The Research On Literatures Analysis And Angiogenesis Signal Transduction About The Ginseng And Sanqi Recipe

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 researchLiterature 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 AnalysisObjective:To evaluate the effect of Chinese Medicine of Tonifying Qi and Activating Blood on unstable angina pectoris.Methods:â‘ Search 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).â‘¡Literature bolting was performed by 2 reviewers independently according to the standard of in and out,different opinions should be solved through negotiation.â‘¢The relation data of literature were extracted and evaluated.â‘£A meta analysis was performed based on qualified literatures.Result:â‘ 15 literatures were found out.â‘¡Through twice blotting,there were 6 clinical study literatures meet the evaluate procedure.â‘¢6 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 HUVECObjective: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 4×10⁴/ml,6×10⁴/ml and 8×10⁴/ml concentration got into the platform period;while the 1×10⁴/ml,2×10⁴/ml and 3×10⁴/ml concentration also showed the trend of proliferation;however,growth rates were less than the first two days.In the 5th day,1×10⁴/ml,2×10⁴/ml and 3×10⁴/ml concentration also continued proliferating,while the 4×10⁴/ml cell proliferating that has saturated the density.From the second day,the 6×10⁴/ml and 8×10⁴/ml cell gradually got into saturation.Conclusion:In the follow-up experiments,vaccinated the of 2×10⁴/ml～3×10⁴/ 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 VEGFObjective: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 recipeObjective: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 recipeObjective: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.SummaryWith 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.