基于“升降开阖”通玄府的引经药诱导骨髓间充质干细胞向神经元样细胞分化  

Neuron-like differentiation of bone marrow mesenchymal stem cells induced by Rehmannia decoction with Cimicifuga heracleifolia Kom.and Achyranthes bidentata Bl.as guiding drugs

作　　者：武密山[1] 赵素芝 高维娟 任立中[4] 王茹[1] 刘营[1] 韩红伟[1] 李彬[5] 

机构地区：[1]河北中医学院基础医学院方剂学教研室,河北省石家庄市050200 [2]石家庄市长安区胜北社区卫生服务中心,河北省石家庄市050041 [3]河北省心脑血管病中医药防治重点实验室,河北省石家庄市050091 [4]河北医科大学第一医院骨科,河北省石家庄市050031 [5]河北医科大学生物化学教研室,河北省石家庄市050017

出　　处：《中国组织工程研究》2015年第19期2965-2972,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81073074;30472200);河北省自然科学基金资助项目(H2013206005);河北省高等学校科学技术研究重点项目(ZD2015053);河北省高等学校高层次人才科学研究项目(GCC2014013)~~

摘　　要：背景:地黄饮子诱导的骨髓间充质干细胞移植对脑缺血再灌注损伤模型大鼠神经功能恢复有明显的影响。目的:探讨应用"升降开阖"通玄府的引经报使药(升麻、牛膝分别置换薄荷)地黄饮子诱导骨髓间充质干细胞向神经元样细胞分化的可行性,并进一步研究在骨髓间充质干细胞向神经元样细胞分化中的调节作用。方法:取4周龄健康清洁级SD大鼠骨髓,采用全骨髓贴壁法体外培养骨髓间充质干细胞,流式细胞仪检测第3代细胞表面的骨髓基质标志CD90和造血细胞标志CD45。取第3代细胞,根据诱导条件不同分为4组:空白对照组、地黄饮子组、牛膝组、升麻组,空白对照组只用含体积分数为1%胎牛血清的DMEM/F12培养,后3组用地黄饮子含药血清、以牛膝为药引子的地黄饮子含药血清、以升麻为药引子的地黄饮子含药血清+体积分数为1%胎牛血清+DMEM/F12培养,倒置相差显微镜观察细胞形态变化,诱导7 d后,采用免疫细胞化学法和Western blot检测神经元特异性烯醇化酶、胶质纤维酸性蛋白的表达,RT-PCR检测胶质细胞源性神经营养因子、脑源性神经营养因子m RNA的表达。结果与结论:第3代骨髓间充质干细胞大致呈单一的长梭形或扁平形,紧密漩涡样排列生长,流式细胞仪示CD90表达高达98%,而CD45表达仅1.3%。诱导7 d后,升麻组、牛膝组和地黄饮子组的细胞收缩变圆,向四周伸出多个明显突起,部分突起间存在连接,表现出典型的神经元样细胞形态,而空白对照组变化不明显。免疫组化染色显示牛膝组及升麻组神经元特异性烯醇化酶、胶质纤维酸性蛋白表达均高于地黄饮子组,牛膝组高于地黄饮子组,升麻组高于牛膝组,差异均有显著性意义(P<0.05)。Western blot检测结果与免疫细胞染色结果一致。RT-PCR检测胶质细胞源性神经营养因子、脑源性神经营养因子m RNA改变类似于相应蛋白改变,升麻组>牛�BACKGROUND： Rehmannia decoction has remarkable effects on the neurologic function recovery of cerebral ischemia-reperfusion model rats undergoing bone marrow mesenchymal stem cell transplantation. OBJECTIVE： To investigate the feasibility of bone marrow mesenchymal stem cells differentiating into neuron-like cells induced by Rehmannia decoction with Cimicifuga heracleifo/ia Kom. and Achyranthes bidentata BI. as guiding drugs and to further explore the regulatory role. METHODS： Bone marrow mesenchymal stem cells were harvested from 4-week-old Sprague Dawley rats and cultured in vitro using the whole bone marrow adhesion method. Flow cytometry assay was used to detect the expression of CD90 and CD45 of bone marrow mesenchymal stem cells at passage 3. Cells at passage 3 were divided into four groups according to different culture conditions： blank control group （1% fetal bovine serum＋DMEM/F-12）, Rehmannia decoction group （serum containing Rehmannia decoction＋l% fetal bovine serum＋DMEM/F-12）, Achyranthes bidentata BI. group （serum containing Rehmannia decoction with Achyranthes bidentata BI. as the guiding drug＋l% fetal bovine serum＋DMEM/F-12）, Cimicifuga heracleifolia Kom. group （serum containing Rehmannia decoction with Cimicifuga heracleifolia Kom. as guiding drug＋ 1% fetal bovine serum＋DMEM/F-12）. Then the cellular morphology was observed under inverted phase contrast microscope. At 7 days after induction, immunocytochemistry staining and western blot assay were used to detect expression of neuron-specific enolase and gliat fibrillary acidic protein; and RT-PCR was used to detect mRNA expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor. RESULTS AND CONCLUSION： Bone marrow mesenohymal stem cells roughly presented a single long spindle or fiat-shaped morphology at passage 3, grew with close whirlpool-like arrangement, and the CD90 expression level was up to 98%, but the expression level of CD45 was only 1.3%. At 7 days after inducti

关 键 词：骨髓 间质干细胞 升麻属 细胞分化 干细胞 骨髓干细胞 药引子 升麻 牛膝 地黄饮子 骨髓间充质干细胞 神经元样细胞 分化 国家自然科学基金 

分 类 号：R304.2[医药卫生—基础医学]