摘要
目的:以丹参水溶性成分丹参素、丹酚酸为干预药物,观察其对胎鼠脑神经干细胞迁移的诱导作用。方法:实验于2006-03/08在广州暨南大学医学院完成。①丹参素(粉剂,纯度大于90%,昆明长春花科技公司生产,批号1108552200203);丹酚酸(粉剂,纯度大于90%,昆明长春花科技公司生产,批号CCH601012);MBC96型趋化小室(NeuroProbe公司)。②取妊娠13.5d昆明种小鼠2只,处死后取出胎鼠,分离其胎脑神经干细胞进行传代培养。取培养至对数生长期的细胞,采用Boyden小室法观察细胞迁移情况。取单细胞悬液50μL,分别加入Boyden小室的12个上室;取分别含有5,10,20,40,80,100mg/L丹参素和5,10,20,40,80,100mg/L丹酚酸的DMEM培养基各450μL,加入Boyden小室的12个下室;另2孔以不含丹参素和丹酚酸的DMEM培养基作为空白对照。上下两室以预先包被Ⅰ型胶原的聚碳酸酯微孔滤膜相隔。将Boyden小室置于37℃、体积分数为0.05的CO2培养箱中培养24h,取出微孔滤膜,用甲醇将迁移至微孔滤膜下层的细胞固定,Nissl染色,显微镜下随机选择3个不同视野,计数膜上不同层次的细胞数。③选取昆明种小鼠18只,随机数字表法分为生理盐水对照组、丹参素组、丹酚酸组,6只/组。丹参素组于侧脑室内连续注射80mg/L丹参素5μL,丹酚酸组注射40mg/L丹酚酸5μL,生理盐水对照组注射等量生理盐水,共3d,术后14d处死,取脑,制备切片,行ABC免疫细胞化学染色,观察丹参素和丹酚酸干预后nestin阳性神经元的分布变化。结果:18只小鼠全部进入结果分析。①不同质量浓度的丹参素对神经干细胞迁移的诱导:与空白对照比较,5,10mg/L的丹参素促神经干细胞迁移的作用并不明显(t=1.037,1.374,P>0.05);20,40mg/L的丹参素可明显促进神经干细胞的迁移(t=2.894,3.468,P<0.01);当丹参素质量浓度为80mg/L时促迁移作用最为强烈(t=4.031,P<0.01),之后有所减弱。②不同浓度的丹酚酸对神经干细胞迁移的诱导:与空白对照比较,5mg/L的丹酚酸促神经干细胞迁移的作用并不明显(t=1.365,P>0.05);10,20mg/L的丹酚酸可明显促进神经干细胞的迁移(t=3.021,3.947,P<0.01);当丹酚酸质量浓度为40mg/L时促迁移作用最为强烈(t=4.342,P<0.01),之后有所减弱。③丹参素和丹酚酸对小鼠室管膜下nestin阳性神经元迁移的影响:丹参素组侧脑室内侧和外侧的nestin阳性细胞的数量多于生理盐水对照组,而且并不局限于侧脑室周围区,在脑皮质和尾壳核可见少量的阳性细胞,细胞的突起明显,伸向皮质方向,侧脑室内侧的阳性细胞有相似的特征。丹酚酸组侧脑室周围的阳性细胞数量多,而且在皮质和尾壳核检测到大量的阳性细胞。结论:丹参素和丹酚酸均具有诱导胎鼠脑神经干细胞迁移的作用,且丹酚酸的作用优于丹参素。
AIM： To investigate inductive effect of danshensu and salvianolic acid, as intervention drugs, on migration of neural stem cells from mouse embryo. METHODS： The experiment was finished in Medical College of Jinan University during March to August 2006. （1） Danshensu （batch number 1108552200203） and salvianolic acid （batch number CCH601012） powders were purchased from Kunming Changchunhua Technology Co. Ltd. Their purities were both more than 90 percent. MBC96 transwell was purchased from Neuro Probe Company. （2）Two Kunming mice and their 13.5-day embryos were used in this experiment, after neural stem cells were segregated and subcultured, the cells in exponential phase of growth were used for studying the migration of neural stem cells by Boyden cave. And 50 μL cell suspension were added in 12 Boyden upper wells individually, then 450 μL DMEM culture medium including 5, 10, 20, 40, 80, 100 mg/L danshensu and saManolic acid was added in 12 Boyden inferior wells respectively. Other two wells were control wells that were added with DMEM culture medium without danshensu and salvianolic acid. Between upper and inferior wells, there were polycarbonate microporous membranes that were primarily coated by collagen type I. Then Boyden wells were cultured in 0.05 volume fraction of CO2 incubator at 37 ℃ for 24 hours. After taking out microporous membranes, we put methanol to stain with Nissl method. Microscopy was used to observe three regions and count the number of cells.（3）A total of 18 Kunming mice were selected to three groups including control, danshensu and salvianolic acid groups randomly. And 6 mice of each group ware injected with normal saline, danshensu （80 mg/L, 5 μl） or salvianolic acid （40 mg/L, 5 μL） in lateral cerebral ventricle for three days. After 14 days, all mice was sacrificed, then their brains were sliced and detected by ABC immunocytechemistry. The nestin-immunopositive neurons were observed to study their changes after intervention. RESULTS： All 18 mice were used in the end results.（1）Migration of neural stem cells induced by danshensU. Compared with controls, 5 and 10 mg/L danshensu showed little effect on cell migration （t =1.037, 1.374, P 〉 0.05）, however, 20 and 40 mg/L danshensu had significant effect （t =2.894, 3.468, 3.952, P 〈 0.01）, and 80 mg/L manifested the most significant effect （t =4.031, P 〈 0.01 ）. But 100 mg/L was slightly decreased.（2）Migration of neural stem cells induced by salvianolic acid： Compared with controls, 5 mg/L had no significant roles （t =1.365, P 〉 0.05）; 10 and 20 mg/L could induce significant migration （t =3.021, 3.947, P 〈 0.01）. The strongest concentration was 40 mg/L （t =4.342, P 〈 0.01）, and 80-100 mg/L continued the similar effect but slightly decreased. （2）Migration of nestin-positive neurons in subependyma induced by danshensu and salvianolic acid： After mice were injected danshensu in lateral cerebral ventricle, nestin-positive neurons were more than those of control group, and the positive neurons were not limited in lateral periventdcular area and were observed in pallium and striatum corpora, with the obvious neurite extended toward cortex, which was identical with the positive cell in lateral ventricle. Compared with danshensu, after the mice were injected with salvianolic acid, more positive neurons of nestin were detected in pallium and striatum corpora. CONCLUSION： Danshensu and salvianolic acid can both induce migration of neural stem cells from mouse embryo, and the effect of salvianolic acid is better than that of danshensu.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第7期1225-1228,I0002,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
广州市科技计划项目(2004J1C0241)~~
