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| 体外共培养条件下毛囊神经嵴干细胞促进神经束膜细胞迁移和增殖 |
| 于皓杰1,杜甑衎2,付靖乔2,肖楚兰3,杨向群1,许家军1*,刘芳1* |
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(1. 海军军医大学(第二军医大学)基础医学院人体解剖学教研室, 上海 200433;
2. 海军军医大学(第二军医大学)基础医学院学员四大队, 上海 200433;
3. 海军军医大学(第二军医大学)基础医学院学员一大队, 上海 200433
*通信作者) |
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| 摘要: |
| 目的 探索体外培养神经束膜细胞的有效方案，并初步研究毛囊神经嵴干细胞（hfNCSC）对神经束膜细胞的激活作用。方法 采用“限时消化-差速贴壁-化学药物”方法对大鼠坐骨神经束膜细胞进行培养和纯化，同时培养大鼠触须垫hfNCSC，并对细胞进行免疫细胞化学染色鉴定。借助Transwell小室建立hfNCSC与神经束膜细胞的共培养模型，在Transwell上室接种神经束膜细胞，下室接种hfNCSC（hfNCSC共培养组）或不接种细胞（对照组），共培养6、12和18 h后进行结晶紫染色，观测神经束膜细胞的迁移情况。取hfNCSC条件培养基（hfNCSC条件培养基组）和含2% FBS的DMEM培养基（对照组）分别作用于神经束膜细胞，24、48和72 h后用CCK-8试剂盒检测细胞增殖情况。结果 本方案可在2周的时间内获得纯度高达（97.66±2.08）%的神经束膜细胞。将神经束膜细胞与hfNCSC共培养6、12和18 h后，可见hfNCSC共培养组神经束膜细胞的迁移数较对照组增多，差异有统计学意义（P<0.05，P<0.01）。以hfNCSC条件培养基作用于神经束膜细胞24 h和48 h后，hfNCSC条件培养基组与对照组神经束膜细胞的细胞活力差异无统计学意义（P均>0.05）；但作用72 h后，hfNCSC条件培养基组神经束膜细胞的细胞活力高于对照组，差异有统计学意义（P<0.01）。结论 “限时消化-差速贴壁-化学药物”方法可成功培养和纯化神经束膜细胞；hfNCSC可激活神经束膜细胞，促进其迁移和增殖。 |
| 关键词: 神经束膜细胞 毛囊神经嵴干细胞 共培养 细胞增殖 细胞迁移 |
| DOI：10.16781/j.0258-879x.2021.05.0512 |
| 投稿时间：2021-03-29修订日期：2021-04-27 |
| 基金项目:国家自然科学基金（81571211）. |
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| In vitro co-culture of hair follicle neural crest stem cells promoting migration and proliferation of perineurial cells |
| YU Hao-jie1,DU Zeng-kan2,FU Jing-qiao2,XIAO Chu-lan3,YANG Xiang-qun1,XU Jia-jun1*,LIU Fang1* |
(1. Department of Anatomy, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. The Fourth Student Team, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
3. The First Student Team, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding authors) |
| Abstract: |
| Objective To explore an effective method for culturing perineurial cells in vitro, and to preliminarily study the role of hair follicle neural crest stem cells (hfNCSCs) in activating perineurial cells. Methods Perineurial cells from rat sciatic nerve were cultured and purified by the method of "limited digestion-differential adherence-chemical drug", hfNCSCs from rat vibrissa were cultured, and the cells were identified by immunocytochemistry staining. hfNCSCs and perineurial cells were co-cultured in Transwell plates, where perineurial cells were seeded in the upper chamber, and hfNCSCs (hfNCSC co-culture group) or acellular grids (control group) were seeded in the bottom chamber. Crystal violet staining was performed after co-culture for 6, 12 and 18 h to observe the migration of perineurial cells. The perineurial cells were treated with hfNCSCs conditioned medium (hfNCSC conditioned medium group) and 2% FBS DMEM medium (control group), respectively, and the proliferation of perineurial cells was detected by cell counting kit 8 (CCK-8) after 24, 48 and 72 h. Results Perineurial cells with purity up to (97.66±2.08)% were obtained within 2 weeks by this method. The migration number of perineurial cells was significantly higher in the hfNCSC co-culture group than in the control group after 6, 12 and 18 h of co-culture (P<0.05, P<0.01). The cell viability of perineurial cells in the hfNCSC conditioned medium group and control group was similar after treated with hfNCSCs conditioned medium for 24 and 48 h (both P>0.05); however, the cell viability of perineurial cells was significantly higher in the hfNCSC conditioned medium group than in the control group after 72 h (P<0.01). Conclusion Perineurial cells can be successfully cultured and purified by the method of "limited digestion-differential adherence-chemical drug"; hfNCSCs can activate perineurial cells and promote their migration and proliferation. |
| Key words: perineurial cells hair follicle neural crest stem cells co-culture cell proliferation cell migration |
