丹参诱导骨髓间充质干细胞向神经元样细胞分化及相关基因的表达

背景：骨髓间充质干细胞在适当的诱导条件下，可在体内外分化为神经细胞与神经前体细胞，但多数研究多以含血清及细胞因子的培养基为诱导剂。目的：以丹参作为诱导剂，观察骨髓间充质干细胞向神经元样细胞分化前后相关基因的表达，拟寻找新的诱导方法。设计：以细胞为观察对象的重复观察测量，对照性体外实验。单位：四川大学基础与法医学院人体解剖教研室。材料：实验于2004-10／2005-12在四川大学基础与法医学院人体解剖教研室完成。为四川省重点实验室。SD大鼠，体质量150～200g，由本校动物中心提供，实验过程中对动物处置符合动物伦理学标准。丹参注射液由安徽天洋药业有限公司提供，批号20050411。方法：采用密度梯度离心和细胞贴壁法培养骨髓间充质干细胞，取第5代细胞进行诱导。用丹参注射液诱导骨髓间充质干细胞向神经元样细胞分化，对照组以不含丹参注射液的无血清培养液进行培养。采用RT-PCR检测诱导前后细胞ngn-1，mash—1的表达，免疫组织化学检测诱导前后的骨髓间充质干细胞NSE和GFAP的表达。主要观察指标：①RT-PCR检测细胞诱导前后ngn-1，mash-1的表达。②免疫组织化学检测诱导前后的骨髓间充质干细胞NSE和GFAP的表达。结果：①骨髓间充质干细胞贴壁生长情况良好，大部分细胞贴壁呈长梭形。加入诱导剂后，细胞体积缩小，部分细胞有突起，形状类似神经元。②RT-PCR反应检测显示，未经诱导的骨髓间充质干细胞ngn-1，mash-1，mRNA为阴性，诱导后呈阳性表达。③细胞免疫组织化学检测显示，诱导后的细胞NSE和GFAP呈阳性反应，对照组为阴性。结论：丹参注射液能够诱导骨髓间充质干细胞向神经元样细胞分化。

Gene expression in differentiation of rat bone marrow-derived mesenchymal stem cells into neurocyte likt cells induced by salvia mitiorrhiza

BACKGROUND:It has been reported that mesenchymal stromal cells(MSCs)are capable of differentiating into cells of multilineage.Different methods and reagents have been used to induce the differentiation of MSCs,but most inducing systems contain serum and cytokines.OBJECTIVE:To investigate the gene expression of mash-1 and ngn-1 in differentiation of SD rat bone marrow-derived mesenchymal stem cells induced by salvia mitiorrhiza. DESIGN:Controlled experiment in vitro with repeated observation and measurement based on cells.SETTING:Department of Anatomy,School of Preclinical and Forensic Medicine,Sichuan University. MATERIALS:This study was performed in the Department of Anatomy,School of Preclinical and Forensic Medicine,Sichuan University from October 2004 to December 2005.SD male rats weighing 160-200 g were purchased from the Animal Center of Sichuan University.The experimental animals were disposed according to ethical criteria.Parenteral solution of salvia mitiorrhiza purchased from Tianyang Medicine Company Limited of Anhui(batch number:20050411).METHODS:The nucleated cells were separated from rat bone marrow through gradient centrifugation and cell adherent method,and then MSCs differentiated into neurcyte-like cells induced by salvia mitiorrhiza.Cells in the control group were cultured with salvia mitiorrhiza-free serum-free culture media.The expression of neuron specific enolase(NSE)and glial fibrillary acidic protein(GFAP)were detected by immunohistochemistry.RT-PCR was used to detect the mRNA of mash-1 and ngn-1. MAIN OUTCOME MEASURES:①mash-1 and ngn-1 expressions were detect by the RT-PCR method.②NSE and GFAP expressions were detected by immunohistochemistry.RESULTS:①The MSCs were well adherent to walls.Most of the cells transformed in dipolar-like or multipolar-like.Axon-like or dentrite-like process was developed and these processes synapse with each other.② RT-PCR showed that ngn-1 and mash-1 mRNA were negative before induction,but positive after induction.③Immunohistochemistry indicated that NSE and GFAP expressions were positive after induction but negative in the control group. CONCLUSION:MSCs can be induced to differentiate into neurocyte-like cells by salvia mitiorrhiza.