| Part I:The role of matrix metalloproteinases in the formation and development of cerebral aneurysms.Objective: To analyze the clinical significance of the expression of MMP-2,MMP-9 and TIMP-2 in the serum of patients with aneurysms through population studies,and to analyze the significance of MMPs in the formation of aneurysms in animal experiments.Methods: 1.Population experiment: 156 patients with cerebral aneurysms were collected as the case group.During the same period,120 health examinees were collected in the hospital health examination center as the control group.The venous blood of each subject was collected for 5 ml in anticoagulant tube at the time of initial diagnosis or physical examination,centrifuged for 10 minutes at 1000 rpm,and the supernatant was taken.The serum levels of MMP-2,MMP-9 and TIMP-2 were detected by ELISA kit.2.Rabbit experiment: 32 adult New Zealand rabbits were purchased from Beijing Vitonlihua Laboratory Animal Technology Co.,Ltd.They were divided into four groups: normal control group,1 week group,2 weeks group and 3 weeks group,with 8 rats in each group.Elastase induces cerebral aneurysms in rabbits.One week group,two weeks group and three weeks group were induced for one week,two weeks and three weeks respectively.After the intervention,the rabbits were given general anesthesia along the neck incision,the right common carotid artery was exposed,and the size of carotid aneurysm was measured with vernier caliper.Western blot was used to detect the expression of MMP-2,MMP-9 and TIMP-2 proteins in aneurysms.The expressions of MMP-2,MMP-9 and TIMP-2 in aneurysms were detected by RT-PCR.HE staining was used to make pathological sections of cerebral aneurysms.3.Rat experiment: 30 SPF male Wistar rats and 30 TIMP-2 deficient Wistar rats were purchased from Beijing Witonglihua Laboratory Animal Technology Co.,Ltd.Thirty rats were randomly divided into three groups: normal control group,model group and doxycycline group.Wistar rats with TIMP-2 deficiency were TIMP-2 deficiency group.In the model group,doxycycline group and TIMP-2 deficiency group,the rat cerebral aneurysm model was induced by renal artery hypertension,and the normal control group was treated with sham operation.After operation,rats in Doxycycline group were subcutaneously injected with a concentration of 25 mg/ml of doxycycline 0.5 ml twice a day.The other three groups were injected with normal saline as control,and the intervention lasted for one week.After 4 weeks of induction,rats were killed,blood was taken,brain was taken by craniotomy,intracranial aneurysms were carefully observed under microscope,and their size was measured.The aneurysm tissue was removed and HE stained for pathological section.The levels of MMP-2,MMP-9 and TIMP-2 in serum were detected by ELISA kit.Results: 1.Population experiments:(1)The expression levels of MMP-2,MMP-9 and TIMP-2 in the serum of patients with aneurysms were significantly higher than those of the normal control group;the ratio of MMP-2/TIMP-2 and MMP-9/TIMP-2 in the serum of patients with aneurysms was significantly higher than that of the normal control group.(2)The expression of serum MMP-2 protein was significantly increased in patients with HuntHess grade IV-V and aneurysm diameter > 2.5 cm;the expression of serum TIMP-2 protein was significantly increased in patients with Hunt-Hess grade IV-V;the levels of MMP-9 protein,MMP-2/TIMP-2 and MMP-9/TIMP-2 ratio were significantly increased in patients with Hunt-Hess grade IV-V,multiple aneurysms,aneurysm diameter 1.2-2.5 cm and > 2.5 cm.Rise.(3)Multivariate logistic regression analysis showed that high levels of MMP-2,MMP-9,MMP-2/TIMP-2 and MMP-9/TIMP-2 were independent risk factors for death of patients with cerebral aneurysms.2.Rabbit experiment:(1)The relative expression order of MMP-2,MMP-9 and TIMP-2 in each group of aneurysms was 6 weeks group > 3 weeks group > 1 week group > control group;MMP-2 and MMP-9 were significantly different in control group,1 week group,3 weeks group and 6 weeks group;TIMP-2 had no significant difference between 3 weeks group and 6 weeks group,and there were significant differences in other groups.(2)The levels of MMP-2,MMP-9 and TIMP-2 protein in 1 week group,3 week group and 6 week group were significantly higher than those in control group,and gradually increased in 1 week group,3 week group and 6 week group.(3)The width(mm,mean)of cerebral aneurysms in 1 week group,3 week group and 6 week group were 2.31,4.37 and 5.46,respectively.There were significant differences among the three groups.The width(mm,mean)of cerebral aneurysms in 1 week group,3 week group and 6 week group were 3.15,6.08 and 6.98,respectively.There were significant differences in the height of cerebral aneurysms among the three groups(F=7.54,P=0.010),but the height of cerebral aneurysms in 3 week group and 6 week group was high.There was no significant difference in degree.(4)The elastic fibers of aneurysm wall were ruptured,the number of smooth muscle cells decreased and the endothelial cells were damaged in 1 week group,3 week group and 6 week group.The most serious damage was found in 6 week group.3.Rat experiment:(1)There were significant differences in the levels of serum MMP-2,MMP-9 and TIMP-2 protein in each group.The levels of MMP-2 and MMP-9 protein in model group,doxycycline group and TIMP-2 deficiency group were higher than those in control group.The levels of MMP-2 and MMP-9 protein in Doxycycline group were significantly lower than those in model group.The levels of MMP-2 and MMP-9 protein in TIMP-2 deficiency group were significantly higher than those in model group.The level of TIMP-2 protein in the model group was significantly higher than that in the control group;the level of TIMP-2 protein in the doxycycline group was significantly lower than that in the model group;and the level of TIMP-2 protein in the TIMP-2 deficiency group was significantly lower than that in the other three groups.(2)The tumorigenic rates of model group,doxycycline group and TIMP-2 deficiency group were 90%,60% and 100%,respectively.The tumorigenic rates of doxycycline group were significantly lower than those of model group and TIMP-2 deficiency group.The width and height of cerebral aneurysms in Doxycycline group were significantly lower than those in model group.The width and height of cerebral aneurysms in TIMP-2 deficiency group were significantly higher than those in model group.(3)In the normal control group,the intima,media and adventitia of arteries were intact without abnormal cell infiltration.The intima of model group and doxycycline group became thinner,elastic fibers were broken and the structure was disordered,but the intima of artery in Doxycycline group was thicker than that in model group.In TIMP-2 defect group,the intimal structure was destroyed or disappeared,the structure was disordered and the fibers were broken.Conclusions: MMP-2,MMP-9 and TIMP-2 have important significance in the formation,progression and prognosis of cerebral aneurysms.Part II: Effects of endovascular embolization on MMPs,inflammatory factors,oxidative stress factors and Caspase 3.Objective: To investigate the effect of interventional embolization on the expression of MMP-2,MMP-9,inflammatory factors,oxidative stress factors and Caspase-3 in order to explore the mechanism of therapeutic effect.Methods: 1.Population study: The subjects were included in the study and the operation method was the same as the first part.After 1,3,5 and 7 days of operation,5 ml of venous blood was taken in anticoagulant tube,centrifuged at 1000 rpm for 10 minutes,and supernatant was taken.The levels of MMP-2 and MMP-9 in serum were detected by ELISA.2.Rabbit experiment: 32 New Zealand rabbits were purchased from Beijing Vitonlihua Laboratory Animal Technology Co.,Ltd.They were divided into four groups: control group,model group,vascular intervention group and craniotomy clipping group,with 8 rats in each group.For model group,vascular intervention group and craniotomy clipping group,the induction method is the same as the second part.In the vascular interventional embolization group and craniotomy clipping group,the cerebral aneurysms were treated with vascular interventional embolization and craniotomy clipping respectively.The operation method was the same as in the first part.The vascular interventional embolization was only treated with single coil.On the 7th day after operation,the rabbits were given general anesthesia along the neck incision,and the right common carotid artery was exposed.Tumor tissues or peripheral artery wall tissues were taken for corresponding treatment to detect the levels of various indicators in the tissues.The relative expression levels of MMP-2,MMP-9,IL-6 and TNF-alpha were detected by RT-PCR.The activity of SOD in tissues was measured by hydroxylamine method.Total nitric oxide synthase activity was detected by colorimetry and Caspase-3 activity was detected by spectrophotometry.Results: 1.Population results:(1)The levels of MMP-2 and MMP-9 protein in serum of intervention group and clamp group decreased gradually after operation.At 5 and 7 days after operation,the expression of MMP-2 protein in serum of clipping group was significantly higher than that of interventional group.On the 3rd,5th and 7th day after operation,the expression of MMP-9 protein in serum of clipping group was significantly higher than that of interventional group.(2)The longer the operation time,the more blood loss during the operation,the higher the levels of MMP-2 and MMP-9 in serum of patients after operation.(3)Multivariate logistic regression analysis showed that interventional embolization was an independent factor affecting low levels of MMP-2 and MMP-9 after operation.4.There was no significant difference in serum MMP-2 and MMP-9 levels between coil group,stent-assisted group and balloon-assisted group.2.Rabbit experiment:(1)The relative expression levels of MMP-2 and MMP-9 in model group,interventional group and clamping group were significantly higher than those in control group,while those in interventional group and clamping group were significantly lower than those in model group,and those in interventional group were significantly lower than those in clamping group.(2)The relative expression levels of IL-6 and TNF-alpha in model group,intervention group and clamping group were significantly higher than those in control group,while those in intervention group and model group were significantly lower than those in clamping group.There was no significant difference between intervention group and model group.(3)The SOD activity of model group,intervention group and clamping group was significantly lower than that of control group,and intervention group was significantly higher than that of clamping group and model group.There was no significant difference between clamping group and model group.INOS activity of model group,interventional group and clipping group was significantly higher than that of control group,interventional group and clipping group were significantly lower than that of model group,interventional group was significantly lower than that of clipping group.(4)Caspase-3 activity in model group,interventional group and clamping group was significantly higher than that in control group,while that in interventional group was significantly lower than that in clamping group and model group.There was no significant difference between clamping group and model group.Conclusions: Compared with craniotomy,interventional embolization can significantly reduce the levels of MMP-2 and MMP-9 in serum,proinflammatory factors,oxidative stress and apoptotic factors. |
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