共培养下脂肪源性干细胞对内皮祖细胞活性的影响及其相关机制  

The effect of ADSC on the activity of EPC under co-culture and its related mechanism

作　　者：曹乐 丁振飞 孙凯 范海涛 杨海涛 Cao Le;Ding Zhenfei;Sun Kai;Fan Haitao;Yang Haitao(Dept of Orthopaedic,Fuyang Hospital Affiliated to Anhui Medical University,Fuyang 236000;Anhui Medical University Graduate School,Hefei 230032)

机构地区：[1]安徽医科大学附属阜阳医院骨科,阜阳236000 [2]安徽医科大学研究生学院,合肥230032

出　　处：《安徽医科大学学报》2023年第4期547-553,共7页Acta Universitatis Medicinalis Anhui

基　　金：安徽省高校自然科学研究项目(编号:KJ2019A0261);安徽医科大学校科研基金(编号:2020xkj059、2020xkj225)。

摘　　要：目的 探讨脂肪源性干细胞(ADSC)与内皮祖细胞(EPC)共培养对EPC活性的影响及ADSC对EPC增殖、迁移、分化及成血管活性产生影响的机制。方法 体外分离、培养、扩增并鉴定大鼠来源的ADSC与EPC。实验分为EPC组、EPC+ADSC共培养组、EPC+ADSC+PI3K-inhibitor组,三组细胞使用Transwell共培养处理48 h后,分别通过CCK-8实验、划痕实验和血管形成实验评估ADSC与EPC共培养和PI3K/AKT通路对EPC活性的影响;通过Western blot检测EPC中血管内皮生长因子A(VEGFA)、内皮型一氧化氮合酶(eNOS)、血管内皮细胞钙粘连蛋白(VE-cadherin)、CD133、磷酸化磷脂酰肌醇3-激酶(p-PI3K)和磷酸化蛋白激酶B(p-AKT)的表达水平来探究ADSC与EPC共培养和PI3K/AKT通路对EPC向成熟内皮细胞分化能力的影响。结果 CCK-8检测结果显示,EPC+ADSC共培养组中EPC在不同时间点吸光度值均高于EPC组和EPC+ADSC+PI3K-inhibitor组,差异有统计学意义(P<0.01);划痕实验结果显示,EPC+ADSC共培养组24 h后划痕相对距离小于EPC组和EPC+ADSC+PI3K-inhibitor组,差异有统计学意义(P<0.01);血管形成实验结果显示,EPC+ADSC共培养组24 h形成管腔样结构平均数量高于EPC组和EPC+ADSC+PI3K-inhibitor组,差异有统计学意义(P<0.01);Western blot检测显示,EPC+ADSC共培养组中EPC的VEGFA、eNOS、VE-cadherin、p-PI3K和p-AKT表达水平高于EPC组和EPC+ADSC+PI3K-inhibitor组,而CD133表达水平低于EPC组和EPC+ADSC+PI3K-inhibitor组,差异有统计学意义(P<0.01)。结论 ADSC与EPC共培养能够提高EPC增殖、迁移、分化和成血管等活性,其机制可能是通过调控PI3K/AKT通路实现的。Objective To investigate the effect of co-culture of adipose-derived stem cells(ADSC)and endothelial progenitor cells(EPC)on the activity of EPC and its related mechanism.Methods Rat ADSC and EPC were isolated,cultured,expanded and identified in vitro.The experiment was divided into three groups:EPC group,EPC+ADSC co-culture group,and EPC+ADSC+PI3K-inhibitor group.After 48 hours of co-culture,the cells of the three groups were treated with Transwell.The effects of ADSC and EPC co-culture and PI3K/AKT pathway on EPC activity were evaluated by CCK-8 assay,scratch assay and angiogenesis assay,respectively.Western blot was used to detect vascular endothelial growth factor A(VEGFA)and endothelial nitric oxide synthase(eNOS),vascular endothelial-cadherin(VE-cadherin),CD133,phospho-phosphatidylinositol 3-kinase(phospho-phosphatidylinositol 3-kinase(p-PI3K)and phospho-protein Kinase B(p-AKT)expression levels in EPC to detect the effects of ADSC and EPC co-culture and PI3K/AKT pathway on the differentiation ability of EPC into mature endothelial cells.Results CCK-8 results showed that the absorbance at 450 nm of EPC in EPC+ADSC co-culture group was higher than that in EPC group and EPC+ADSC+PI3K-inhibitor group at different time points,and the difference was statistically significant(P<0.01).The scratch test showed that the relative scratch distance of EPC+ADSC co-culture group was smaller than that of EPC group and EPC+ADSC+PI3K-inhibitor group after 24 hours,and the difference was statistically significant(P<0.01).Tube formation assay showed that the average number of tube-like structures formed in EPC+ADSC co-culture group was higher than that in EPC group and EPC+ADSC+PI3K-inhibitor group,and the difference was statistically significant(P<0.01).Western blot showed that the expression levels of VEGFA,eNOS,VE-cadherin,p-PI3K and p-AKT of EPC in EPC+ADSC co-culture group were higher than those in EPC group and EPC+ADSC+PI3K-inhibitor group.The expression level of CD133 in EPC group was lower than that in EPC+ADSC+PI3K-in

关 键 词：脂肪源性干细胞 内皮祖细胞 PI3K/AKT 

分 类 号：R681.8[医药卫生—骨科学]