| Objective 1. The preliminary experimental study found yiqi huoxue traditional Chinese medicine(TCM) before and after the intervention of miR- 223-3 p have difference expression in ischemic myocardial Microvascular Endothelial Cells, and found that miR- 223-3 p may be affected by RPS6KB1 / HIF- 1 a signaling pathway inhibition of ischemic myocardial Microvascular Endothelial Cells(Cardiac Microvascular Endothelial Cells, CMECs) of the migration and proliferation, thus inhibiting angiogenesis.In this study, on the basis of preliminary experiment, through the gene transfection technology, further demonstrates miR- 223-3 p the mechanism of inhibiting angiogenesis. 2. Observing yiqi huoxue medicine intervention in Acute Myocardial Infarction(Acute Myocardial Infarction, AMI) in patients before and after plasma miR- 223-3 p, miR- 208, miR- 133- a dynamic change, miR- 223-3 p feasibility as a marker of Acute Myocardial Infarction and sensitivity, provide more basis for the diagnosis of Acute Myocardial Infarction.Methods 1. The experimental research(1) On the basis of preliminary experiment, obtain virus carrier, establish experimental animal model of acute myocardial infarction(ami), observation(ZC) normal group, model group(MX), transfection empty virus(AD- EGFP),transfection miR- 223-3 p interference genome(AD- miR- 223-3 p) biology characteristics of angiogenesis, thiazole blue cell growth curve drawing method(determined by MTT) cell proliferation ability and detection, scratch method cell migration ability, inverted phase contrast microscope observation tube cavity structure formation;Determine the CMECs angiogenesis window period of migration, proliferation, into a tube.(2) according to the four groups of cell proliferation, migration, into the pipe window, take the best MOI, best window, Trizol four groups of total RNA extracted, using rt-pcr test groups the expression of miR- 223-3 p.(3) Western blot detection target genes and HIF- 1 alpha, VEGF, MAPK, PI3 K, and AKT protein expression. 2. Clinical research(1) select the AMI patients with 28 cases, were randomly divided into treatment group and control group, each group of 14 cases, and selected 10 cases of healthy volunteers into groups.Given conventional western medicine treatment group with yiqi huoxue traditional Chinese medicine therapy, the control group given conventional treatment of western medicine.1 h, 7 days, 14 days after admission tests in the two groups the expression of miR- 223-3 p.(2) to retrieve the documents, find and found that miR-208 and miR- 133- a specific miRNA expression rule of acute myocardial infarction(ami), compare the expression of time window.Results 1 experimental study(1) The research production of 60 rat model of acute myocardial infarction, among them, the successful survival rats 54, death 6, model making success rate of 90%.(2)According to the results of adenovirus transfection vaccination adenovirus after 24 hours, 6 group and 60 group is only a small amount of green fluorescent expression, transfection rate was 5% and 5%;More than three groups all have a large number of green fluorescence expression, transfection rate were 89%, 98% and 99% respectively.Inoculation of adenovirus after 48 hours, 6 group and 60 fluorescence expression did not see increased;Express strong fluorescence 600 group, 6000 group, 60000 group reduced fluorescence expression, 6000 group and 60000 hole cell membrane rupture, a large number of suspension cell death.(3) the ZC group, MX, AD- EGFP group, AD- miR- 223-3 p group cell proliferation window respectively for 3 days, 6 days, 6 days, 3 days;Four groups of cells migration and pipe window of 1 and 2 days respectively.ZC group and AD- miR- 223-3 P, mobility, and hence tube rate were higher than MX and AD- EGFP group(P < 0.05).(4) the rt-pcr test results showed that miR- 223-3 p expression level, compared with ZC group, MX, AD- EGFP group, AD- miR- 223-3 p group were raised;Compared with MX group, ZC, AD- miR- 223-3 p group, expression level down.Compared with ZC group, MX group Rps6kb1 m RNA expression has no obvious difference(P > 0.05), the AD-EGFP group Rps6kb1 m RNA expression level(P < 0.01), the AD- miR- 223-3 P group expression reduced(P < 0.05);Compared with MX group, AD- miR- 223-3 P group Rps6kb1 m RNA expression level lowered(P < 0.05), while the AD- EGFP group Rps6kb1 m RNA expression level(P < 0.01)., according to the results of four groups of cell signaling pathways m RNA expression compared with ZC group, MX and AD- EGFP group MAPK, AKT and VEGF m RNA expression were significantly lower(P < 0.05), the AD- miR-223-3 P group HIF- 1 alpha, PI3 K, MAPK and VEGF gene expression were significantly lowered(P < 0.05), the difference was statistically significant.Compared to the MX group, AD- EGFP group there was no statistically significant difference AKT, MAPK and VEGF expression, ZC and AD- miR- 223-3 P group of HIF- 1 alpha, PI3 K, and AKT and VEGF gene were significantly higher(P < 0.01). Back to(5) Western blot test result shows, compared with ZC group, MX, AD- EGFP group and AD- miR- 223-3 P set of target genes protein levels were lower(P < 0.01 or P < 0.05).Compared with MX group, target gene protein expression in the AD- miR- 223-3 p group increased significantly, the AD- EGFP group no significant difference.Four groups of cell signaling pathways, according to the results of protein expression compared with ZC group, MX and AD-EGFP group of MAPK, AKT and VEGF protein expression were significantly lower(P < 0.05), the AD- miR- 223-3 P group HIF- 1 alpha, PI3 K, MAPK and VEGF gene expression were significantly lowered(P < 0.05), the difference was statistically significant.Compared to the MX group, AD- EGFP group there was no statistically significant difference AKT, MAPK and VEGF expression, ZC and AD- miR-223-3 P group of HIF- 1 alpha, PI3 K, and AKT and VEGF gene were significantly higher(P < 0.01).Conclusion 1.The best MOI adenovirus CMECs transfection ischemia between 300-600, transfection time in 12 to 18 hours can achieve maximum transfection efficiency, and damage to the cell is smaller. 2. Mi R-223-3p silence, than normal ischemia CMECs proliferation, migration, into a tube window period ahead of time, prove that miR-223-3p has affected the cell proliferation, migration, the tube forming process, which affect the angiogenesis. 3. Proves that miR-223-3p is through Rps6kb1/ HIF-1 alpha key molecular signaling pathways that angiogenesis, VEGF, p38 lightning MAPK, PI3 K, and AKT expression levels, promote ischemic myocardial angiogenesis. 4. Yiqi huoxue medicine combined conventional western medicine treatment of AMI is relatively simple routine western medicine treatment can achieve good clinical curative effect, its mechanism may be related to regulating the expression of miR- 223-3p.And on the pathogenesis of myocardial infarction, initial, miR-223-3p miR-133 and miR-208 May be more early into the window. |
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