| The increasingly serious aging of China’s population,fast-paced lifestyle,unhealthy diet,etc.have become the predisposing factors of acute ischemic stroke（AIS）,and the onset population is gradually becoming younger,and stroke has become the first cause of death in China,causing a great burden to individuals,families and society.Clinical manifestations and imaging remain the mainstay of diagnosing and assessing the severity of AIS.Inadequate understanding of the disease,atypical pathogenesis,and delayed imaging findings lead to untimely diagnosis and missed optimal treatment time.Therefore,it is of great significance to explore the biological markers in serum as auxiliary diagnosis or therapeutic intervention targets for acute ischemic stroke,which is conducive to early detection and treatment of diseases,improve vascular recanalization rate,reduce the mortality of brain neurons,save ischemic semi-dark bands to the greatest extent,reduce neurological damage,and thus improve the prognosis of patients.In recent years,with the deepening understanding of miRNA,a large number of studies have found that miRNA can participate in the mechanism regulation pathway of disease through the up-regulation and down-regulation of its expression in the pathogenesis of some diseases,which can provide reference for clinical diagnosis based on the change of its expression level.Other studies have shown that the expression levels of miRNA vary at different stages of the same disease.In the first part of this experiment we observed whether there is any difference in the expression of miRNAs（including miR-133b and miR-296-5p）in the serum of healthy people and patients with AIS within 24 hours of onset（acute phase）and the 7th day（softening phase）,analyze whether it is related to the severity of nerve injury,and discusses the correlation with blood routine and biochemical related indicators,and explore the possible related factors affecting the long-term prognosis of patients in3 months.In the second part,the expression changes of miRNA-133b under the condition of ischemia and hypoxia were verified in vitro by modeling PC12 highly differentiated cells,and the relationship between the expression levels of miRNA-133b in vivo and in vitro was further explored.Part Ⅰ Analysis of serum miR-133b and miR-296-5p expression levels in patients with acute ischemic strokeObjective:1.Compare the expression levels of miR-133b and miR-296-5p in serum of healthy people and patients with acute ischemic cerebral infarction within 24 hours and 7 days.2.To analyze the correlation between the expression levels of miR-133b and miR-296-5p in serum of patients with cerebral infarction,nerve injury,inflammatory factors in blood routine and 3-month prognosis,and evaluate their clinical diagnostic value for AIS.Method:1.Select 35 patients with acute ischemic cerebral infarction who were admitted to the Department of Neurology of the 981 Hospital of the Joint Service Support Force from December 1,2021 to June 25,2022,and divide them into acute cerebral infarction admission group and acute cerebral infarction hospitalization group for 7 days according to the different onset stages of the same AIS patient.In addition,select 33 healthy people who were examined in our hospital at the same time as the control group,collect the peripheral blood of all patients,record their gender,age,blood pressure Body mass index;history of chronic diseases such as hypertension,diabetes and coronary heart disease;personal history of smoking,drinking,etc.and the National Institute of Health Stroke Scale（NIHSS）scores were given to the enrolled patients at the time of admission and on the 7th day of hospitalization.The etiology of cerebral infarction was differentiated according to the trial of Org10172 in Acute Stroke Treatment（TOAST）combined with imaging,symptoms and signs.Blood routine,biochemical,coagulation and other related tests were carried out for all patients upon admission.All patients standard antithrombotic therapy and targeted functional rehabilitation therapy were carried out according to the guidelines.2.Quantitative real-time qR-PCR was used to detect the expression of miR-133b and miR-296-5p in the serum of all participants,and the stem ring method was used for relative quantitative detection by adding external reference.Comparative analysis of relative values calculated by 2-ΔΔCT.Then draw the receiver operating characteristic curve（ROC）and calculate the area under curve（AUC）using SPSS.26 statistical software to analyze the diagnostic evaluation value of AIS.3.Analyze the relationship between miR-133b and miR-296-5p in the cerebral infarction group and the NIHSS score of admission and hospitalization for 7 days.4.Analyze the relationship between miR-133b and miR-296-5p on the day of admission of cerebral infarction and blood routine（white blood cell count,neutrophil count,lymphocyte absolute value etc）,blood biochemistry,coagulation function index and admission blood pressure（systolic blood pressure,diastolic blood pressure）etc.5.The patients in the cerebral infarction group were followed up 3months after discharge by telephone or face to face,and their neurological recovery after 3 months was evaluated with the Modified Rankin Scale（m RS）,and the related factors affecting the prognosis of 3 months were analyzed.According to the m RS≤2 and m RS>2,the patients were divided into good and bad prognosis,and the independent risk factors affecting the prognosis of AIS were discussed with binary logistic regression analysis.Result:1.The AIS patients included in this experiment include 24 males and 11 females;the average age was 64（54.5,71.5）years old,the systolic blood pressure at admission was 161.1±20.2mm Hg,the diastolic blood pressure at admission was 90.8±13.8mm Hg,and the body mass index was 22.8（20.2,26.8）.The NIHSS scores at admission and 7 days were 4.89±3.64,3.43±2.82,respectively.According to TOAST etiology,17 cases were major atherosclerotic stroke,17 cases were small artery occlusive,and 1 case was cardiogenic stroke;in the healthy control group,there were 24 males and 9 females,with an average age of 22（22,24）years.Compared with the baseline data of the acute cerebral infarction group,only gender and body mass index had no statistical difference（P>0.05）.The healthy control group met the experimental requirements.2.The detection level of miR-133b in patients with acute cerebral infarction was 0.05（0.02,0.20）at the time of admission,which was significantly lower than 0.56（0.37,1.03）in the healthy group（P<0.05）;on the 7th day of hospitalization,the expression level increased to 0.40（0.29,3.54）,which was statistically significant（P<0.05）compared with that at admission,but there was no statistically significant difference compared with the healthy group（P>0.05）;the AUC under the ROC curve of miR-133b was 0.867（P<0.05）,95%CI:（0.771-0.062）,specificity was 0.879,sensitivity was 0.743.3.There was no significant difference between the expression of miRNA-296-5p at the level of 0.33（0.11,4.07）in the healthy group and0.96（0.42,2.39）in the admission of acute cerebral infarction（P>0.05）;the expression of 1.98（0.49,11.66）increased at 7 days of hospitalization,which was significantly different from that of the healthy group and the acute group（P<0.05）;the area under the ROC curve was 0.446,P>0.05 had no statistical difference.4.The miR-133b of AIS patients at admission was negatively correlated with the NIHSS score at admission（r=-0.409,P<0.05）,and the miR-133b of 7 days in hospital was also negatively correlated with the NIHSS score at 7 days（r=-0.388,P<0.05）;there was no significant correlation between miR-296-5p and NIHSS score at admission and 7 days after hospitalization in acute cerebral infarction group（P>0.05）.5.The miR-133b in the admission serum of the acute cerebral infarction group was negatively correlated with the absolute value of neutrophils and fibrinogen degradation products,and the correlation coefficients were-0.339 and-0.344,respectively,with statistical significance（P<0.05）.However,there was no significant correlation between miR-296-5p and neutrophils,fibrinogen degradation products,and admission blood pressure（P>0.05）.6.The results of the 3-month follow-up of prognosis:the average m RS was 1.31±1.41,included 8 patients had poor prognosis（22.9%）,27 patients had good prognosis（77.1%）,and there were no deaths.Spearman analysis of the relevant factors affecting the prognosis of neurological function found that age,NIHSS score at admission,homocysteine,activated partial thrombin were positively correlated with the prognosis,and the correlation coefficients were 0.47,0.592,0.348,0.336,respectively;there was a negative correlation coefficient with miR-133b,cholinesterase and Ca2+,which was-0.363,-0.382 and-0.386,with statistical significance（P<0.05）;there was no significant correlation between miR-296-5p,admission blood pressure and TOAST typing.The above related factors were analyzed by binary logistic analysis.The results showed that only NIHSS score on admission was an independent risk factor for prognosis（P<0.05）.Other indicators were related to prognosis,but not independent risk factors.Conclusion:1.miR-133b may be used as a potential biomarker for diagnosis of AIS,and it may participate in the pathological process of AIS through anticoagulation or anti-inflammatory mechanisms.It is negatively correlated with the severity of the disease.2.miR-296-5p may become the target of AIS treatment intervention.Part Ⅱ Expression of miRNA-133b in PC12 cellsObjective:Analyze the expression level of miR-133 b in vitro cells after reoxygenation of highly differentiated PC12 cells derived from rat adrenal pheochromocytoma cell line stimulated by nerve growth factor（NGF）for 24 h after glucose and oxygen deprivation（OGD）model,and verify whether it is consistent with the expression in peripheral blood of AIS patients.Method:1.The highly differentiated PC12 cell line was purchased from Wuhan Punosa Life Technology Co.,Ltd.,and the cells were transferred to the post-seeded plate for 6 to 7 generations,and then cultured in CO2 incubator for 24 hours,and then oxygen glucose deprivation/reoxygenation（OGD/R）was performed.The cells were divided into control group and experimental group according to the pre-and post-modeling.2.Check whether the modeling is successful or not:（1）Observe the changes of cell morphology before and after learning;（2）Cell proliferation and toxicity test kit（CCK-8）was used to detect the cell viability of the experimental group and the control group.3.The relative expression of miRNA-133 b was calculated and analyzed by real-time fluorescent PCR technology and simultaneous detection of intracellular reference U6.Results:1.After the model of PC12 cells with glucose and oxygen deprivation（OGD）,the cells close to the culture dish fell off and floated loosely in the culture medium,the neural synapses interwoven into a network disappeared,and the cells became oval;the survival rate detected by CCK-8 reagent after modeling was 40% of that before modeling.2.After 24 hours of reoxygenation,the expression level of miR-133 b in the experimental group and the control group was 0.005 ±0.002 and 1.51 ± 1.22,respectively,which was significantly lower in the experimental group than in the control group（P<0.05）.Conclusion:The expression of miR-133 b was significantly reduced after 24 h of OGD/R modeling in PC12 cells.It is consistent with expression in the peripheral blood of AIS patients with a course of 24 hours. |
PDF Full Text Request