黄芪和三七对下肢缺血患者骨髓间充质干细胞体外分化的影响

背景：目前临床观察已经证实中药黄芪、三七能够改善下肢缺血症状，假设这种治疗作用与其促进骨髓间充质干细胞向血管内皮细胞分化有关。目的：验证黄芪、三七是否能够有效促进骨髓间充质干细胞增殖并向血管内皮细胞分化。设计、时间及地点：骨髓间充质干细胞的体外分化实验，于2005—04／06在北京中医药大学附属东直门医院教育部重点实验室完成。材料：抽取北京中医药大学东商门医院收治的下肢血管缺血性病变患者23例骨髓血，密度梯度法分离骨髓单个核细胞，即骨髓间充质干细胞。相当于生药材2g／mL的黄芪注射液为成都地奥九泓制药厂产品，主要成分为三七总皂苷的血塞通粉针剂黑龙江珍宝岛制药有限公司产品，CD34抗体为美国BD公司产品，FACS Calibur流式细胞仪为美国BD公司产品。方法：将骨髓间充质干细胞与3mL低、中、高剂量含生药4，12，36g/L的黄芪注射液，3mL低、中、高剂量含三七总皂苷50，100，200mg／L血塞通混悬液进行共培养，并设未加入药物的单纯培养干细胞为空白对照组；隔日换液一次，连续培养3周。主要观察指标：培养3周后，倒置相差显微镜下观察细胞形态；流式细胞仪检测CD34^＋细胞百分比。结果：①骨髓间充质干细胞培养3周后贴壁细胞大部分呈梭形，束状排列，具有内皮细胞形态和特性。②与空白对照组比较，黄芪低、中剂量组和三七中、高剂量组CD34^＋细胞百分比显著增加（P〈0．05～0．01）。结论：黄芪、三七有促进骨髓间充质干细胞增殖并向血管内皮细胞分化的作用，此作用呈剂量依赖性。

Effects of astragalus mongholicus and radix notoginseng on in vitro differentiation of bone marrow mesenchymal stem cells in patients with lower limb ischemia

BACKGROUND: It has been confirmed that Chinese medicine astragalus mongholicus and radix notoginseng can improve symptoms of lower limb ischemia. It is supposed that this kind of therapy is associated with the promotion effect of astragalus mongholicus and radix notoginseng on differentiation of bone marrow mesenchymal stem cells (BMSCs) into vascular endothelial cells.OBJECTIVE: To identify that whether astragalus mongholicus and radix notoginseng are effective in promoting the proliferation and differentiation of BMSCs into vascular endothelial cells. DESIGN, TIME AND SETTING: The in vitro differentiation experiment was performed at the Key Laboratory of Education Ministry, Dong Zhimen Hospital Affiliated to Beijing University of Chinese Medicine from April to June 2005. MATERIALS: Bone marrow blood from 23 patients with lower limb ischemia was collected routinely at the Dong Zhimen Hospital Affiliated to Beijing University of Chinese Medicine. The density gradient centrifugation was used to separate bone marrow mononuclear cells, I.e. BMSCs. Astragalus mongholicus injection (equal'to 2 g/mL crude drug, Diaojiuhong, China), Xuesaitongfen injection (main component radix notoginseng, Zhenbaodao, China), CD34 antibody (BD, USA) and FACS Calibur flow cytometer (BD, USA) were used in this study. METHODS: BMSCs were incubated with 3 mL low, middle and high doses of astragalus mongholieus (containing 4, 12 and 36 g/L crude drug), and 3 mL low, middle and high doses of Xuesaitong suspension (50, 100 and 200 mg/L radix notoginseng). BMSCs only cultured without drugs served as a blank control group. Medium was changed every other day, for successively 3 weeks.MAIN OUTCOME MEASURES: Three weeks later, cell morphology was observed under an inverted phase contrast microscope. CD34+ cell percentage was detected by flow cytometry.RESULTS: Three weeks after BMSC culture, most cells were spindle-shaped, fascicular, with shape and characteristics of endothelial cells. Compared with the blank control group, the percentage of CD34+ cells were significandy increased in the low and middle doses of astragalus mongholicus group and in the middle and high doses of radix notoginseng group (P < 0.05-0.01). CONCLUSION: Astragalus mongholicus and radix notoginseng have the function of promoting proliferation and differentiation of BMSCs into vascular endothelial cells in a dose dependent effect.