| Objective:To explore the microcirculation disturbance of brain and analyse clinical application value by abserving the change and influence of hemorheologic indexes in patients with acute intracerebral hemorrhage. Furthermore, to discuss the biological function, effect in different cell signaling pathways and regulation function network by quantitative proteomics analysis on differential proteomics of plasma in the patients with acute intracerebral hemorrhage. Expecting to take new effeetive measure s to improve the prognosis of patients with intracerebra lhemorrhage.Objects and methods1. Objects1.1 Eighty diagnosed acute intracerebral hemorrhage patients from neurological department in the Hospital Affiliated Jiangsu University from January 2014 to May 2015 were divided into case group and by the method of clinical epidemiological investigation.Thirty normal persons were as control group. The correlation of hemorheology index with the history of hypertension(hemorrhage and non-hypertensive hemorrhage), hemorrhage quantity(<10ml, 10~20ml, 20~30ml and>30ml) and the hemorrhage location(thalamus, brainstem, lobe, basal ganglia and cerebellum) were analyzed.1.2 From Nov 2013 to May 2015, 8 cases with acute intracerebral hemorrhage patients from neurological department in the Hospital Affiliated Jiangsu University and Jiangsu Provincial Hospital of Traditional Chinese Medicine were included(4male and 4female). Choose 4 healthy persons as control group(2male and 2female). Analyse and explore the change and biological function of differential display proteomics of plasma in two groups.2. Methods2.1 Hemorheology indexes detection:The hemorheology indexes were detected within 24 hours.Collectd 4ml biood(heparin anticoagulation) of all patiens before trentment and healthy persons. Using Beijing LBY- N6 B full-automatic blood rheological analyzer and reagent detect whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate, erythrocyte aggregation index and erythrocyte transformation index. Hematocrit was evaluated by wintrobe method and ESR was determined of by westergren method.2.2 Differential display proteomics measurement:Collect anticoagulant blood 4ml, then separated plasma and packed in200 ul eppendorf tubes and freezed in-80 degrees; Remove the peripheral blood high abundance proteins; Protein digestion and peptides purification; i TRAQ marker protein; SCX strong cation exchange column separation of proteins; Reversed-phase liquid chromatography separation, point target and mass spectrometry; Qualitative and quantitative proteomics.3. Statistical processing and bioinformatics analysis3.1 All hemorheology indexes data were analyzed by q-test with IBM SPSS Statistics 22 package, P < 0.05 for the difference was statistically significant.3.2 Participation ratio compared in each group samples to obtain relative expression of protein for quantitative calculation. Measurement data x expessed;The comparison data of two groups with one dependent variable variance analysis, take protein expression is greater than 1.3 times between the two groups and P < 0.05 for differences in protein.3.3 Apply IPA Bioinformatics software to the analysis of classical pathway, disease and function, upstream regulation factors and protein interaction network analysis.Results:1. Compared with control group, the indexes of whole blood viscosity(200 s-1, m Pa·s; 50 s-1, m Pa·s; 5s-1 m, Pa·s), plasma viscosity and ESR were increased in acute intracerebral hemorrhage patients and there were significant statistical differences(P<0.05).2. The whole blood viscosity(200 s-1, m Pa·s) and ESR in hypertensive cerebral hemorrhage group were significant higher than those in the non-hypertensive cerebral hemorrhage group(P<0.05).3. The whole blood viscosity and plasma viscosity increased as the hemorrhage quantity increased, compared the index of whole blood viscosity(200 s-1, m Pa·s and 50 s-1, m Pa·s) among four groups(all P<0.05). The erythrocyte aggregation index significantly decreased as the hemorrhage quantity increased(P<0.05) in each groups. There were no statistical differences compared the indexes of whole blood viscosity(5 s-1, m Pa·s and 1s-1 m, Pa·s), plasma viscosity and ESR in four groups(P>0.05).4. There were statistical differences compared the indexes of whole blood viscosity(200 s-1, mPa·s and 1 s-1, mPa·s), plasma viscosity, erythrocyte aggregation index and ESR among each groups(all P<0.05). The whole blood viscosity was the highest in brainstem group and the lowest in cerebellum group. Plasma viscosity was the highest thalamus group and the lowest in brainstem group. The erythrocyte aggregation index was the highest in lobe group and the lowest in cerebellum group. The index of erythrocyte sedimentation rate was the highest in cerebellum group and the lowest in basal ganglia group. There were no statistical differences compared the indexes of whole blood viscosity(50 s-1, m Pa·s and 5 s-1, m Pa·s),erythrocyte transformation index and hematocrit among each groups(P>0.05).5. In proteomics experiments, 498 proteins were identified with detailed quantitative information, then make use of Uniprot protein database with GO annotation. Results showed that 498 proteins produced forty-three categories in molecular function, in components and biological process.6. Compared with the control group, there were 22 differential display proteomics of plasma in ICH group and 14 proteins was downgrade regulation-expressed.7. Seven candidate biomarkers were selected respectively:APCS 、 FGB 、LBP、MGMT、IGFBP2、LYZ、APOA4.8. IPA On-line analysis software was used in cell signaling pathway analysis process and the results showed that 36 differential plasma proteins in the acute phase of cerebral hemorrhage involved in the several classic signaling pathways and functional network.Conclusions1. The blood of acute cerebral hemorrhage patients shows a state of concentrated, sticky, aggregation and coagulation that severely affects the prognosis. Detecting the hemorheologic indexes is of important significance to the positive control of blood pressure and takes effective measures to the prevention and treatment of acute intracerebral hemorrhage and improves the prognosis.2. Compared with healthy controls, 36 proteins with identification and quantitative information were determined by the proteomics methods based on mass spectrometry in plasma of cerebral hemorrhage patients, by analyzing the differences or disorder of proteins, they all involed in the occurrence and development process of acute brain injury after intracerebral hemorrhage. Also it is potential to become targets of prognostic molecular markers and stratification treatment in the pathological process of cerebral hemorrhage. |
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