三七总皂甙通过细胞外信号调节蛋白激酶1/2信号通路促进大鼠骨髓基质细胞向神经样细胞的增殖与分化

Effects of panax notoginseng saponins on proliferation and differentiation of bone marrow stromal cells into neuron-like cells mediated by extracellular signal-regulated kinase 1/2 signal transduction pathway

背景：三七总皂甙对骨髓基质细胞向神经样细胞分化的效应及相关信号通路目前尚不明确。目的：探讨细胞外信号调节蛋白激酶1，2信号通路对三七总皂甙调节大鼠骨髓基质细胞向神经细胞增殖、分化的作用。设计、时间及地点：细胞学体外观察，于2008-05／10在汕头大学医学院分子生物学实验室完成。材料：4-6周龄雄性SD大鼠9只，由汕头大学医学院实验动物中心提供。三七总皂甙为，一两梧州制药集团股份有限公司产品，细胞外信号调节蛋白激酶的抑制剂PD98059为CellSignalingTech公司产品。方法：全骨髓法体外分离纯化大鼠骨麓基质细胞，取传至第3代细胞进行诱导分化，设立3组：单纯诱导组向培养液中加入10ug／L碱性成纤维细胞生长因子，预诱导24h后全量换为正式诱导液（含10P-g／L碱性成纤维细胞生长因子、2％DMSO、200μmol／L丁羟回醚的α-MEM培养基），每24h半量换诱导液1次；三七总皂甙组向预诱导液和正式诱导液内加入终浓度100mg／L三七总皂甙；PD98059组在三七总皂甙组培养液基础上加入10μmol／LPD98059。主要观察指标：细胞形态学观察，MTT法检测细胞增殖情况，免疫组织化学方法鉴定细胞Nestin的表达。结果：诱导6h后，少数细胞呈锥形，细胞突起增多伸长，类似神经元，继续诱导培养后细胞突起增多，突起相互连接成网状。与单纯诱导组、PD98059组比较，诱导6，12，24h三七总皂甙组细胞均明显增殖（P〈005），且随诱导时间延长呈递增趋势（P〈0．05）：单纯诱导组、PD98059组间比较无明显差异（P〉0．05）。与单纯诱导组比较，诱导24h后三七总皂甙组Nestin阳性率明显升高（P〈0．05），PD98059组Nestin阳性率明显降低（P〈0．05）。结论：三七总皂甙可以促进骨髓基质细胞的神经分化和分化后增殖，细胞外信号调节蛋白激酶1／2的特异性抑制剂PD98059能够拈抗三七总皂甙促增殖分化作用，提示细胞外信号调节蛋白激酶1，2信号通路是三七总皂甙促进骨髓基质细胞向神经细胞分化和增殖的重要环节。

BACKGROUND： The mechanisms and relevant signal pathway of panax notoginseng saponins （PNS） on the differentiation of bone marrow stromal cells （BMSCs） into neuron-like ceils were not clear. OBJECTIVE： To study the effects of extracellular signal-regulated kinase （ERK）1/2 signal transduction pathway on the proliferation and differentiation of rat BMSCs into neuron-like cells. DESIGN, TIME AND SETTING： The cytological in vitro study was performed at the Laboratory of Molecular Biology of the Medical College of Shantou University from May to October 2008. MATERIALS： A total of 9 male Sprague Dawley rats aged 4-6 weeks were supplied by Experimental Animal Center, Medical College of Shantou University. PNS was produced by Guangxi Wuzhou Group Co., Ltd. ERK inhibitor PD98059 was produced by Cell Signaling Tech. METHODS： Rat BMSCs were isolated and purified in vitro by the whole bone marrow method. At the third passage, BMSCs were induced and assigned into 3 groups. BMSCs in the induction group were incubated with medium supplemented with 10 μg/L basic fibroblast growth factor for 24 hours preindcution, and then incubated with formal α-MEM medium, containing 10μg/L basic fibroblast growth factor, 2% dimethyl sulfoxide, 200μ mol/L butyl hydroxy anisol. A half of inductor was changed once every 24 hours. BMSCs in the PNS group were treated with preinductor and formal inductor supplemented with 100 mg/L PNS. BMSCs in the PD98059 group were additionally treated with 10 μ mol/L PD98059 based on the medium in the PNS group. MAIN OUTCOME MEASURES： Morphology was observed. Cell proliferation was measured by MTT assay. Nestin expression was determined by immunohistochemistry. RESULTS： Following 6 hours of induction, a few BMSCs were cone-shaped, with an increased number of long processes, similar to neurons. Cell processes became more over time, and connected each other. Compared with the induction and PD98059 groups, BMSCs in PNS group significantly proliferated at 6, 12 and 24 hours （P 〈 0.05）, and increased with prolonged time （P 〈 0.05）. No significant difference was detected between the induction and PD98059 groups （P 〉 0.05）. Compared with the induction group, Nestin-positive rate was significantly increased in the PNS group at 24 hours （P 〈 0.05）, but Nestin-positive rate was significantly decreased in the PD98059 group （P 〈 0.05）. CONCLUSION： PNS can promote proliferation and differentiation of BMSCs. ERK1/2 specific inhibitor PD98059 can resist PNS effects, which indicated that ERK1/2 is the key signal pathway of the promotion effects of PNS on proliferation and differentiation of BMSCs into neurons.