摘要目的 观察血管生成素-2(Ang2)上调后对血管瘤干细胞(HemSCs)生物学功能的影响.方法 通过酶消法结合免疫磁珠分选技术分离,纯化提取HemSCs;通过慢病毒转染的方法获得Ang2上调稳定表达的HemSCs;酶联免疫吸附试验(ELISA)法检测Ang2上调的HemSCs中分泌型Ang2、血管内皮生长因子(VEGF)表达水平;EDU法、细胞迁移(Transwell)法观察Ang2上调的HemSCs细胞增殖,迁移能力变化;通过蛋白质印迹法(Western blot)检测磷脂酰肌醇3激酶(PI3K)/丝氨酸-苏氨酸蛋白激酶(Akt)通路关键靶蛋白表达变化.结果 Ang2上调的HemSCs细胞增殖能力增强(空白组:117.000±3.786,对照组:126.000±3.464,实验组:179.667±2.603,P＜0.01)、迁移能力增强(空白组:102.667±2.028,对照组:108.000±2.646,实验组:308.667±2.603,P＜0.01);HemSCs Ang2上调组中Ang2(空白组:60.103±0.361,对照组:69.343±0.526,实验组:1962.440±43.205,P＜0.01)、VEGF(空白组:92.667±3.480,对照组:91.000±1.523,实验组:455.000±15.100,P＜0.01)分泌蛋白表达上调;PI3K-Akt信号通路相关蛋白:Akt1(空白组:0.612±0.163,对照组:0.624 ±0.163,实验组:0.907±0.162,P＜0.01)、PI3K(空白组:0.624±0.163,对照组:0.474±0.163,实验组:0.766±0.162,P＜ 0.01)、PI3KR5(空白组:0.505±0.163,对照组:0.405±0.163,实验组:0.890±0.162,P＜0.01)、PAkt(空白组:0.549±0.163,对照组:0.644±0.163,实验组:0.684 ±0.162,P＜0.01)、p38(空白组:0.618 ±0.163,对照组:0.759±0.163,实验组:0.947±0.162,P＜0.01)在HemSCs Ang2上调组中表达增强.结论 Ang2上调可通过PI3K-Akt信号通路增强HemSCs增殖,迁移能力.

abstractsObjective To investigate the effect of Angiopoietin-2 (Ang2) up-regulation on the biological function of hemangioma stem cells (HemSCs).Methods HemSCs was isolated and purified by enzyme digestion and immunomagnetic beads sorting.HemSCs was stably up-regulated by Ang2 by lentiviral transfection.The expression level of secreted Ang2 and vascular endothelial growth factor (VEGF) in HemSCs up-regulated by Ang2 was detected by enzyme linked immunosorbent assay (ELISA) method.EDU method The Trangswell method was used to observe the proliferation and migration ability of HemSCs cells up-regulated by Ang2.The tumor formation of nude mice was observed by single cell suspension.The tumorigenic ability of HemSCs was up-regulated by Ang2.The expression of key target proteins in phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (Akt) cell signaling pathway was detected by Western blotting.Results The proliferation of HemSCs cells up-regulated by Ang2 was enhanced (blank group:117.000 ± 3.786,control group:126.000 ± 3.464,experimental group:179.667 ± 2.603,P ＜ 0.01),and migration ability was enhanced (blank group:102.667 ± 2.028,controlgroup:108.000 ± 2.646,experimental group:308.667 ± 2.603,P ＜ 0.01);Ang2 in HemSCs Ang2 ↑group (blank group:60.103 ± 0.361,control group:69.343 0.526,experimental group:1 962.440 ± 43.205,P ＜ 0.01),VEGF (The blank group:92.667 ± 3.480,the control group:91.000 ± 1.523,the experimental group:455.000± 15.100,P ＜ 0.01) the expression of secreted protein was up-regulated;PI3K-Akt signaling pathway-related protein:Akt1 (blank group:0.612 ± 0.163,control group:0.624 ± 0.163,experiment Group:0.907 ± 0.162,P ＜ 0.01),PI3K (blank group:0.624 ± 0.163,control group:0.474 ± 0.163,experimental group:0.766 ± 0.162,P ＜ 0.01),PI3 KR5 (blank group:0.505 ± 0.163,control group:0.405 ± 0.163,Experimental group:0.890 ± 0.162,P ＜ 0.01),PAkt (blank group:0.549 ± 0.163,control group:0.644 ± 0.163,experimental group:0.684 ± 0.162,P ＜0.01),p38 (blank group:0.618 ±0.163,control Group:0.759 ±0.163,experimental group:0.947 ±0.162,P ＜ 0.01) Enhanced expression in HemSCs Ang2 ↑ group.Conclusion Upregulation of Ang2 can enhanced HemSCs proliferation,migration,in vivo angiogenesis;through PI3 K-Akt signaling pathway Development has an impact.