The Study On Correlation Between COMT And Cerebral Vasospasm After Subarachnoid Hemorrhage In Early Stage

Section one.The changes and significance of COMT expression in early period after SAHObjectives:To observe the changing pattern of mRNA and protein expression of COMT,CA content in blood plasma,diameter and wall thickness of basilar artery in early period after SAH,and to discuss the significance of COMT expression in early period after SAH.Methods:Rat SAH model was estabished by single blood injection to prechiasmatic cistern.RT-PCR and Western Blotting were adopted to detect COMT's mRNA and protein expression respectively in striatum corpora at 6h,12h,24h,48h,72h after SAH.CA content in rat's plasm was determined by high performance liquid chromatography and the diameter and wall thickness ofbasilar artery in rats were measured after SAH in corresponding time points.Results:Compared with the control group,there was no statistic difference in mRNA and protein expression of COMT in sham operation group(P＞0.05).After SAH modeling,COMT expression in rats in SAH group began to rise at 6h,reached peak at 12h,then decreased continually at 24h and 48h,returned close to the level of control group at 72h.The expressions of COMT at 6h,12h,24h,48h after SAH were significantly higher than that of control group(P＜0.01) and those of sham group at corresponding time points(P＜0.01).Compared with control group,there was no statistic difference in plasma CA content in sham operation group(P＞0.05).After SAH modeling,CA content in SAH group began to rise 6h after SAH and reached peak at 24h,and decreased after 24h to 72h.The contents in each time points were higher than that in control group and those in sham operation group at corresponding time points(P＜0.01).Compared with control group,there were no statistic difference in caliber and wall thickness of basilar artery in rats in sham operation group. The caliber began to shrink and vessel wall began to thicken in rats' basilar artery in SAH group 6h after SAH modeling but without statistical difference in comparison with control group and sham operation group at corresponding time points(P＞0.05).Compared with control group and sham operation group,the caliber constriction and wall thickening aggravated obviously 12h after modeling(P＜0.01),reached the peak at 24h (P＜0.01) and last to 48h(P＜0.01).The caliber and wall thickness of basilar artery reached nearly normal size 72h after SAH modeling(P＞ 0.05).Conclusion:(1) Increased COMT expression can be induced in early stage after experimental SAH.(2) CA contents increased obviously accompanyed with CVS in early stage after experimental SAH.(3) CVS and increased CA contents in early stage after experimental SAH may be associated with insufficient elevated expression of COMT.Section two.Consturction,packaging,amplification and purification of recombinated pAdxsi-GFP-rCOMTObjectives:To construct pAdxsi-GFP-rCOMT recombinated adenovirus vector system for further study on gene transfection of COMT.Methods:The target gene segments obtained by amplification were cloned to pGEM-T Easy vector to gain rCOMT-T clone vector,from which rCOMT gene was obtained by EcoRI restriction enzyme rection and was connected to pShuttle-GFP-CMV vector to get pShuttle-GFP-rCOMT.By transferring pShuttle-GFP-rCOMT to pAdxsi vector,Ad-GFP-rCOMT virus plasmid was obtained.293T cell was transfected with Ad-GFP-rCOMT virus plasmid mediated by liposome after PacⅠrestriction enzyme rection, pholchloroform extracting and ethanol precipitation.The recombinated adenovirus was amplificated,purified with CsCl density gradient ultracentrifugation and added to 10%sterilization glycerin and finally packaged to preserve for use below 80℃.Results:Results of enzyme digestion and PCR of rCOMT-T cloning vector plasmid,recombinated shuttle vector plasmid and recornbinated adenovirus vector plasmid were correct.After transfected into 293-T cells, recombinant plasmid could be expressed successfully in 293-T cells.Conclusion:pAdxsi-GFP-rCOMT recombinated adenovirus vector system is successully constructed which lay a foundation for the study of gene transfection.Section three.The impact of pAdxsi-GFP-rCOMT recombinated adenovirus transfection on COMT expression,plasma CA content, diameter and wall thickness of barilar artery of SAH ratsObjectives:To probe into gene transfection's impact on rats' COMT expression,plasma CA content,diameter and wall thickness ofbasilar artery in early stage after experimental SAH.Methods:Rat SAH model was established by same method as described at section one and pAdxsi-GFP-rCOMT recombinated adenovirus was stereotaxisly injected into rats' striatum corpora after modeling instantly. The same methods as mentioned in section one were adopted to detect COMT's mRNA and protein expression in rat's striatum corpora,CA content in rat's plasm,diameter and wall thickness of basilar artery of rats in early period after transfection respectively(6h,12h,24h,48h,72h after transfection).Results:The expression of COMT mRNA and protein in rats in adenovirus injection group began to rise 6h after transfection but without statistical difference in comparison with SAH group and empty vector injection group(P＞0.05).They increased much obviously 12h after transfection and reached the peak at 24h,and then decreased from 24h to 72h,but the expression level in each time points were much higer than those in SAH group and empty vector injection group in corresponding time points(P＜0.01).The plasm CA content in rats in adenovirus injection group began to rise 6h after transfection but without statistical difference(P＞0.05) in comparison with SAH group and empty vector injection group.They increased much obviously 12h after transfection and reached the peak at 24h, and then decreased 24h after transfection and closed to the normal level at 72h,the CA levels in each time points were obviously lower than those in SAH group and empty vector injection group in corresponding time points (P＜0.01).The caliber began to shrink and vessel wall began to thicken in rats' basilar artery in adenovirus injection group 6h after transfection but without statistical difference in comparison with SAH group and empty vector injection group(P＞0.05).The caliber constriction and wall thickening aggravated 12h after transfection,reached the peak at 24h and last to 45h,the levels in each time points ameliorated obviously in comparison with SAH group and empty vector injection group in corresponding time points(P＜0.01).The caliber contraction and vessel wall thickening relieved obviously to nearly normal level 72h after adenovirus injection(P＞0.05).Conclusion:(1) pAdxsi-GFP-rCOMT recombinated adenovirus can obviously enhance COMT expression in each time points(12h after transfection) after transfection with longer duriation of higher expression. (2) Compared with SAH group and empty vector injection group in corresponding time points,CA contents decreased obviously and CVS alleviated remarkably 12h after transfection.(3) Decreased CA contents and alleviated CVS may be related to further enhanced COMT expression induced by adenovirus transfection.(4) It was proved again that early CVS after SAH and increased plasm CA contents is associated with insufficient elevated COMT expression after SAH.Section four.The idetification of correlation between COMT gene polymorphism and CVS in early period after SAHObjectives:To analyze the correlation between COMT gene polymorphism of SAH patients and CVS in early period after SAH.Methods:One hundred and sixty-seven patients with spontaneous SAH in early stage in the department of neurosurgery of the First Affiliated Hospital of Chongqing Medical University were selected in this study. Venous bood samples and clinical data of the patients were collected. Polymerase chain reaction-restriction fragment length polymorphism was used to analyze COMT genetype and allele frequency of SAH patients.The degree of CVS was identified by TCD.Hunt-Hess classification was used to evaluate the severity of the patients' condition,the bleeding amount was evaluated by means of Fisher classification of head CT.x~2 test(SPSS13.0 software) and Logistic regression were done to analyze the correlation of COMT gene polymerization and other clinical data with CVS in early period after SAH.Results:The distribution of each allele matched Hardy-Weinberg law and research samples were heredity equilibrium population.CVS incidence of patients with COMT-A allele(51.7%) was much higher than those with COMT-G allele(38.5%).CVS incidence of patients with COMT A/A genetype(66.7%) was obviously higher than those with COMT G/G genetype(35.9%).Univariate Logistic regression demonstrated that COMT-A allele,A/A genetype and Grade 3 to 5 of Hunt-Hess classfication were all associated with CVS.After adjustment of general information, further multivariate Logistic regression hinted that COMT-A allele,A/A genetype were risk factors of CVS in early stage after SAH.Conclusion:COMT-A allele,A/A genetype were risk factors of CVS in early stage after SAH.