEGFR-2 blocked by SU5416 of IC50 of Western-blotting detection method,after ginseng and Sanqi recipe intervention.
Results:The intervention of the ginseng and Sanqi recipe can increase the downstream signaling protein Ras,MAPK expression,when the HUVEC VEGFR-2 was blocked by SU5416 of IC50.
Conclusion:The ginseng and Sanqi recipe has a direct role in angiogenesis in the Ras protein signaling pathway,to promote its expression,but it can not explain the direct role of Chinese medicine on MAPK.
The 7~(th) Experiment:The effect of FPP on the downstream signal protein and the role of the ginseng and Sanqi recipe
Objective:To study the role targets of the ginseng and Sanqi recipe on angiogenesis signaling pathway,after ginseng and Sanqi recipe intervention.
Methods:To observe the expression of the downstream signaling protein MAPK, with the HUVEC Ras blocked by FPP of IC50 of Western-blotting detection method.
Results:The intervention of the ginseng and Sanqi recipe can increase the downstream signaling protein,MAPK expression,when the HUVEC Ras was blocked by FPP of IC50.
Conclusion:The ginseng and Sanqi group has a direct role on the MAPK protein of angiogenesis signaling pathway.
The 8~(th) Experiment:The research of umbilical vein endothelial cells forming vessel-like structure in three-dimensional culture.
Objective:To compare the differences of the two-dimensional model and three-dimensional model of cell culture,and analog the real environment of angiogenesis.
Method:A 24 well culture board and Transwell Inserts were adopted for two-dimensional and three-dimensional cell culture respectively,and the inverted phase contrast microscope was adopted for compare the difference of cell culture between two and three-dimensional,and the laser scanning confocal microscopy (LSCM) was adopted to observate the formation of HUVEC in the vascular liked structure of collagen.
Results:In two-dimensional cell culture,a classical paving stones and the lumen-like appearance of HUVEC were observed;in three-dimensional cell culture, the structure of sporadic incomplete mesh evolving into the lumen-like gradually was observed.
Conclusion:With the Transwell Inserts,the three-dimensional culturing system of HUVEC was successfully constructed,and the process of angiogenesis was simulated,and thus we can accurately assess the function of angiogenesis.
Summary
With the Transwell Inserts,we successfully constructed the three-dimensional culture system of HUVEC,and observated the differences of two-dimensional and three-dimensional cell culture,with inverted phase contrast microscope and LSCM.
With the immunohistochemistry,ELISA,Western-blotting and realtime-PCR method,we found:
1 The ginseng and Sanqi recipe can promote HUVEC proliferation.
2 The ginseng and Sanqi recipe can promote HUVEC to secrete VEGF.
3 The ginseng and Sanqi recipe can directly role in VEGFR-2,to promote its expression.
4 The ginseng and Sanqi recipe can direct role in the Ras protein,and not because the upper VEGFR-2 expression increased the expression of downstream signal.
5 The ginseng and Sanqi recipe can directly role on MAPK protein,but not because the upper Ras expression increased the expression of downstream signal.
By the above studies,we clarified that the molecular mechanism of Chinese Medicine of Tonifying Qi and Activating Blood can promote angiogenesis,and revealed the intervention role of angiogenesis and molecular targets.
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