| 杨紫菡,潘乙怀,邹多宏.一种诱导牙髓干细胞神经向分化的有效方法和诱导时间的探索[J].口腔材料器械杂志,2023,32(1):32-39. |
| 一种诱导牙髓干细胞神经向分化的有效方法和诱导时间的探索 |
| An effective method and induction time for inducing neural differentiation of dental pulp stem cells |
| 投稿时间：2022-09-10 修订日期：2022-12-21 |
| DOI：10.11752/j.kqcl.2023.01.06 |
| 中文关键词: 牙髓干细胞 神经向分化 单层培养 神经修复 |
| 英文关键词:Dental pulp stem cell Neural differentiation Monolayer culture Nerve repair |
| 基金项目:国家自然科学基金（编号：31870969，32171347）；中国医学科学院医学与健康科技创新工程项目资助（编号：2019-I2M-5-037）；上海交通大学医学院“双百人”人才计划（编号：20191816） |
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| 中文摘要: |
| 目的 建立一种简单而高效的诱导牙髓干细胞（DPSC）向神经向分化的方法。方法 使用酶消化法分离原代DPSC，通过流式细胞术表征表面标志物以及三系诱导分化鉴定其多向分化潜力。添加20 ng/mL bFGF和20 ng/mL EGF的单层培养方法诱导DPSC向神经向分化，通过Western Blot、q-PCR检测神经相关标志物随诱导时间增加发生的改变，通过免疫荧光染色检测表面标志物改变。通过止血钳夹闭的方式构建大鼠坐骨神经损伤模型，胰酶消化收集诱导6d的dDPSC，注射到损伤处，4周后通过HE染色和免疫荧光染色检测神经修复情况。结果 获取的DPSC经流式细胞术、三系诱导分化鉴定，符合间充质干细胞特征。Western Blot、q-PCR结果表明Nestin、GFAP随诱导时间增加而上升，在6~9 d趋于稳定。细胞免疫荧光染色结果显示dDPSC的CD73、CD146表达下降，而Nestin、GFAP的表达升高。HE染色显示，PBS组的坐骨神经损伤严重，发生神经变性，而dDPSC组的神经组织结构与对照组类似。NF200/S100β的免疫荧光染色结果显示dDPSC组轴突和髓鞘再生情况优于PBS组。结论 酶消化法分离得到的DPSC纯度良好，通过添加20 ng/mL bFGF和20 ng/mL EGF培养的单层培养法培养6 d可以诱导DPSC向神经前体细胞分化，dDPSC对大鼠坐骨神经损伤的恢复有一定的促进作用。 |
| 英文摘要: |
| Obiective The study aimed to establish a simple and efficient method for inducing neural differentiation of Dental pulp stem cell (DPSC). Methods Primary DPSC were isolated by enzymatic digestion. The surface markers were characterized by flow cytometry and the multi-directional differentiation potential was identified by three-line induction. Monolayer culture supplemented with 20 ng/mL bFGF and 20 ng/mL EGF was used to induce the neural differentiation of DPSC. Western Blot and q-PCR were used to detect the changes of neural markers with the increase of culture time. Surface marker expression changes were detected by immunofluorescence staining. A rat model of sciatic nerve injury was established by clamping the sciatic nerve with hemostatic forceps. The dDPSC induced for 6 days was collected and injected into the injured site. After 4 weeks, the recovery of injured sciatic nerve was detected by HE staining and immunofluorescence staining. Results The obtained DPSC was identified by flow cytometry and three-line induction differentiation, which conformed to the characteristics of mesenchymal stem cells. Western Blot and q-PCR analysis showed that Nestin and GFAP were increased with the prolonged culture time, and tended to be stable at 6-9d. Immunofluorescence staining showed that the expressions of CD73 and CD146 decreased in dDPSC group, while the expressions of Nestin and GFAP increased. The results of HE staining showed that the sciatic nerve in PBS group was severely injured and degenerated, while the structure of sciatic nerve in dDPSC group was similar to that in control group. Immunofluorescence images of NF200/S100 β showed that axon and myelin regeneration in dDPSC group was better than that in PBS group. Conclusion The purity of DPSC isolated by enzyme digestion is good. DPSC could be induced to differentiate into neural precursor cell by monolayer culture with 20 ng/mL bFGF and 20 ng/mL EGF for 6 days. dDPSC could promote the recovery of injured sciatic nerve. |
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