Detection Of Molecular Markers MiRNA-208a In Acute Myocardial Infarction

Acute myocardial infarction(AMI)is one of the main causes of death for people in the world.Due to the large individual clinical differences and the lack of obvious symptoms,the molecular markers of early AMI have become the focus of research by scholars.MiRNA-208 a,as one of the internal contents of exosomes released by damaged myocardial cells within 0-3 h after the onset of AMI,has the advantages of myocardial specificity,high abundance,and earlier as molecular markers.Therefore,it is expected to be used in clinical or POCT to detect AMI.In this paper,a lipid-modified magnetic bead was prepared and optimized as a capture probe for exosomes from cardiomyocytes,and a miRNA isothermal amplification and fluorescence immunochromatographic assay was established and verified for the first time.The blood samples of 31 AMI patients and 23 healthy people were selected for verification,which proved that miRNA-208 a has a very significant positive correlation with the current clinical "gold standard" cTnI.This study has laid the foundation for clinical application of miRNA-208 a as an AMI molecular marker.The main work is as follows:1)Establishment of separation method based on magnetic beads to extract exosomes.Two different lipid magnetic bead capture probes were prepared.The experimental results show that the lipid magnetic bead capture probe has excellent dispersibility,stability and strong cell capture ability,and the magnetic response time is less than 20 s.In the capture experiment,by optimizing the ratio of lipids and magnetic beads,the magnetic bead capture environment and other factors,it is shown that the magnetic beads: NH2-PEG-CLS: NH2-m PEG feed mass ratio of 10:1:3 is the optimal preparation scheme.The prepared MB@PEG-CLS/m PEG magnetic beads(cholesterol magnetic beads)and MB@PEGDSPE/m PEG magnetic beads(DSPE magnetic beads)exhibited different capture effects for cells and exosomes.The exosomes captured by the lipid magnetic beads were immunofluorescently labeled with the exosomes surface marker protein CD9,which verified that the exosomes were captured by DSPE magnetic beads,laying the foundation for the subsequent extraction and detection of miRNA-208 a using exosomes.2)The establishment of the methodology for the determination of miRNA-208 a by the combination of strand exchange isothermal amplification and lateral flow fluorescence immunoassay(SEA-LFIA)test strips.A strand exchange isothermal amplification method based on a pair of primers was established.By optimizing conditions such as reaction temperature and reaction time,the isothermal amplification of miRNA-208 a could be achieved within 30 minutes.The accuracy of the SEA method was further verified by restriction digestion.Using lateral flow fluorescence immunoassay test strips and fluorescence quantitative analyzer,the established SEA-LFIA method can detect miRNA-208 a sensitively and specifically,and the detection results could be digitally quantified,with a detection sensitivity of 1 pmol.3)Clinical sample verification and correlation analysis of SEA-LFIA detection method.Using clinically collected fresh blood within 6 hours after the occurrence of AMI,the consistency and correlation between the cTnI concentration of the sample and the fluorescence signal value measured by the SEA-LFIA method were analyzed.The quantitative detection results of the two methods show that there is a positive correlation between the fluorescence signal value and the concentration of cTnI,and the correlation coefficient r reaches 0.677,which is one of the molecular markers with the highest correlation with cTnI in the reported studies.Through ROC curve analysis,the established SEA-LFIA method to detect miRNA-208 a has an AUC of 0.986,indicating that its authenticity is very high.When the fluorescence signal value is greater than 1543,the sensitivity and specificity of miRNA-208 a detection is 100% and 91.3% respectively.In addition,miRNA-208 a was not detected in blood samples with high cTnI concentration but not derived from AMI patients,suggesting its better diagnosis value.These results verify the reliability of the established SEA-LFIA method and the feasibility of miRNA-208 a as a molecular marker for AMI.