西洛他唑促进小鼠急性脑梗死区域血管内皮增殖相关研究

Cilostazol Promotes Vascular Endothelial Proliferation in Mice with Acute Cerebral Infarction

目的观察西洛他唑(CLZ)促进小鼠脑梗死急性期新生血管增殖效果并探讨其作用机制。方法12只8周龄雄性Icr小鼠建立左侧大脑中动脉永久闭塞模型,分为CLZ组和羟乙基-β-环糊精(HPβCD)组,分别给予CLZ悬浊液及HPβCD溶液进行每日腹腔内注射治疗,7天时处死小鼠使用福尔马林溶液进行灌流固定后,将小鼠大脑取出进行冰冻切片并进行血小板-内皮细胞粘附分子(CD31)和4,6-二氨基-2-苯基吲哚(DAPI)免疫荧光染色,通过使用imagaJ软件测定小鼠大脑半暗带区域内CD31标记区域所占面积,评估CLZ是否具有促进小鼠脑梗死半暗带区域血管内皮增殖以及是否具有促进半暗带区域血管新生能力。另将小鼠大脑由来血管内皮细胞用血管内皮培养液进行培养,1天后分别加入含等量西洛他唑悬浊液(10μg/L)和HPβCD溶液的血管内皮培养液继续进行培养,培养开始第4天时,各组培养皿中加入5-溴脱氧尿嘧啶核苷(BrdU),于培养开始后第5天时固定细胞并进行相关染色,观察BrdU阳性标记细胞数、DAPI细胞数以及两者比例,评估西洛他唑是否具备促进血管内皮增殖效能。结果CLZ组小鼠注射药物后半暗带区域CD31阳性标记细胞所占区域面积(0.101±0.027)较HPβCD组(0.049±0.013)有明显增高。加入CLZ组培养的小鼠大脑由来血管内皮细胞中,CLZ组的BrdU阳性标记细胞所占比例(0.72±0.03)较HPβCD组(0.39±0.05)有明显增加。以上差异均有统计学意义(P<0.05)。结论CLZ可通过促进小鼠血管内皮增生,从而增加小鼠脑梗死后半暗带新生微小直径血管数目,从而达到改善半暗带区域脑血流量效果。

Objective To observe the effect of cilostazol(CLZ)on the proliferation of neovascularization in mice with acute cerebral infarction and explore its mechanism.Methods Twelve 8-week-old male Icr mice were used to establish a permanent occlusion model of the left middle cerebral artery.Model mice were divided into a CLZ group and a hydroxyethyl-β-cyclodextrin(HPβCD)group,each with 6 animals.CLZ suspension and HPβCD solution were injected intraperitoneally every day.At 7 days,the mice were sacrificed and perfused with formalin solution for fixation.The brains were taken out for frozen section and platelet endothelium immunofluorescence staining of cell adhesion molecule(CD31)and 4,6-diamino-2-phenylindole(DAPI)was used to determine the area of CD31-labeled area in the penumbra region of mice by imagaJ software,and to evaluate whether CLZ can promote the proliferation of vascular endothelium in penumbra area of cerebral infarction and whether it can promote angiogenesis in penumbra area.In addition,vascular endothelial cells derived from mouse brain were cultured in vascular endothelial culture medium.After 1 day,the vascular endothelial culture medium containing the same amount of cilostazol suspension(10μg/L)and HPβCD solution was added to continue the culture.On the 4th day,5-bromodeoxyuridine(BrdU)was added to the culture dishes of each group,and the cells were fixed and stained on the 5th day after the start of culture.The number of BrdU-positive labeled cells,DAPI cells and the ratio of the two were observed to evaluate whether cilostazol could promote the proliferation of vascular endothelial cells.Results The area occupied by CD31-positive labeled cells in the penumbra area[(0.101±0.027)]of CLZ group mice was significantly higher than that of HPβCD group[(0.049±0.013)]after drug injection.In the mouse brain-derived vascular endothelial cells cultured in the CLZ group,the proportion of BrdU-positive labeled cells in the CLZ group[(0.72±0.03)]was significantly higher than that in the HPβCD group[(0.39±0.05)].The above differences were statistically significant(P<0.05).Conclusion CLZ can increase the number of new microvessels in penumbra after cerebral infarction by promoting the proliferation of vascular endothelium in mice,so as to improve the cerebral blood flow in penumbra.