摘要
目的 研究巨噬细胞来源含Scr同源区2 结构域蛋白酪氨酸磷酸酶 2(SHP2)通过磷脂酰肌醇-3-激酶(PI3K)/磷酸酯酶与张力蛋白同源物(PTEN)通路促进氧化应激和血管生成对子宫内膜异位症凋亡表型的影响.方法 收集2019 年1 月至2022 年12 月50 例子宫内膜异位症异位子宫内膜组织和50 例正常子宫内膜组织.选取健康雌性C57BL/6 小鼠12 只,采用子宫组织自体移植腹膜壁的方法建立小鼠子宫内膜异位症模型,将小鼠随机分为SHP2-NC组和SHP2-shRNA组,每组6 只,利用慢病毒感染的方法构建稳定敲低SHP2 基因的RAW264.7 细胞,经小鼠尾静脉注射.使用 HE 染色、Western blot、TUNEL 染色、CCK-8、成管实验分别检测子宫内膜组织的病理改变、CD68+巨噬细胞相关蛋白表达情况、内膜细胞凋亡、内膜细胞增殖活性以及内皮细胞血管生成情况.结果 相较于正常子宫内膜组织,异位子宫内膜组织CD68+巨噬细胞SHP2 表达水平升高(P＜0.05).而在SHP2-shRNA组子宫内膜异常病变区域间质细胞出现,但腺体和血管形成减少,同时SHP2、NADPH氧化酶 2(NOX2)、NADPH氧化酶 4(NOX4)、环氧化酶2(COX2)、血管内皮生长因子(VEGF)和Bcl-2 表达水平低于SHP2-NC组,而p53、Caspase-3、Bax表达水平高于SHP2-NC组(P＜0.05).与SHP2-NC组比较,SHP2-shRNA组内膜细胞凋亡数目增加,细胞增殖活性降低,内皮细胞血管生成数量减少(P＜0.05).与 SHP2-NC 干预比较,SHP2-shRNA 干预使 p-PI3K、NOX4、COX2 和VEGF表达水平降低,而PTEN和p53 表达水平升高(P＜0.05).在LY294002 干预后,p-PI3K、NOX4、COX2 和VEGF表达水平较干预前下降,而PTEN和p53 表达水平较干预前上升(P＜0.05).在740 Y-P干预后,p-PI3K、NOX4、COX2和VEGF表达水平较干预前上升,而PTEN和p53 表达水平较干预前下降(P＜0.05).结论 SHP2 通过促进PI3K/PTEN通路活性来增强氧化应激和内皮细胞血管新生,同时抑制子宫内膜细胞凋亡.
Abstract
Objective To study the effect of Src homology 2-containing protein tyrosine phosphatase 2(SHP2)in promoting oxidative stress and angiogenesis through phosphatidylinositol-3-kinase(PI3K)/phosphate and tension homology de-leted on chromosome ten(PTEN)pathway on apoptotic phenotype of endometriosis.Methods The ectopic endometrial tis-sues of 50 patients with endometriosis and 50 normal endometrial tissues were collected from January 2019 to December 2022.Twelve healthy female C57BL/6 mice were selected to establish a mouse endometriosis model by autologous peritoneal wall transplantation of uterine tissue,and the mice were randomly divided into SHP2-NC group(n=6)and SHP2-shRNA group(n=6).RAW264.7 cells with stable SHP2 gene knockdown were constructed by lentivirus infection.It was injected through a mouse tail vein.HE staining,Western blot,TUNEL staining,CCK-8 and tube formation tests were used to detect the patho-logical changes of endometrial tissue,CD68+macrophage-related protein expression,endometrial cell apoptosis,endometrial cell proliferation and endothelial cell angiogenesis,respectively.Results Compared with normal endometriosis tissues,the expression level of SHP2 in CD68+macrophages in endometriosis tissues was increased(P＜0.05).In the SHP2-shRNA group,mesenchymal cells appeared in endometrial abnormal lesions,but glandular and vascular formation decreased.Mean-while,the expression levels of SHP2,NADPH oxidase-2(NOX2),NADPH oxidase-4(NOX4),cyclooxygenase-2(COX2),vascular endothelial growth factor(VEGF)and Bcl-2 were lower than those of SHP2-NC group,while the protein expression levels of p53,Caspase-3 and Bax were higher than those of SHP2-NC group(P＜0.05).Compared with the SHP2-NC group,the number of apoptosis of intima cells in the SHP2-shRNA group was increased,the cell proliferation activity was decreased,and the number of endothelial cell angiogenesis was decreased(P＜0.05).The protein expression levels of p-PI3K,NOX4,COX2 and VEGF after SHP2-shRNA intervention were decreased compared with SHP2-NC intervention(P＜0.05),while the expression levels of PTEN and p53 were increased(P＜0.05).After LY294002 intervention,the expression levels of p-PI3K,NOX4,COX2 and VEGF were decreased(P＜0.05),while the expression levels of PTEN and p53 were increased,as compared with those before intervention(P＜0.05).However,after 740 Y-P intervention,the expression levels of p-PI3K,NOX4,COX2 and VEGF were increased(P＜0.05),while the expression levels of PTEN and p53 were decreased,as compared with those before intervention(P＜0.05).Conclusion SHP2 enhances oxidative stress and endothelial angio-genesis by promoting PI3K/PTEN pathway activity,while inhibiting endometrial cell apoptosis.
基金项目
2021年度江苏省妇幼健康科研项目(F202141)
维普
万方数据