中药复方诱导骨髓间充质干细胞向神经元样细胞分化的实验研究
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摘要
目的：本研究旨在通过补肾填精的中药复方(左归丸)诱导骨髓间充质干细胞分化,并通过免疫组化鉴定诱导分化后的细胞为神经元样细胞,从而为“肾藏精、生髓”的传统中医理论提供相应的实验依据。
方法：实验一利用全骨髓贴壁法分离大鼠骨髓间充质干细胞,并在体外扩增、纯化,倒置显微镜下观察其形态,利用左归丸药液对分离后的细胞进行诱导分化,通过阿利新蓝染色、改良钙钴法碱性磷酸酶染色、神经元特异性烯醇化酶免疫组织化学染色以鉴定分化后的细胞是否为成骨细胞、软骨细胞、神经元样细胞。实验二利用全骨髓贴壁法分离大鼠骨髓间充质干细胞,利用左归丸拆药后的各组药液分别对获得的细胞进行诱导分化,并进行神经元特异性烯醇化酶免疫组织化学染色,计其阳性表达的细胞。
结果：实验一显示,分离纯化后的细胞,经倒置显微镜观察其形态,再左归丸药液诱导分化后,经阿利新蓝染色、改良钙钴法磷酸酶染色、神经元特异性烯醇化酶免疫组织化学染色,证明获得的细胞向软骨细胞、成骨细胞、神经元样细胞进行了分化,获得的细胞的形态符合骨髓间充质干细胞的形态表现。实验二将获得的骨髓间充质干细胞,用左归丸拆药后的各组药液对其进行诱导分化,经NSE染色后计阳性表达细胞,结果显示撤出熟地NSE表达明显增高；撤除枸杞后阳性表达率下降最多,其次是山茱萸、山药、牛膝；撤出菟丝子不影响该酶的表达。
结论：实验一：采用全骨髓贴壁法获得的细胞为大鼠骨髓间充质干细胞；中药复方左归丸对大鼠骨髓间充质干细胞有多向诱导分化作用。实验二：撤出熟地神经元烯醇化酶表达增高,提示熟地可能对BMSC向神经元样细胞方向分化有一定抑制作用(或调控作用)；枸杞对骨髓间充质干细胞向神经元样细胞分化的诱导作用最强,其次是山茱萸、山药、牛膝：菟丝子对骨髓间充质干细胞向神经元样细胞分化无明显的促进作用。
Objective To investigate tonifying kidney formula-ZuoGui Wan induct differentiation of bone marrow mesenchymal stem cells (BMSC), it was identified that the cells differentiated into neuron-like cells by immunohistochemical method. So, the traditional theory "the kidney stores essence, marrow" was provided experimental evidence
Methods (1) Rat BMSC were isolated by the whole bone marrow adherent method, which were amplified, purified in vitro and the morphology was observed under inverted microscope, then, the differentiation of cells were induced by ZuoGui Wan's liquid. Alcian blue, phosphatase modified Gomori phosphatase and neuron specific enolase staining were used to identify the cellularity (osteoblast, cartilage cell or neuron-like cells).(2) Rat BMSC were isolated by the whole bone marrow adherent method, the separated-liquid of Zuo Gui Wan was used to induct the cells in the different group, neuron specific enolase staining were used to calculate the positive expression cells.
Results to the first experiment, The purified cells induced into osteoblast, cartilage cell and neuron-like cells under the inverted microscope, the cellular morphology accord with the BMSC. To the second experiment, the separated liquid of formula induced different group'cells, and it was showed the expression was significantly high by removing Shu Di, and the expression is lower after removed Gou Qi, then was Shan Zhu YU, Shan Yao, Niu Xi. The Tu Si Zi didn't effect the expression.
Conclusion (1)the cells were Rat BMSC, which were gotten by the whole bone marrow adherent method; Zuo Gui Wan has multi-directional differentiation effect on inducting to BMSCs;(2) the expression of NSE is higher after removing Shu Di, it was identified the Shu Di has inhibition effect (or controlling effect) on inducting BMSCs into neuron-like cells; Gou Qi has strongest effect, then is Shan Zhu YU, Shan Yao, Niu Xi; Tu Si Zi has no effect on induction.
方法：实验一利用全骨髓贴壁法分离大鼠骨髓间充质干细胞,并在体外扩增、纯化,倒置显微镜下观察其形态,利用左归丸药液对分离后的细胞进行诱导分化,通过阿利新蓝染色、改良钙钴法碱性磷酸酶染色、神经元特异性烯醇化酶免疫组织化学染色以鉴定分化后的细胞是否为成骨细胞、软骨细胞、神经元样细胞。实验二利用全骨髓贴壁法分离大鼠骨髓间充质干细胞,利用左归丸拆药后的各组药液分别对获得的细胞进行诱导分化,并进行神经元特异性烯醇化酶免疫组织化学染色,计其阳性表达的细胞。
结果：实验一显示,分离纯化后的细胞,经倒置显微镜观察其形态,再左归丸药液诱导分化后,经阿利新蓝染色、改良钙钴法磷酸酶染色、神经元特异性烯醇化酶免疫组织化学染色,证明获得的细胞向软骨细胞、成骨细胞、神经元样细胞进行了分化,获得的细胞的形态符合骨髓间充质干细胞的形态表现。实验二将获得的骨髓间充质干细胞,用左归丸拆药后的各组药液对其进行诱导分化,经NSE染色后计阳性表达细胞,结果显示撤出熟地NSE表达明显增高；撤除枸杞后阳性表达率下降最多,其次是山茱萸、山药、牛膝；撤出菟丝子不影响该酶的表达。
结论：实验一：采用全骨髓贴壁法获得的细胞为大鼠骨髓间充质干细胞；中药复方左归丸对大鼠骨髓间充质干细胞有多向诱导分化作用。实验二：撤出熟地神经元烯醇化酶表达增高,提示熟地可能对BMSC向神经元样细胞方向分化有一定抑制作用(或调控作用)；枸杞对骨髓间充质干细胞向神经元样细胞分化的诱导作用最强,其次是山茱萸、山药、牛膝：菟丝子对骨髓间充质干细胞向神经元样细胞分化无明显的促进作用。
Objective To investigate tonifying kidney formula-ZuoGui Wan induct differentiation of bone marrow mesenchymal stem cells (BMSC), it was identified that the cells differentiated into neuron-like cells by immunohistochemical method. So, the traditional theory "the kidney stores essence, marrow" was provided experimental evidence
Methods (1) Rat BMSC were isolated by the whole bone marrow adherent method, which were amplified, purified in vitro and the morphology was observed under inverted microscope, then, the differentiation of cells were induced by ZuoGui Wan's liquid. Alcian blue, phosphatase modified Gomori phosphatase and neuron specific enolase staining were used to identify the cellularity (osteoblast, cartilage cell or neuron-like cells).(2) Rat BMSC were isolated by the whole bone marrow adherent method, the separated-liquid of Zuo Gui Wan was used to induct the cells in the different group, neuron specific enolase staining were used to calculate the positive expression cells.
Results to the first experiment, The purified cells induced into osteoblast, cartilage cell and neuron-like cells under the inverted microscope, the cellular morphology accord with the BMSC. To the second experiment, the separated liquid of formula induced different group'cells, and it was showed the expression was significantly high by removing Shu Di, and the expression is lower after removed Gou Qi, then was Shan Zhu YU, Shan Yao, Niu Xi. The Tu Si Zi didn't effect the expression.
Conclusion (1)the cells were Rat BMSC, which were gotten by the whole bone marrow adherent method; Zuo Gui Wan has multi-directional differentiation effect on inducting to BMSCs;(2) the expression of NSE is higher after removing Shu Di, it was identified the Shu Di has inhibition effect (or controlling effect) on inducting BMSCs into neuron-like cells; Gou Qi has strongest effect, then is Shan Zhu YU, Shan Yao, Niu Xi; Tu Si Zi has no effect on induction.
引文
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