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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。
BACKGROUND: Bone marrow mesenchymal stem cells can differentiate into neural cells and neural progenitor cells in vitro and in vivo under appropriate inducing conditions. However, in most studies, medium containing serum and cytokines is the inducer. OBJECTIVE: To observe the expression of related genes before and after the differentiation of bone marrow mesenchymal stem cells into neuron-like cells with Salvia miltiorrhiza as an inducer, and to find out a new induction method. Design: Repetitive observation measurements with cells as observational, controlled in vitro experiments. Unit: Department of Human Anatomy, School of Basic and Forensic Medicine, Sichuan University. MATERIALS: Experiments were performed at Department of Human Anatomy, School of Fundamental and Forensic Medicine, Sichuan University from October 2004 to December 2005. For the Sichuan Provincial Key Laboratory. SD rats, body weight 150 ~ 200g, provided by the school animal center, the process of animal handling in line with animal ethical standards. Danshen injection by the Anhui days Yang Pharmaceutical Co., Ltd., batch number 20050411. Methods: Bone marrow mesenchymal stem cells were cultured by density gradient centrifugation and cell adherent method, and the cells of passage 5 were induced. Danshen injection was used to induce bone marrow mesenchymal stem cells to differentiate into neuron-like cells. The control group was cultured in serum-free medium without Salvia miltiorrhiza injection. The expression of ngn-1 and mash-1 were detected by RT-PCR before and after induction. The expression of NSE and GFAP in bone marrow-derived mesenchymal stem cells before and after induction was detected by immunohistochemistry. MAIN OUTCOME MEASURES: ① RT-PCR was used to detect the expression of ngn-1 and mash-1 before and after induction of cells. ② Immunohistochemistry was used to detect the expression of NSE and GFAP in bone marrow-derived mesenchymal stem cells before and after induction. Results: ① The adherent growth of bone marrow mesenchymal stem cells was good, most of the cells were long fusiform. After adding inducer, the cell volume is reduced, some cells have protrusions, and the shape is similar to neurons. ② RT-PCR assay showed that the expression of ngn-1 and mash-1 mRNA in bone marrow-derived mesenchymal stem cells was negative and induced positive after induction. ③ Immunohistochemistry showed that NSE and GFAP of the induced cells were positive and negative in the control group. Conclusion: Salvia miltiorrhiza injection can induce bone marrow mesenchymal stem cells to differentiate into neuron-like cells.