Effect of Tongxinluo on neurocyte lineage derived from rat embryonic neural stem cells / 中国组织工程研究

ABSTRACT

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 12th-14th 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 (+), ?tubulin (+) or GFAP (+) differentiated with each other (P 0.05). ③The ratios of NSCs labeled with nestin (+) decreased soon after administered Tongxinluo, and increased after that. The ratios of ?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.