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

文题释义：PI3K/Akt信号通路：Akt是PI3K/Akt信号转导通路的核心，其分子质量约为60 kD，是原癌基因c-akt表达编码的一种丝氨酸/苏氨酸蛋白激酶。外源性抑制剂LY294002或wortmannin均可负向调节PI3K/Akt信号通路，导致第二信使PIP3逆行转化为PIP2，阻断其调节作用。 低氧：氧体积分数是机体微环境的重要组成部分，直接影响着细胞的成活和生物学行为。在体外培养过程中氧体积分数为1%-5%时即可为脂肪干细胞的增殖和功能提供足够的刺激，同时维持细胞形态和多向分化能力不变，因此可以认为低氧环境可以更好地为干细胞特性的维持提供信号传导。 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%O₂培养箱中培养24 h，然后低氧组转移至体积分数为2%O₂培养箱中进行培养，常氧组继续在体积分数为20%O₂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。 ORCID: 0000-0001-8454-263X(殷令妮) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

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+LY294002 group(P<0.05).The proliferation of adipose-derived stem cells in the hypoxia+LY294002 group was significantly lower than that in the hypoxia group(P<0.05).The results of qRT-PCR and anti-CD31 immunofluorescence staining showed that the expression of endothelial cell gene and specific protein CD31 in the hypoxia group was significantly higher than that in the normoxia and hypoxia+LY294002 groups(P<0.05).The expression of CD31 in the hypoxia+LY294002 group was significantly lower than that in the hypoxia group(P<0.05),but it was significantly higher than that in the normoxia group(P<0.05).These results suggest that the PI3K/Akt pathway plays an important role in hypoxia-induced cell proliferation and vascular endothelial cell differentiation of adipose-derived stem cells.