摘要
目的:探讨PTEN/PI3K/Akt信号通路在血管壁干细胞(VRSCs)参与血管损伤后新内膜形成中的作用。方法:①从42只健康SD大鼠中随机选取18只制备颈动脉内皮损伤动物模型,分别于0、7、28 d时随机取6只大鼠,各取损伤部位的左颈总动脉1 cm左右,制备血管组织冰冻切片,观察动脉内膜厚度的变化;行组织免疫荧光染色,计算PTEN、p-PI3K及p-Akt阳性细胞占CD34^(+)细胞的比例,观察损伤后PTEN/PI3K/Akt信号通路在CD34^(+)VRSCs中的激活情况。②另取18只大鼠随机分为假手术组、赋形剂组和抑制剂组3组,每组6只。假手术组只分离血管,赋形剂组和抑制剂组均制备颈动脉内皮损伤模型,然后通过血管外膜分别给予磷酸盐缓冲液(赋形剂组)和PI3K/Akt通路阻断剂LY294002(抑制剂组),测定各组损伤血管组织新内膜面积、中膜面积及其比值。③余6只大鼠随机分为对照组和小干扰RNA(siRNA)-PTEN组,每组3只,均取颈总动脉外膜,通过免疫磁珠分选法筛选并纯化CD34^(+)VRSCs,siRNA-PTEN组的CD34^(+)VRSCs体外培养后转染PTEN siRNA;两组CD34^(+)VRSCs均应用定量聚合酶链反应及Western blot方法检测PTEN/PI3K/Akt基因及其蛋白表达水平。结果:血管新内膜随损伤后时间的延长而逐渐增厚。在损伤修复早期(损伤后7 d),CD34^(+)VRSCs p-PI3K、p-Akt表达比例明显升高,PTEN的表达比例未见明显升高;在损伤修复晚期(损伤后28 d)后,CD34^(+)VRSCs细胞中的PTEN表达量明显升高,p-PI3K、p-Akt表达量下降。在血管内皮损伤术后,通过外膜给药阻断PI3K/Akt通路,新生血管内膜面积和内膜/中膜面积比均较赋形剂组下降。体外分离纯化后的模型大鼠CD34^(+)VRSCs经转染PTEN siRNA后,PTEN基因和蛋白表达下调,PI3K/Akt基因和蛋白表达水平上调。结论:PTEN/PI3K/Akt信号通路在血管外膜CD34^(+)干细胞参与血管损伤后新生内膜的形成中发挥着重要作用。
Objective:To investigate the role of PTEN/PI3K/Akt signaling pathway in the involvement of VRSCs in neointima formation after vascular injury.Methods:1)To prepare the animal model of carotid endothelial injury,18 rats were randomly selected from 42 healthy SD rats.On the 0,7th and 28th day,6 rats were randomly selected,about 1 cm of the left common carotid artery was taken from each injury site.The frozen sections of vascular tissue were prepared to observe the changes of intima thickness.Tissue immunofluorescence staining was performed to calculate the proportion of PTEN,p-PI3K and p-Akt positive cells in CD34^(+)cells,and the activation of PTEN/PI3K/Akt signaling pathway in CD34^(+)VRSCs after injury was observed.2)Another 18 rats were randomly divided into sham operation group,excipient group and inhibitor group,with 6 rats in each group.Only blood vessels were separated in the sham operation group,and the carotid endothelial injury models were prepared in the excipient group and the inhibitor treatment group.Then phosphate buffer and PI3K/Akt pathway blocker LY294002 were given through the vascular adventitia respectively,and the neointima area,media area and their ratios of the injured blood vessels were measured in each group.3)The remaining 6 rats were randomly divided into control group and siRNA-PTEN group,with 3 rats in each group and the adventitia of common carotid artery was taken.CD 34^(+)VRSCs were screened and purified by immunomagnetic beads sorting method.CD34^(+)VRSCs in the small interfering RNA(siRNA)-PTEN group were cultured in vitro and transfected with PTENsiRNA.The PTEN/PI3K/Akt gene and their protein expression levels were detected by quantitative polymerase chain reaction and Western blot in CD34^(+)VRSCs in both group.Results:The neointi⁃ma thickened gradually with the extension of time after injury.In the early stage of injury repair(7 days after injury),the ex⁃pression ratio of p-PI3K and p-Akt in CD34^(+)VRSCs was significantly increased,while the expression ratio of PTEN did not increase significantly.In the late stage of injury repair(28 days after injury),the expression of PTEN in CD34^(+)VRSCs cells in was significantly increased,while the expression of p-PI3K and p-Akt was decreased.After vascular endothelial injury,com⁃pared with the excipient group,the PI3K/Akt pathway was blocked by adventitia administration,and the ratio of neovasculariza⁃tion intima area and intima/media area decreased.When the isolated and purified CD34^(+)VRSCs in vitro were transfected with PTEN siRNA,the expression levels of PTEN gene and protein were down-regulated,the expression levels of PI3K/Akt gene and protein were up-regulated.Conclusions:PTEN/PI3K/Akt signaling pathway plays an important role in the involvement of CD34^(+)stem cells in the formation of neointima after vascular injury.
作者
张佳
沈艳
Zhang Jia;Shen Yan(No.1 School of Clinical Medicine,Kunming Medical University,Kunming,Yunnan 650500,China)
出处
《感染．炎症．修复》
2023年第1期7-12,F0002,共7页
Infection Inflammation Repair
基金
昆明医科大学大学生创新性实验项目(2021JXD07)。
