摘要
目的观察创伤性脑组织匀浆条件下,川芎嗪诱导大鼠骨髓间充质干细胞(BMSCs)向神经元样细胞的分化,并寻找川芎嗪的最佳诱导浓度。方法①Wistar幼鼠麻醉后,取其股骨、胫骨和肱骨,从骨髓中分离BMSCs,接种于培养瓶培养并传代,于倒置显微镜下观察细胞形态。②采用Feeney自由落体撞击法制作中度脑损伤大鼠模型,取伤后24h大鼠创伤脑组织制作匀浆,用含1.00,1.15,1.30,1.45g/L 4种剂量盐酸川芎嗪的达氏修正伊氏培养基(DMEM)对体外培养的第4代BMSCs诱导12h。③倒置显微镜下观察细胞诱导后形态学变化,应用免疫细胞化学染色技术检测细胞内神经元特异性烯醇化酶(NSE)和细胞巢蛋白(Nestin)的表达,并比较4种剂量川芎嗪诱导后各组神经元样细胞抗原的表达率。结果原代细胞接种3d后多数细胞贴壁,传代后细胞贴壁速度和增殖更快,诱导后细胞出现类似神经元细胞样形态;免疫细胞化学方法检测显示多数细胞巢蛋白、NSE阳性表达,1.30g/L浓度组细胞的NSE阳性表达率最高,表达量为(66.4±4.7)%,细胞巢蛋白表达量为(62.23±4.47)%。结论川芎嗪可诱导BMSCs分化为神经元样细胞,在创伤性脑组织匀浆条件下1.30g/L为其最适诱导剂量。
Objective To investigate the effects that ligustrazine induces rat bone marrow mesenchymal stem cells （BMSCs） differentiate into neuron-like cells under the traumatic brain tissue extracts environment ,and to search for the optimal inductive concentration .Methods ①After anaesthetized ,to separate the femoral ,tibia and humerus of the rat .The rat BMSCs were isolated and cultured by the method of full marrow culture method . After expansion ,it was observed under an inverted phase microscope .②Rat models of moderate brain injury were established by Feeney′s free fallers hit method .The traumatic brain tissue extracts which acquired on the point a-bout 24 hours after the injury were used to form an induced environment .DMEM medium contains 1.00 ,1.15 , 1.30 and 1.45 g/L of ligustrazine concentrations were used to induce the 4th passage of BMSCs in vitro .③ The changes of BMSCs were observed by the inverted phase microscope .The expression of neuron-specific enolase （NSE） and nestin was identified by immunocytochemical technique ,and the expression ratio of neuron-like cells′surface antigens induced by different concentrations of ligustrazine were compared .Results ①Most primarily cul-tured BMSCs adhered to the wall at 3 days after culture ,which proliferated faster after passaged .Neuron-like cells with prominence and bifurcation could be seen after induction .The immunocytochemical method showed that nestin and NSE were positively expressed in most induced cells ,especially received a highest ration of NSE ex-pressing in the induced group with 1 .30 g/L concentration of ligustrazine . The immunocytochemical method showed in the 12 hours that the ratio of NES expressing was （66.4 ± 4.7 ）% and the expressing of nestin was （62.23 ± 4.47）% .Conclusion It reveals that BMSCs can be induced differentiating into neuron-like cells by the ligustrazine .Under the traumatic brain tissue extracts environment ,1.30 g/L ligustrazine concentrations is the optimal inductive concentration .
出处
《山西医药杂志（上半月）》
CAS
2013年第11期1224-1227,共4页
Shanxi Medical Journal
关键词
骨髓
间质干细胞
神经元
磷酸丙酮酸水合酶
巢蛋白
川芎嗪
Bone marrow
Mesenchymal stem cells
Neurons
Phosphopyruvate hydratase
Nestin Ligustrazine
