| Part one：KLF2expression in rabbit basilar artery of induced aneurysm modelObjective: Investigate the Kruppel-like factor2(KLF2) expression in differentmethods construct hemodynamics induced aneurysm models and the correlation of basilarartery flow velocity changes and klf2expression was analyzed.Methods: One hundred forty-four male New Zealand white rabbits were randomlydivided into three groups: sham group(n=48), unilateral carotid artery ligation group(n=48),bilateral carotid artery ligation groups(n=48), each group is divided into postoperative1W,2W,3W and4W subgroups, each subgroup12rabbits. Sham operation group onlyexposed carotid artery but not ligated, unilateral ligation group’s right common carotidartery were ligated unified, bilateral ligation group’s bilateral common carotid artery wereligated. After procedured1week,2weeks,3weeks and4weeks, the blood flow velocity ofthe basilar artery were measured in each group by transcranial Doppler ultrasound. Then theanimals were sacrificed at each time point,6basilar artery bifurcation for the pathologicalexamination, including HE staining, EVG staining, Masson staining and KLF2immunohistochemical staining, the other6KLF2protein were detectived by western blot.Results: Each group basilar artery mean flow velocity measurements: in the shamgroup did not change significantly at different time points after surgery, unilateral commoncarotid artery ligation group were increased compared with the sham group, but nostatistically significant difference (P>.05). In the bilateral common carotid artery ligationgroup mean flow velocity was significantly faster than the sham group and unilateralcommon carotid artery ligation group, there was statistically significant difference (p <0.05).Pathological examination results suggest that in accordance with the evaluation criteriaaneurysm, sham basilar artery bifurcation lumen smooth, endothelial cells intact, the elasticmembrane integrity, no breakage and thinning, no aneurysm formation. Unilateral common carotid artery ligation group,1has slightly, but score small relatively. Bilateral commoncarotid artery ligation group had12bulging outward, statistical analysis showed thatstatistically significant differences among the three groups (p <0.05). Bilateral commoncarotid artery ligation group KLF2protein expression a significant increase frompostoperation1W, and then maintained at a high level, statistics showed statisticallysignificant (P <0.05) compared with sham group and unilateral ligation group. KLF2protein expression changes and blood flow velocity correlation analysis showed havesignificant positive correlation (r=0.845, P <0.05).Conclusion: Established hemodynamic induced aneurysm rabbit model by usingcommon carotid artery ligation caused the hemodynamic most significant, more suitable asan animal model for the study of intracranial aneurysms. Between KLF2expression andblood flow velocity showed a significant positive correlation, suggesting that KLF2isregulated by lumen hemodynamic. Part two： KLF2upstream regulatory factor miR-92a expression in hemodynamicinduced aneurysm in rabbitsObjective: To explore miRNA-92a role in the regulation of expression of KLF2byusing RT-PCR method detect differential miRNA-92a expression of the basilar arterybifurcation in each group of hemodynamics induced Aneurysm in Rabbits.Methods: Seventy-two male New Zealand white rabbits were randomly divided intothree groups: sham group(n=24), unilateral carotid artery ligation group(n=24), bilateralcarotid artery ligation groups(n=24), each group is divided into postoperative1W,2W,3Wand4W subgroups, each subgroup6rabbits. Then the animals were sacrificed at each timepoints, The miRNA-92a expression of the basilar artery bifurcation were detected byreal-time quantitative PCR. Correlation analysis of miRNA-92a expression results andbasilar artery blood flow velocity was performed.Results: Unilateral ligation common group and bilateral ligation common group of thebasilar artery bifurcation mir-92a mRNA expression level was significantly lower than thenormal control group. Bilateral ligation group decreased most significantly, compared withthe sham group and unilateral ligation group there was statistically significant differences(P <0.05). Mir-92a mRNA expression levels and KLF2protein expression correlationanalysis showed that were significantly negatively correlated (r=-0.768, P <0.01), and thecorrelation analysis with basilar artery blood flow velocity showed that were alsonegatively correlated (r=-0.709, P <0.01).Conclusion: MiRNA-92a mRNA expression was decreased accompany the flowvelocity increased and with the blood flow velocity has a negative correlation; Presume thatmiRNA-92a mRNA expression was regulated by hemodynamic. MiRNA-92a expressionand KLF2expression trend contrary, showed a significant negative correlation. Part three： Effects of simvastatin on KLF2expression and downstream factor inhemodynamic induced aneurysm in rabbitsObjective: To explore the mechanism of KLF2/eNOS, VCAM-1, MMP-9pathwaypromote the growth of the aneurysmn and simvastatin suppressed the progression of theaneurysm.Methods: Seventy-two male New Zealand white rabbits were randomly divided intothree groups: unilateral carotid artery ligation group(n=36), bilateral carotid artery ligationgroups(n=36), each group is divided into postoperative1W and4W subgroups, eachsubgroup18rabbits. Simvastatin group of animals were administered the simvastatin25mg/kg/d the day after the bilateral common carotid artery were ligated, free diet.Thereafter administered the same dose of simvastatin intervals24h, until postprocedure oneweek, four weeks. Bilateral ligated group in addition to no drug treatment, other treatmentsame as simvastatin group. After one week, four weeks each group were taken six rabbitsbasilar artery were detected pathology sliced, another12were detected by real-timequantitative PCR and Western blot. Another six sham group rabbits basilar artery weretaken as control eNOS, VCAM-1MMP-9mRNA expression detected by real-timequantitative PCR.Results: There were no significant differences occurred in number in unilateralligation common group and bilateral ligation common group on1W and4W, but in thesimvastatin group basilar artery bifurcation the elastic membrane weak length shorten (P<0.05), the thinning degree of medial smooth muscle (P <0.05) and the longest diametershorten compared with bilateral ligation group, with statistically significant differences (P<0.05). Simvastatin group basilar artery bifurcation, VCAM-1and MMP-9mRNAexpression decreased in postoperative1W,4W, while the eNOS mRNA expressionincreased, and compared with bilateral ligation group the same time points with statisticallysignificant significance (P <0.05). KLF2Western-blot protein semi-quantitative resultsfound: KLF2expression increased, more significante in4W, compared with bilateralligation group with significant statistical difference (P <0.05)Conclusion: In25mg/kg/d dose of simvastatin can significantly upregulated KLF2expression of bilateral ligation group. KLF2/eNOS, VCAM-1, MMP-9pathway is involvedin the initiation of intracranial aneurysms. Simvastatin can reduce the pathological changes in the basilar artery bifurcation aneurysm. |
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