通心络对大鼠胚胎神经干细胞源性神经细胞谱系的影响

Effect of Tongxinluo on neurocyte lineage derived from rat embryonic neural stem cells

目的：前期体内动物模型实验说明，通心络可能具有影响神经干细胞增殖及向各种神经细胞分化的作用。实验拟观察通心络对大鼠胚胎神经干细胞源性神经细胞谱系的影响以及时效、量效的关系。方法：实验于2007—06／10在南方医科大学机能学实验室完成。①实验材料：孕12～14dSD大鼠由南方医科大学实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。通心络，主要成分为人参、水蛭、全蝎、土鳖虫、蜈蚣、蝉蜕、赤芍、冰片等，由石家庄以岭药业股份有限公司生产，国药准字Z19980015。通心络含药血清的制备：按大剂量组1g／（d·kg）、小剂量组0．5g／（d·kg）分别予大鼠通心络混悬液灌胃，7d后抽血离心，吸取血清，过滤消毒，分装，-70℃冻存备用。②实验方法：自孕12～14d大鼠胚胎中分离培养神经干细胞，取第3代细胞分别给予大、小剂量组通心络含药血清干预。以添加普通血清干预的为对照。③实验评估：于培养1，3，7d通过免疫荧光染色观察各种类型神经细胞所占比例。结果：①神经干细胞经通心络含药血清干预后1d，仅在大剂量组海马齿状回有极少数细胞呈BrdU（＋）GFAP（＋），其余细胞都呈BrdU（＋）Nestin（＋），小剂量组和对照组均为BrdU（＋）Nestin（＋）细胞。②经通心络含药血清干预后3d，大剂量组、小剂量组和对照组Nestin（＋）、13tubulin（＋）、GFAP（＋）细胞比例差异有显著性意义（P〈0．01），GalC（＋）细胞比例差异无统计学意义（P〉0.05）。③大鼠胚胎神经干细胞经通心络含药血清干预后：各组Nestin（＋）细胞比例先降后升：13tubulin（＋）细胞比例大剂量组持续上升，小剂量组和对照组先升后降；GFAP（＋）细胞比例大剂量组先升后降，小剂量组和对照组持续上升，GalC（＋）细胞比例大、小剂量组均较平稳，对照组7d时明显上升。结论：通心络有促进大鼠胚胎神经干细胞增殖及向神经元分化的效应，并存在时效、量效关系，剂量越大效应越明显及持久。

AIM： Tongxinluo had showed effects of inducing proliferation and differentiation on neural stem cells in previous observations on animal models. The experiment would investigate the time-effect and dose-effect relationship of Tongxinluo on neurocyte lineage development from rat embryonic neural stem cells （NSCs）. METHODS： Experiments were performed at the Laboratory of Functional Science of Southern Medical University from June to October 2007. ①Pregnant rats of 12-14 days were provided by Experimental Animal Center of Southern Medical University. The experiment was performed in strict compliance with all standards for ethics. Tongxinluo contained ginseng, leech, scorpion, ground beetle, centipede, pellicula cicadae, red peony root, borneol and so on. Tongxinluo was produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd., No. Z19980015. Preparation of serum containing Tongxinluo： 1 g/kg per day Tongxinluo suspension was given by lavage in rats of high-dose Tongxinluo group, and 0.5 g/kg per day in rats of low-dose Tongxinluo group. 7 days later, sera were extracted from all the rats and sterilized with filtrating, followed by separate-loading and cryopreservation at -70 ℃. ②NSCs cultured were separated from 12^th- 14^th day SD rat embryo. The third passage of cells was treated with serum containing Tongxinluo in the high-dose and low-dose Tongxinluo group. Rats were treated with common serum as a control. ③Proportion of various kinds of nerve cells was observed by immunofluorescence staining at days 1, 3 and 7. RESULTS： ①At day 1, there was few cells labeled with 5-bromodeoxyuridine （BrdU） （＋） and glial fibrillary acidic protein （GFAP） （＋） in high-dose Tongxinluo group, with rest cells all labeled with BrdU （＋） and Nestin （＋）. While in low-dose Tongxinluo group and control group, all cells were BrdU（＋）Nestin（＋）. ②At day 3, the ratios of cells labeled with Nestin （＋）, 13 tubulin （＋） or GFAP （＋） differentiated with each other （P 〈 0.01）. The ratios of GalC （＋） cells had no difference between each other group （P 〉 0.05）. ③The ratios of NSCs labeled with nestin （＋） decreased soon after administered Tongxinluo, and increased after that. The ratios of 13 tubulin （＋） cells increased in the high-dose Tongxinluo group while increased firstly then decreased in the low-dose Tongxinluo group and control group. The ratios of GFAP （＋） cells increased in the low-dose Tongxinluo group and control group while increased firstly then decreased in the high-dose Tongxinluo group. The ratios of GalC （＋） cells had no remarkable change in the high-dose and low-dose Tongxinluo group, but increased significantly at day 7 in the control group. CONCLUSION： Tongxinluo may induce proliferation and neuronal differentiation of rat embryonic NSCs with time-effect and dose-effect relationship. The more Tongxinluo is administered, the more significant and permanent effects will be achieved.