| 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. |
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