黄芪对低氧环境下血管内皮细胞生长因子/基质细胞衍生因子-1诱导人骨髓间充质干细胞分化、迁移的干预作用的研究

【作者】 沈凌；

【导师】 杨博华；

【作者基本信息】 北京中医药大学 ， 中医外科学， 2010， 博士

【摘要】 目的自体骨髓干细胞移植是将外周血或骨髓中的干细胞移植到缺血的肢体肌肉或闭塞的血管中,使其分化、形成新生毛细血管,改善甚至恢复下肢血流以达到治疗下肢缺血的目的。如何在下肢缺血缺氧的环境中,提高干细胞向VECs分化和迁移能力,成为研究的重点。目前以黄芪为代表的益气活血中药在中西医结合治疗下肢PAOD方面,发挥了重要作用。本研究模拟人体内低氧环境,研究黄芪和VEGF、SDF-1重要细胞因子对hBMSCs向VECs分化及迁移能力的影响,探讨中药干预的主要靶点、作用时机等关键因素,为临床运用提供科学依据。方法1黄芪对低氧环境下VEGF诱导hBMSCs分化的干预作用的研究随机将hBMSCs分为七组,分别为VEGF+常氧组、黄芪+常氧组、VEGF+黄芪+常氧组、低氧对照组、VEGF+低氧组、黄芪+低氧组、VEGF+黄芪+低氧组。将常氧三组置于普通C02细胞培养箱(21%02)中；低氧四组置于三气培养箱(5%O2)中培养。培养7天后,免疫细胞化学法检测CD34、CD31、vWF、VEGFR-2. HIF-la标记物。2 AMI对低氧条件下SDF-1诱导hBMSCs迁移的干预作用的研究随机分为7组,分别为SDF+常氧组、黄芪+常氧组、SDF+黄芪+常氧组、低氧对照组、SDF+低氧组、黄芪+低氧组、SDF+黄芪+低氧组共七组。将hBMSCs细胞悬液放入Transwell系统的上室,将含不同药物的细胞培养液加入到Transwell系统的下室中。将常氧三组Transwell系统置于普通CO2细胞培养箱(21%02)中；低氧四组Transwell系统置于三气培养箱(5%O2)中培养。培养48h后,取上室,酒精棉球擦掉上室膜上未迁移的细胞,95%酒精固定10min,苏木素染色5min,随机选择3个连续显微镜视野(×200)计数迁移到上室膜下的细胞,取平均数。结果1黄芪对低氧条件下VEGF诱导hBMSCs分化的干预作用的研究(1)CD34阳性率(%)1)在常氧培养三组中,VEGF+黄芪+常氧组与其他两组相比较,显著升高(p<0.01,p<0.05)。2)在低氧培养四组中,VEGF+黄芪+低氧组与其他三组相比较,显著升高(p<0.01,p<0.05)；低氧对照组与其他三组相比较,显著降低(p<0.01)。3)常氧和低氧相对应组间比较,低氧各组显著升高(p<0.01)。(2)CD31阳性率(%)1)在常氧培养三组中,VEGF+黄芪+常氧组与其他两组相比较,显著升高(p<0.01)。2)在低氧培养四组中,VEGF+黄芪+低氧组组与其他三组相比较,显著升高(p<0.01)；低氧对照组组与其他三组相比较,显著降低(p<0.01,p<0.05)。3)常氧和低氧相对应组间比较,低氧各组显著升高(p<0.01,p<0.05)(3)vWF阳性率(%)1)在常氧培养三组中,VEGF+黄芪+常氧组与其他两组相比较,显著升高(p<0.01)。2)在低氧培养四组中,VEGF+黄芪+低氧组与其他三组相比较,显著升高(p<0.01)；低氧对照组组与其他三组相比较,显著降低(p<0.01)。3)常氧和低氧相对应组间比较,低氧各组显著升高(p<0.01)。(4) VEGFR-2阳性率(%)1)在常氧培养三组中,VEGF+黄芪+常氧组与其他两组相比较,显著升高(p<0.01)。2)在低氧培养四组中,VEGF+黄芪+低氧组与其他三组相比较,显著升高(p<0.01)；低氧对照组与其他三组相比较,显著升高(p<0.01)。3)与VEGF+常氧组相比较,VEGF+低氧组显著升高(p<0.05)；与黄芪+常氧组相比较,黄芪+低氧组显著升高(p<0.01)；VEGF+黄芪+常氧组与VEGF+黄芪+低氧组组间无统计学意义(p>0.05)。(5)HIF-1α阳性率(%)1)在常氧培养三组中,各组间无统计学意义(p>0.05)。2)在低氧培养四组中,各组间无统计学意义(p>0.05)。3)常氧和低氧相对应组间比较,低氧各组显著升高(p<0.01)。2黄芪对低氧条件下SDF-1诱导hBMSCs迁移的干预作用的研究(1)在常氧培养三组中,SDF+黄芪+常氧组与其他两组相比较,显著升高(p<0.01,p<0.05)。(2)在低氧培养四组中,SDF+黄芪+低氧组与其他三组相比较,显著升高(p<.01)；与SDF+低氧组相比较,低氧对照组显著升高(p<0.01)；低氧对照组与黄芪+低氧组无统计学意义(p>0.05)。(3)与SDF+黄芪+常氧组相比较,SDF+黄芪+低氧组组间显著升高(p<0.05)；SDF+常氧组与SDF+低氧组、黄芪+常氧组与黄芪+低氧组组间无统计学意义(p>0.05)。结论1低氧环境下黄芪可能对BMSCs向VECs分化有促进作用,可能与对HIF-1α和VEGF的影响有关。2在常氧和低氧环境中黄芪与SDF-1联合使用可促进BMSCs迁移：而在低氧环境中,单独应用黄芪没有明显的促进作用。更多还原

【Abstract】 ObjectiveTo improve or even recover blood flow through transplanting BMSCs into the ischemic appendicular muscle or occlusive vessel contributing to the formation of new blood vessels is the purpose of Autologous Bone Marrow Stem Cell Transplantation. The current focus of the study on stem cell is how to improve the migration and differentiation capabilities. TCM drugs based on replenishing qi and activating blood represented by Astragalus have played an important role in treatment of lower extremity PAOD with therapies of Integrated Traditional and Western medicine. We study the effect on the capabilities of migrating to the lesion sites and differentiating into vascular endothelial cells influenced by Astragalus, VEGF and SDF-1 by simulating hypoxic environment of human body, to explore the key factors, such as the main target and timing of the intervention of TCM drugs for providing scientific data for clinical use.Methods1 The study on the intervention of Astragalus on the role of VEGF which induced hBMSCs to differentiate into vascular endothelial cells in hypoxic environmentHBMSCs were randomly divided into 7 groups:VEGF+Normoxia group, Astragalus+ Normoxia group, VEGF+Astragalus+Normoxic group, Hypoxic control group, VEGF+Hypoxia group, Astragalus+Hypoxia group, Astragalus+VEGF+Hypoxia group. The three normxic groups were placed in ordinary cell culture incubator (21%O2); The four hypoxic groups in the special cell culture incubator (5%O2). Cultured for 7 days, cells were evaluated with CD34, CD31, vWF, VEGFR-2, HIF-la markers by immunocytochemistry.2 The study on the intervention of Astragalus on the role of SDF-1 which induced hBMSCs to migrate to the lesion sites in hypoxic environmentHBMSCs were randomly divided into 7 groups:SDF+Normoxia group, Astragalus+ Normoxia group, SDF+Astragalus+Normoxia group, Hypoxic control group, SDF+Hypoxia group, Astragalus+Hypoxia group, SDF+Astragalus+Hypoxia group. hBMSC cell suspensions were placed into the upper chamber of the Transwell system, cell culture medium with different drugs into the lower chamber.The three normxic groups were placed in ordinary cell culture incubator (21% O2); The four hypoxic groups in the special cell culture incubator (5% O2). After cultured for 48h, we removed the upper chamber of the Transwell system, wiped off cells on the membrane of the upper chamber that did not migrate, immersed the membrane with 95% alcohol in 10min, stained cells with hematoxylin in 5min, at last randomly selected three consecutive microscope visual field (×200) to count cells below the membrane, taking the average.Results1 The study on the intervention of Astragalus on the role of VEGF which induced hBMSCs to differentiate into vascular endothelial cells in hypoxic environment(1) the positive rate of CD34 (%)1) Among the three normoxic groups, the positive rate of CD34 in VEGF+Astragalus+ Normoxia group was significantly higher than that of others (p<0.01, p<0.05).2) Among the four hypoxic groups, the positive rate of CD34 in VEGF+Astragalus +Hypoxia group was significantly higher than that of others (p<0.01,p＜0.05); The positive rate of CD34 in Hypoxic control group was significantly lower than that of others (p<0.01).3) The positive rate of CD34 in each hypoxic group was significantly higher than that of the corresponding normoxic group (p<0.01). (2)The positive rate of CD31(%)1)Among the three normoxic groups, compared with the other two groups, the positive rate of CD31 in VEGF+Astragalus+normxia group was significantly higher than that of others (p <0.01).2)Among the four hypoxic groups, the positive rate of CD31 in VEGF+Astragalus+hypoxia group was significantly higher than that of others (p<0.01); The positive rate of CD31 in hypoxic control group was significantly lower than that of others (p<0.01, p<0.05).3) The positive rate of CD31 in each hypoxic group was significantly higher than that of the corresponding normoxic group (p<0.01,p＜0.05).(3) The positive rate of vWF (%)1)Among the three normoxic groups, the positive rate of vWF in VEGF+astragalus+ normoxia group was significantly higher than that of others(p<0.01).2)Among the four hypoxic groups, the positive rate of vWF in VEGF+astragalus+hypoxia group was significantly higher than that of others(p<0.01); The positive rate of vWF in hypoxic control groups was significantly lower than that of others(p<0.01).3) Each hypoxia group was significantly higher than that of the corresponding normoxic group (p<0.01).(4)The positive rate of VEGFR-2(%)1)Among the three normoxic groups, the positive rate of VEGFR-2 in VEGF+Astragalus+ normoxia group was significantly higher than that of others (p<0.01).2) Among the hypoxia groups, the positive rate of VEGFR-2 in VEGF+Astragalus+Hypoxia group was significantly higher than that of others(p<0.01); the positive rate of VEGFR-2 in hypoxic control group was significantly higher than that of others(p<0.01).3)The positive rate of VEGFR-2 in VEGF+Hypoxia group was significantly higher than that ofVEGF+Normoxia group(p<0.05); The positive rate of VEGFR-2 in Astragalus+Hypoxia group was significantly higher than that of Astragalus+Normoxia group (p<0.01); The positive rate of VEGFR-2 in VEGF+Astragalus+Hypoxia group was no significantly highe than that of VEGF+Astragalus+Normoxia group(p>0.05).(5)The positive rate of HIF-1α(%)1)Among the three normoxic groups, the positive rate of HIF-1αin each group was not significantly higher than that of others(p>0.05).2)Among the four hypoxic groups, the positive rate of HIF-1αin each group was not significantly higher than that of others (p>0.05).3) The positive rate of HIF-1αin each hypoxic group increased significantly than that of the corresponding normoxic groups(p<0.01).2 The study on the intervention of Astragalus on the role of SDF-1 which induced hBMSCs to migrate to the lesion sites in hypoxic environment(1)Among the three normoxic groups, the ability of migration of hBMSCs in SDF+Astragalus+Normoxia group was significantly higher than that of others (p<0.01, p<0.05).(2)Among the four hypoxic groups, the ability of migration of hBMSCs in SDF+Astragalus+hypoxia group was significantly higher than that of others (p<0.01); the ability of migration of hBMSCs in SDF+Hypoxia group was significantly higher than that in hypoxic control group (p＜0.01); the ability of migration of hBMSCs in hypoxia+Astragalus group was not significantly higher than that of Hypoxic control group(p> 0.05).(3)The ability of migration of hBMSCs in SDF+Astragalus+Hypoxia group was significantly higher than that in SDF+Astragalus+Normoxia group(p<0.05); The ability of migration of hBMSCs in SDF+Normoxia group was not significantly higher than that in SDF+ Hypoxia group (p<0.05); The ability of migration of hBMSCs in Astragalus+Normoxia group was not significantly higher than that in Astragalus+Hypoxia group (p<0.05);Conclusions1 Astragalus may promote the ability of differentiating into the VECs of BMSCs in hypoxic environment, which may be related to HIF-1 a and VEGF.2 In normoxic and hypoxic environment,Astragalus combined with SDF-1 can promote the migration of BMSCs.In the hypoxic environment, only Astragalus can not promote the migration of BMSCs.更多还原