川芎嗪对大鼠大脑皮层神经细胞的体外存活及其钙超载的影响

Effects of Tetramethylpyrazine on the Survival and Intracellular Calcium Overload of Rat Cerebral Cortex Neurons in Vitro

目的:研究川芎嗪对体外培养的大鼠大脑皮层神经细胞的保护作用及其钙超载的影响。方法:取出生1d内的乳鼠大脑皮层制成细胞悬液,接种于经多聚赖氨酸包被的培养板。于培养6d后,加入不同浓度的川芎嗪,继续培养1d,采用MTT比色法测量其存活细胞的吸收值,同时行NSE检查。采用激光扫描共聚焦显微镜(LSCM)技术检测药物对谷氨酸(500μmol·L-1)、KCl(50mmol·L-1)诱导培养6d的大脑皮层神经细胞内钙超载的影响,计算荧光强度变化率。结果:NSE检查结果表明,培养6d的存活细胞大部分均为神经元。MTT比色检查结果表明,川芎嗪浓度为1.6～200.0μg·mL-1时,存活细胞数均明显增加(P<0.05)。LSCM测定结果表明,100μg·mL-1川芎嗪能明显降低荧光强度变化率(P<0.01)。结论:川芎嗪能促进体外培养的大脑皮层神经细胞存活,且可抑制谷氨酸、KCl诱导的神经细胞内钙超载,这可能是其治疗缺血性脑血管病的作用机制。

OBJECTIVE：To observe the effect of tetramethylpyrazine （TMP） on the survival and intracellular calcium overload of rat cerebral cortex neurons. METHODS： The cerebral cortex of suckling mouse with 1 day after birth were collected and dissoci- ated into cell suspension. The cell suspension was incubated in culture plates covered with polylysine in each well. After culturing for 6 days, TMP were added into the well cultures in order to continually culture for 1 day. The OD of living cells in each well was measured by MTT assay, and neuronal specific nolase （NSE） assay was conducted. After culturing for 6 days with glutamic acid （500 μmol. L^-1） and KC1 （50 μmol. L^-1）, intracellular calcium concentration changes were measured using LSCM. RESULTS： Most of the living cells were cerebral neurons by NSE. MTT assay indicated that the viable count increased significantly when the concentrations of TMP were 1.6-200.0 μg.mL^-1（P〈0.05）. LSCM assay indicated that the change of fluorescence intensity was re- duced when the concentrations of TMP was 100 μg.mL^-1（P〈0.01）. CONCLUSION： These findings indicate that TMP can pro- mote cerebral cortex neurons to live in vitro and inhibit intracellular calcium overload induced by KC1 solution or glutamate, which is possibly one of its mechanisms in the treatment of ischemic cerebrovascular disease.