Yiqihuoxue recipe induces differentiation of rat bone marrow mesenchymal stem cells towards neurons in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND: Previous studies have demonstrated that Yiqihuoxue recipe (Buyanghuanwu decoction) can inhibit cell apoptosis and antagonize free radical injury in cerebral ischemia/reperfusion models. However, there have been few reports regarding the effects of Yiqihuoxue recipe on stem cell differentiation. OBJECTIVE: To observe the effects of Yiqihuoxue recipe on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) towards neurons. DESIGN, TIME AND SETTING: The present controlled observational expedment based on cells was performed at the Henan University of Traditional Chinese Medicine, i.e., the Third-Grade Laboratory of the State Administration of Traditional Chinese Medicine between March 2006 and February 2008.MATERIALS: Twenty clean Sprague-Dawley (SD) rats, aged 1-2 months, weighing 100-150 g, of either gender, were included in this study. The Chinese medicine compound Yiqihuoxue recipe was made in the Manufacturing Laboratory of the First Affiliated Hospital of Henan College of Traditional Chinese Medicine as follows. 60 g Danggui (Radix Angelicae Sinensis), 30 g Taoren(Semen Persicae), 30 g Chuanxiong(Szechwan Lovage Rhizome) were selected and mixed in 720 mL water for extract volatile oil for later use. The remaining gruffs and extract were supplemented with 1 200 g Huangqi(Radix Astragali), 60 g Chishao (Radix Paeoniae Rubra), 30 g Dilong(Lumbricus), 30 g Honghua(Flos Carthami) before 920 mL water was added. After I hour of decoction and filtering, the gruffs were decocted for another 1 hour after adding 8 640 mL water. Then all decocted liquid was merged and condensed to 600 mL and mixed with 3 mL of volatile oil and tween-80. After high-temperature and high-pressure stedlization, the products were preserved for future use. 1 mL of drug was equal to 2.4 g raw herbs. METHODS: Bone marrow mononuclear cells were isolated by density gradient centrifugation. After culture, bone marrow MSCs were amplified and purified. Passage 10 cell suspension was inoculated into a 40 mm-diameter plastic culture dish. Dulbecco's modified eagle's medium supplemented with 0.1 volume fraction of fetal bovine serum and basic fibroblast growth factors was added for 24 hours of culture when adherent cells reached 60%-90% confluence in each group. Thereafter, the "Yiqihuoxue" group was incubated for 5 hours using medium containing DMSO, butylated hydroxyanisole, 13-mercaptoethanol, and Yiqihuoxue recipe; simultaneously, the control group was treated for 5 hours with fluid containing DMSO, butylated hydroxyanisole, and β- mercaptoethanol.MAIN OUTCOME MEASURES: Identification of bone marrow MSCs by flow cytometry; Detection of Nestin-, and neuron specific enolase(NSE)-, and glial fibrillary acidic protein(GFAP)-positive expression by immunohistochemistry. RESULTS: Flow cytometery results demonstrated that cell surface marker CD90 and CD106 expression was positive, while CD45 and CD34 expression was negative. Immunohistochemistry results showed that the numbers of Nestin-positive cells (1 and 3 hours after induction) and NSE- and GFAP-positive cells (5 hours after induction) were significantly greater in the Yiqihuoxue group than in the control group (P < 0.01). CONCLUSION: Yiqihuoxue recipe can induce the differentiation of bone marrow MSCs towards neurons in vitro.