番茄红素对人内皮祖细胞增殖、迁移、成管能力的影响观察及其机制探讨

Effects of lycopene on proliferation,migration,and tube-forming capacity of human endothelial progenitor cells

目的 观察番茄红素(Lyc)对人内皮祖细胞(EPCs)增殖、迁移、成管能力的影响,并探讨其作用机制。方法 取EPCs,培养24 h后分为4组;A组加入含10%胎牛血清的培养基和ox-LDL(10 mg/L)的培养基培养24 h后弃去上清,再加入含10%胎牛血清和Lyc(10 mg/L)的培养基培养24 h;B组加入含10%胎牛血清和ox-LDL(100 mg/L)的培养基培养48 h;C组加入含10%胎牛血清和Lyc(10 mg/L)的培养基培养48 h;D组加入含10%胎牛血清的培养基培养48 h;比较各组细胞增殖活力、细胞划痕愈合率、形成小管直径及p62、LC3-Ⅱ、Beclin-1、p-AMPK/AMPK、p-mTOR/mTOR蛋白相对表达量。另取EPCs,培养24 h后分为4组;E组首先加入含10%胎牛血清和AMPK抑制剂小分子化合物C(CC)的培养基温箱孵育30 min,然后加入含10%胎牛血清和ox-LDL(10 mg/L)的培养基培养24 h后弃去上清,最后再加入含10%胎牛血清和Lyc(10 mg/L)的培养基培养24 h;F组加入含10%胎牛血清的培养基和ox-LDL(10 mg/L)的培养基培养24 h后弃去上清,再加入含10%胎牛血清和Lyc(10 mg/L)的培养基培养24 h;G组加入含10%胎牛血清和ox-LDL(100 mg/L)的培养基培养48 h;H组加入含10%胎牛血清的培养基培养48 h;比较各组细胞增殖活力、细胞划痕愈合率、形成小管直径及p62、LC3-Ⅱ、Beclin-1蛋白相对表达量。结果 与B组比较,A组细胞增殖活力、细胞划痕愈合率、形成小管直径及LC3-Ⅱ、Beclin-1、p-AMPK/AMPK蛋白相对表达量增加,p62、p-mTOR/mTOR蛋白相对表达量减小(P均<0.05);与B组比较,D组细胞增殖活力、细胞划痕愈合率、形成小管直径及p-AMPK/AMPK蛋白相对表达量增加,p62、LC3-Ⅱ、Beclin-1、p-mTOR/mTOR蛋白相对表达量减小(P均<0.05)。与F组比较,E组和G组细胞增殖活力、细胞划痕愈合率、形成小管直径减小(P均<0.05);与G组比较,E组p62蛋白相对表达量增加,E组LC3-Ⅱ、Beclin-1蛋白相对表达量和H组p62、LC3-Ⅱ、Beclin-1蛋白相对表达量减小(P均<0.05)。结论 Lyc可以促进EPCs增殖、迁移和成管能力并增强自噬,其作用机制可能是上调AMPK磷酸化水平,下调mTOR的磷酸化水平,进而抑制mTOR信号通路。

Objective To investigate the effects of lycopene(Lyc)on the proliferation,migration and tube-formingcapacity of human endothelial progenitor cells(EPCs)and to explore their mechanisms.Methods EPCs were culturedfor 24 h and then were divided into 4 groups. In the group A,the medium containing 10% fetal bovine serum and ox-LDL(10 mg/L)were added;after 24 h of culture,the supernatant was discarded,and the same serum and Lyc(10 mg/L)were added for 24 h of culture. In the group B,EPCs were cultured in the medium containing the same fetal bovine serumand ox-LDL(100 mg/L)for 48 h. EPCs in the group C were cultured with the same serum and Lyc(10 mg/L)for 48 h.EPCs in the group D were cultured in the same serum for 48 h. Cell proliferation activity,scratch healing rate,tubule di-ameter and relative protein expression levels of p62,LC3-Ⅱ,Beclin-1,p-AMPK/AMPK and p-m TOR/m TOR were com-pared among groups. Some other EPCs were cultured for 24 h and then were divided into four groups. In the group E,wefirst added the medium containing 10% fetal bovine serum and AMPK inhibitor compound C(CC)to incubate for 30 minin a warm chamber,and then the same serum and ox-LDL(10 mg/L)was added for 24 h of culture. After the supernatantwas discarded,we finally added the serum and Lyc(10 mg/L)to culture for 24 h. In the group F,we first added the medi-um containing the same serum and ox-LDL(10 mg/L)to culture for 24 h,then the supernatant was discarded,and finallywe added the serum and Lyc(10 mg/L)to culture for 24 h. EPCs in the group G were cultured with the same serum andox-LDL(100 mg/L)for 48 h. EPCs in the group H were cultured with the medium containing 10% fetal bovine serum for48 h. The cell proliferation activity,scratch healing rate,tubule diameter and relative protein expression levels ofp62,LC3-Ⅱ and Beclin-1 were compared among groups.Results Compared with the group B,cell proliferation activi-ty,cell scratch healing rate,tubular diameter and relative expression levels of LC3-Ⅱ,Beclin-1 and p-AMPK/AMPK in-creased in the group A,while the relative expression levels of p62 and p-m TOR/m TOR decreased(all P<0. 05). Com-pared with the group B,the cell proliferation activity,cell scratch healing rate,tubular diameter and p-AMPK/AMPK pro-tein relative expression levels increased in the group D,while the protein relative expression levels of p62,LC3-Ⅱ,Be-clin-1 and p-m TOR/m TOR decreased(all P<0. 05). Compared with the group F,the cell proliferation activity,cellscratch healing rate and tubular diameter decreased in the groups E and G(all P<0. 05). Compared with the group G,therelative expression level of p62 protein increased in the group E,but the relative expression levels of LC3-Ⅱ and Beclin-1protein and the relative expression levels of p62,LC3-Ⅱ and Beclin-1 protein in the group H decreased(all P<0. 05).Conclusion sLyc can promote the proliferation,migration and tube formation of EPCs,and enhance autophagy.The mechanism may be that it up-regulates AMPK phosphorylation and down-regulates m TOR phosphorylation,thereby in-hibiting m TOR signaling pathway.