| Objective1. Observing the intervention of supplementing Qi and activating blood circulation on rats with ischemic myocardial microvascular endothelial cells(CMECs) proliferation, migration and tube cavity structure form different links, and the effect on the related extracellular signal-regulated kinase(ERK) cell proliferation and cell death related molecules(p53) m RNA expression, so that can definite the specific biological effects and the best dose to promoting angiogenesis.2. Study of the intervention effects of supplementing Qi and activating blood circulation in patients with myocardial infarction on plasma mi R-223-3p, the dynamic change of mi R-132-5p, provide clinical basis for traditional Chinese medicine treatment of myocardial infarction.Methods1. Experimental study Select adult male SD rats weight 220-280 g, Selected adult male SD rats(220-280g) to establish the model of myocardial ischemia with ligation method and cultured the ischemic CMECs by the method of planting myocardium tissue. Use animal lavage method to preparation medicated serum and separate them into high dose(Q- G), middle-dose(Q-Z), low dose(Q- D), co-incubation with rat ischemic CMECs respectively. To prepare blank serum at the same time, co-incubation with myocardial ischemia model of rats(MX) and normal rats(ZC) respectively which source of the original generation CMECs. Observing s the biological characteristics of angiogenesi, MTT were used to draw cell growth curve and detect the proliferation, the cell migration ability were measured by wod healing assay, tube-like structure formation were observed by inverted phase contrast microscope; To determine the window period of migration, proliferation, tube formation of CMECs angiogenesis, Real-time PCR were used to detect m RNA expression of ERK and p53.2.Clinical researchTo choose 30 patients with AMI and 10 cases of healthy volunteers. 30 patients with AMI were divided into a treatment group and a control group at random, there were 15 cases of each group. The treatment group was treated with conventional western medicine combined Tonifying and Activating Blood medicine, the control group was treated with conventional western medicine. Real-time PCR were used to detect the expression of mi R-223-3p and mi R-132-5p in venous blood of the three groups that volunteers in the time of hospitalization and two treatment groups one hour after hospitalization, treatment after three days, treatment after one week and treatment after two weeks.Results1. Experimental Study(1) This study involved 39 male SD rats, only 20 lavage and 19 myocardial infarction model. Each lavaged rats eventually extract serum 2 ~ 5 ml by abdominal aort. The success rate of acute myocardial infarction(AMI) surgical in rat model was 89.47%. There was only one model rats died within 24 h to 72 h, acute survival rate was 94.12%.(2) The myocardial cell culture results said that the window period of proliferation is 2 days for Q-Z、Q-D group, 3 days for Q- G, ZC group, 6 days for MX; The proliferation rate, mobility and tube rate of Q-G、Q-Z、Q-D group were significantly higher than MX groups, including the Q- D group increased most significantly(P < 0.01); Real- time PCR detection results show that compared with MX group, the expression of ERK m RNA in Q-G、Q-Z、Q-D group was increased, the expression of p53 m RNA in Q-G、Q-Z、Q-D group was reduced(P < 0.05).2.Clinical research(1) Treatment group and control group before treatment TCM symptom scores were 19.73±5.21 and 20.62±4.34, no significant difference(P>0.05). TCM symptom scores after treatment in the control group was 11.47±4.26,the treatment group was 6.34±3.56, the difference was statistically significant(P <0.01).(2) ECG treatment group total effective rate was 78.57%; the control group, the total effective rate was 64.29%. By rank sum test, the difference was statistically significant(P = 0.038,P <0.05).(3) Mi R-223-3P, mi R- 132-5 P expression characteristics Compared with healthy volunteers, mi R-223-3P expression level increased significantly and there was a significant statistical significance(P = 0.000, P < 0.01) when AMI patients admitted to hospital after 1 h, then it reduced gradually after treatment, it tends to slow in the seventh days decline and basic into the plateau and continue to 14 days; mi R-132-5P expression level decreased obviously and it has significant statistical significance(P = 0.000, P < 0.01), continue to lower after hospitalization and almost reach the peak on 3 days, then after rising and continues to 14 days.(4) The quantity of plasma mi R-223-3p expression before and after treatment Before the treatment, the expression of mi R-223-3P was(13.126 ± 0.452) in treatment group and(13.015±0.522) in control group, no statistical difference(P = 0.12, P > 0.05); After treatment, the expression of mi R-223-3P was(2.172±0.756) in treatment group and(5.630±1.120) in control group. Compared with before treatment, both groups of varying degrees of decline, have statistical difference(P = 0.000, P < 0.01). Compared with control group, treatment group mi R-223-3P levels drop more significantly(P = 0.000, P < 0.05).(5) The quantity of plasma mi R-132-3p expression before and after treatment Before the treatment, the expression of mi R-132-3p was(0.159±0.019) in treatment group and(0.160±0.025) in control group, no statistical difference(P=0.878,P>0.05); After treatment, the expression of mi R-132-3p was(0.507±0.077)in treatment group and(0.298±0.061) in control group. Compared with before treatment, both groups of varying degrees of decline, have statistical difference(P = 0.000, P < 0.01). Compared with control group, treatment group mi R-132-3p levels drop more significantly(P = 0.000, P < 0.05).Conclusion1. The supplementing Qi and activating blood circulation can promote the neovascularization function of the ischemic CMECs of rats and make the proliferation, migration, tubulate window period in advance. The study results is low-dose drug-containing serum had the greatest effect on promoting angiogenesis, but not sure it’s best dose.2. The effect of supplementing Qi and activating blood on promote rat ischemia CMECs angiogenesis, may relate to the differential expression of p53 and ERK.3. Compare with simple routine western medicine treatment, supplementing Qi and activating blood combined conventional western medicine treatment of AMI can achieve better clinical curative effect, its mechanism may be related to regulating the expression of mi R-223-3p and mi R-132-5p.In the initial of acute myocardium infarction, mi R-223-3p may be earlier to enter the time window than mi R-223-3p. |
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