| Purpose To isolate and identify the serum exosomes in patients with ischemic stroke and healthy controls,and to explore its relationship with ischemic stroke as a candidate predictor of risk element.Methods From April through October,2015,twenty volunteers of ischemic stroke(10 acute patients and 10 stroke patients at recovery stage)and 10 healthy controls were collected.Patients within seven days after stroke occurrence were classified as acute stage,while for those occurred between 7-30 days were classified as convalescent group.All Cases were diagnosed by clinical medicine practitioners in Department of Neurology,Second People's Hospital,Chengdu,Sichuan Province.The diagnosis reference was recruited to the Chinese Society of Neurological Society of the fourth national cerebrovascular disease conference revised "all kinds of cerebrovascular disease diagnosis(1995)" as a diagnostic standard,and referring to the inclusion criteria and discharge criteria.Extraction of elbow vein blood,separation and isolation of exosomes by PEG precipitation.Scanning electron microscopy was used to identify exosomes.The qualitative detection of CD9 and CD63 markers of exosomes was detected by specific antibody Coomassie brilliant blue staining.Quantitative detection of total protein in exosomes was measured by BCA protein quantification.Extraction of mi RNA from serum exosomes by Trizol.The expression levels of mi R-15 a,mi R-497 and mi R-181 a in the peripheral blood of the three groups were detected by real-time fluorescence quantitative PCR.All the data in the experiment were analyzed by SPSS21.0,All measurement data are expressed as mean standard deviation,and analyzed by a completely randomized variance analysis(one-way ANOVA analysis.Classification data were compared using two-way disorder R * C table data independent test,and to evaluate the value of mi RNAs associated with ischemic stroke in predicting the risk of ischemic stroke using ROC curve analysis.Results1.Describing the general clinical situation of volunteers in each groupA total of 30 serum samples were collected,including samples of 10 normal controls,samples of 10 patients with acute ischemic stroke(within 7 days of ischemic stroke),samples of 10 patients with ischemic stroke at recovery stage(within 7-30 days of ischemic stroke).In normal group,the ratio of male to female was 4:6,the ratio of male to female in the acute stage was 4:6,and the ratio of male to female in convalescence group was 7:3(P > 0.05).There is no significant difference and no statistical significance.The mean age and standard deviation of the normal group,the acute phase group and the recovery group were 62.70 + 1.557,70.70 + 4.480,66.30 + 4.284(P > 0.05),There is no significant difference and no statistical significance.There was no significant difference in the distribution of smoking,drinking,hypertension,diabetes history and heart history between the normal,acute and convalescent groups(P > 0.05).The levels of fasting blood glucose,high-density lipoprotein,low-density lipoprotein,glycerol three and uric acid in the normal group,acute phase group and recovery group were not significantly different(P > 0.05),and there was no statistical significance.2.Successful isolation and extraction of serum exosomes for mi RNA analysisThe peripheral serum of normal group,acute phase group and recovery group were extracted repeatedly by PEG sedimentation method,and the solid particles of pale yellow or yellow could be isolated,namely the exosomes.Electron microscopy,whether it is normal group or stroke group can be seen uniform size,consistent shape of the oval or round membranous vesicles,and some vesicles have fusion phenomenon,the diameter between 30-100 nm,but part Peripheral vesicles are relatively scarce and precipitates show very high background signals.As the exosomes by the double lipid membrane coated,it can be seen in the central color shallow,peripheral color depth,clear edge of the cystic structure.3.Detection of endogenous protein expression in serum exosomesThe sample concentration(diluted 8 times)obtained by BCA protein was between 250.8868-902.9671 ug / ml,which was in accordance with the linear range of BCA protein standard curve.The actual concentration of sample was between2007.0946-7223.7367 ug / ml.Kaumas blue staining results indicated that 55 KD in the vicinity of the molecular mass of CD63(the first marke band below)can be observed below the belt distribution in the vicinity of 34 KD,the molecular weight of CD9(marke below third bands)can also be observed below the belt distribution.Western blot results showed that CD63 exposure in 2 minutes,developing 8minutes can see the blurred edge bands;when the exposure for 12 minutes,8minutes after the developer,you can see the clear edge and shape of protein bands.CD9 exposure time of 40 minutes,9 minutes to develop,you can see a lighter color,smaller distribution of linear ribbon.4.The expression and trend of mi RNA related to ischemic strokeThe expression levels of mi R-15 a,mi R-497 and mi R-181 a in peripheral blood of three groups were detected by real-time fluorescence quantitative PCR,Mi R-15 a in ischemic stroke in acute stage group relative expression of mean standard deviation was 0.3397 + 0.9810,lower than the healthy control group the relative expression of 0.7132 + 0.3693,lower than the recovery group relative expression of0.4998 + 0.2829.There was no significant difference between the normal control group,the acute stroke group and the stroke recovery group(P > 0.05).Mi R-181 a in ischemic stroke in acute stage group relative expression of mean standard deviation was 0.0360 + 0.0081,lower than the healthy control group the relative expression of0.7227 + 0.2555,lower than the recovery group relative expression of 0.3462 +0.1330.There was significant difference between the acute stage group and the normal control group(P <0.01),There was a significant difference between the acute stage and the convalescent group(P <0.05),There was no significant difference between the normal control group and the recovery group(P> 0.05).Mi R-497 in ischemic stroke in acute stage group relative expression of mean standard deviation was 0.0852 + 0.1781,lower than the healthy control group the relative expression of0.4357 + 0.1473,lower than the recovery group relative expression of 0.3863 +0.1526.Compared with the normal control group,the acute ischemic stroke group had significant difference(P<0.01),The difference between acute and acute stage of ischemic stroke is different(P<0.05),But there was no difference between the normal control group and the recovery group(P > 0.05).5.ROC curve analysis of ischemic stroke-related exosomeal mi RNAsThe ROC curves of mi R-15 a,mi R-181 a and mi R-497 were analyzed.The area under the curve was 0.494,0.963 and 0.775 respectively.Mi R-15 a,0.494(0.264-0.724),mi R-181 a,0.963(0.898-1.0)and mi R-497,0.775(0.597-0.953).Which indicate that those mi RNAs can be use as potential biomarkers for the diagnosis of stroke.Conclusion The expression levels of mi R-181 a and mi R-497 in peripheral blood were significantly correlated with the occurrence of ischemic stroke,which is expected to be a biomarker for predicting the risk of ischemic stroke. |
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