PI3K/Akt通路在低氧诱导脂肪干细胞增殖和向内皮细胞分化中的作用  

The role of PI3K/Akt pathway in hypoxia-induced cell proliferation and endothelial cell differentiation of adipose-derived stem cells

作　　者：殷令妮 陈德宣 Yin Lingni;Chen Dexuan(Department of Geriatrics,Jiading Central Hospital,Shanghai University of Medicine&Health Sciences,Shanghai 201800,China)

机构地区：[1]上海健康医学院附属嘉定区中心医院老年病科

出　　处：《中国组织工程研究》2020年第19期3004-3009,共6页Chinese Journal of Tissue Engineering Research

摘　　要：背景:大量研究证明低氧可以促进干细胞的增殖和分化,但目前尚不清楚其通过何种途径发挥作用。目的:观察低氧对脂肪干细胞增殖和向内皮细胞分化的影响,并探讨PI3K/Akt通路在此过程中的作用。方法:取培养至第3代的Wistar大鼠脂肪干细胞,分为常氧组、低氧组、低氧+LY294002组,3组均在体积分数为20%O 2培养箱中培养24 h,然后低氧组转移至体积分数为2%O 2培养箱中进行培养,常氧组继续在体积分数为20%O 2培养箱中培养,低氧+LY294002组在低氧培养环境下的细胞培养基中加入终浓度为25 mmol/L的PI3K/Akt通路抑制剂LY294002。培养24 h后,Western blot检测p-Akt的表达明确PI3K/Akt通路的激活情况;CCK-8法检测各组脂肪干细胞的增殖情况;各组脂肪干细胞加入血管内皮细胞诱导培养基进行诱导分化10 d,分别通过qRT-PCR及抗CD31免疫荧光染色检测各组脂肪干细胞分化为内皮细胞的情况。结果与结论:①第3代脂肪干细胞呈现出典型的梭形结构,流式细胞仪检测结果显示CD34和CD45表达阴性,CD105表达阳性,并可向成骨及成脂细胞方向分化;②低氧培养条件下p-Akt的表达明显升高,加入LY294002后p-Akt的表达受到明显抑制;③低氧组细胞增殖率明显高于常氧组和低氧+LY294002组(P<0.05),低氧+LY294002组脂肪干细胞的增殖率低于低氧组(P<0.05);④低氧组内皮细胞基因及特异性蛋白CD31的表达明显高于常氧组和低氧+LY294002组(P<0.05),低氧+LY294002组内皮细胞基因及特异性蛋白CD31的表达低于低氧组(P<0.05),但仍高于常氧组(P<0.05);⑤结果证实PI3K/Akt通路在低氧诱导的脂肪干细胞增殖及向内皮细胞分化过程中发挥重要作用。BACKGROUND:A large number of studies have confirmed that hypoxia can promote the proliferation and differentiation of stem cells,but the pathway by which it plays its role remains unclear.OBJECTIVE:To investigate the effects of hypoxia on the proliferation of adipose-derived stem cells and their differentiation into endothelial cells and investigate the role of PI3K/Akt pathway in this process.METHODS:Passage 3 adipose-derived stem cells of Wistar rats were divided into three groups according to the different culture conditions:normoxia group,hypoxia group,and hypoxia+LY294002 group.Three groups of cells were cultured for 24 hours in an incubator containing 20%O2.Cells in the hypoxia group were cultured in an incubator containing 2%O2,and those in the normoxia group were still cultured in an incubator containing 2%O2.Cells in the hypoxia+LY294002 group were cultured in medium supplemented with 25 mmol/L PI3K/Akt pathway inhibitor LY294002 under the hypoxic culture conduction.After 24 hours of culture,p-Akt expression was detected by Western blot assay to indicate the activation of PI3K/Akt pathway.The proliferation of adipose-derived stem cells was measured by CCK-8 method.Three groups of adipose-derived stem cells were induced to differentiate into vascular endothelial cells for 10 days.The differentiation of adipose-derived stem cells into endothelial cells was detected by qRT-PCR and anti-CD31 immunofluorescence staining.RESULTS AND CONCLUSION:Adipose-derived stem cells at passage 3 exhibited elongated fibroblast-like morphology.Cytometry analysis showed most of adipose-derived stem cells at passage 3 were negative for CD 34 and CD45 and positive for CD105 and could be induced towards osteogenic and adipogenic differentiation.The expression of p-Akt was significantly increased after hypoxic culture,which was inhibited obviously after adding LY294002.CCK-8 showed that the proliferation of adipose-derived stem cells in the hypoxia group was significantly higher than that in the normoxia group and hypoxia+LY29400

关 键 词：脂肪干细胞 低氧 细胞增殖 细胞分化 PI3K/AKT通路 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]