| Objective1.To screen core microRNA of the CMECs of ischemic rats in the processof angiogenesis, clarify the microRNA regulation mechanism of ischemic CMECsangiogenesis.2.To observe the influence of Tonifying and Activating Blood medicine oncore microRNA in the plasma of patients with AMI.Methods1.Experimental Study (1)Selected adult male SD rats(220-280g), toestablish the model of myocardial ischemia with ligation method, and culturedthe ischemic CMECs by the method of planting myocardium tissue, and observedthe morphology of CMECs by inverted phase contrast microscope, and thenidentified its specific antigen by immunocytochemistry.(2)CMECs of ischemicrats were divided as the ischemic group, CMECs of normal rats as the normalgroup; To observe the biological characteristics of angiogenesis, MTT were usedto draw cell growth curve and detect the proliferation, the cell migrationability were measured by wod healing assay, tube-like structure formation wereobserved by inverted phase contrast microscope;To determine the window periodof migration, proliferation, tube formation of CMECs angiogenesis, Real-time PCR were used to detect mRNA expression of ERK and p53.(3) According to thewindow period of migration, proliferation, tube formation of two groups of cells,detected dynamic expression changes of microRNA in the process of ischemicCMECs angiogenesis using microRNA chip, screened differentially expressedsignificant microRNA and used real-time PCR to verify it. According to expressionpattern of differentially expressed microRNA in three periods of migration,proliferation and tube formation, acquired main microRNA related to migration,proliferation and tube formation. and worked out the core microRNA of theischemic CMECs. Then predicted the target genes of core microRNA and usedreal-time PCR to verify it. At the same time, used the Western-blot to detectthe protein expression of target gene,and the key protein expression of MAPK,PI3K, Akt and VEGF in angiogenesis.2.Clinical research30patients with AMI were divided into a treatmentgroup and a control group at random, there were15cases of each group. Thetreatment group was treated with conventional western medicine combinedTonifying and Activating Blood medicine, the control group was treated withconventional western medicine. To detect the expression of core microRNA ofthe two groups in one hour after hospitalization and treatment after two weeks.Results1.Experimental Study (1) Model of myocardial ischemia was madesuccessfully, and part of breakaway CMECs from tissue block were astroid orpolygonal observed by microscope, the cells were short shuttle-like orflagstone appearance when growing into sub-confluence, and forming tube-likeor vascular reticular structure in partial region; The CMECs of rats has thetypical characteristics of microvascular endothelial cells. Factor Ⅷ, CD31related antigen were all positive by immunocytochemical stain identification,which showed that the cultured cells were microvascular endothelial cells.(2)The migration window period was the first day, and the tube formation windowperiod was the second day of the two groups, while the proliferation window period was the third day of the normal group, the sisth day of ischemic group;To compare the rate of migration, proliferation and tube formation of the twogroups, the ischemic group were lower than normal group, the rate of migrationwas reduced the most obviously(P<0.01); The expression of ERK and p53mRNAof ischemic group were higher than normal group(P<0.05).(3) According to thesignificance of the expressional difference and its relationship withangiogenesis, which determined the key microRNA of ischemic CMECs migration wasmir-223-3p; the key microRNA of ischemic CMECs proliferation was mir-142-5p;the key microRNA of ischemic CMECs tube formation was mir-221-5p, andultimately determined the core microRNA in the process of ischemic CMECsangiogenesis was mir-223-3p. From real-time PCR verified that the results ofmir-223-3p were consistented with the chip. Bioinformatics predicted thatRps6kb1is the target genes of mir-223-3p, the pathway analysis reported thatRps6kb1regulated angiogenesis by participating in HIF-1signal pathways.Further research revealed, the genes and protein expression of VEGF,MAPK,PI3K,and Akt which were the downstream molecules of Rps6kb1/HIF-1signal pathways,were significantly reduced in ischemic CMECs stage migration andproliferation.2.Clinical research One hour after hospitalization, the mir-223-3pexpression of the treatment group and control group are (32.139±8.867),(32.938±15.808)respectively; After treatment for2weeks, the expression ofmir-223-3p of the treatment group and control group are(2.172±0.756),(5.630±1.120)respectively. To compare the treatment group before and aftertreatment, the expression of mir-223-3p was obviously decreased(P<0.01); comparedwith the control group, the expression of mir-223-3p of treatment group wasreduced(P<0.05), which stated that Tonifying and Activating Blood medicinecan reduce expression of mir-223-3p in the plasma of the AMI patients.Conclusion1.The success rate to make the model of myocardial ischemia with ligation method is higher than others, the method of planting myocardium tissue canprovide highly purified ischemic CMECs with good structure and function, andit establishes the base for the research of ischemic cardiovascular diseases.2.The angiogenesis capacity of ischemic CMECs is decreased obviously, andthe angiogenesis proliferation window period has changd, which may be relatedto the change of the expression of mRNA that are ERK and p53, and provide theexperimental basis for the further study of genomics and drug intervention.3.Clarify that the mir-223-3p is the core microRNA of ischemic CMECsangiogenesis.4.To clear up mir-223-3p regulates Rps6kb1/HIF-1signal pathway,inhibits the process of migration and proliferation of ischemic CMECsangiogenesis, and eventually reduces the angiogenic ability. Mir-223-3p islikely to be the core target for the treatment of angiogenesis of ischemicheart disease.5.Tonifying and Activating Blood medicine may regulate Rps6kb1/HIF-1signal pathway by reducing the expression of mir-223-3p of the AMI patients,which plays a role in promoting the angiogenesis of ischemic myocardial. |
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