基于神经干细胞微环境研究川芎嗪对OGD损伤下神经元新生的促进作用  

Research on ability of tetramethylpyrazine on promoting neurogenesis after OGD injury based on neural stem cell microenvironment

作　　者：邢婉青 宋孝雄 王雪晴 卢金福[1] 吕高虹[1] 阮鸣 喻斌[1] XING Wan-qing;SONG Xiao-xiong;WANG Xue-qing;LU Jin-fu;LYU Gao-hong;RUAN Ming;YU Bin(Jiangsu Key Lab for Pharmacology and Safety Evaluation of Chinese Materia Medica,Nanjing University of Chinese Medicine,Nanjing 210023,China;School of Food Science,Nanjing Xiaozhuang University,Nanjing 211171,China)

机构地区：[1]南京中医药大学江苏省中药药效与安全性评价重点实验室,江苏南京210023 [2]南京晓庄学院食品科学学院,江苏南京211171

出　　处：《中国药理学通报》2023年第4期731-738,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81973726)。

摘　　要：目的 在构建体外神经干细胞微环境的基础上研究川芎嗪(tetramethylpyrazine, TMP)对氧糖剥夺(oxygen-glucose deprivation, OGD)损伤下神经元新生的促进作用。方法 分别原代培养神经干细胞(NSCs)、星形胶质细胞(ACs)、脑微血管内皮细胞(BMECs),建立NSCs微环境体外模型后,以NSCs活性对OGD造模条件进行优化,以尼氏染色对TMP给药浓度进行优化,随后采用CCK-8、Nestin+/DAPI+免疫荧光法检测NSCs增殖情况;放射性迁移、划痕修复实验检测NSCs迁移情况;β3-Tubulin+/DAPI+、GFAP+/DAPI+检测NSCs分化;以跨膜电阻阻值反应体外血脑屏障结构的完整性。结果 NSCs活性随着OGD造模时间延长而降低,选择OGD 6 h为后续造模时间,此时细胞活性降低至60.72%。在TMP给药浓度优化中发现5~50 mg·L^(-1)范围具有明显量效关系,故以此为后继实验浓度。与模型组相比,3个浓度的TMP均可明显增加NSCs活性、Nestin+/DAPI+阳性细胞数以及β3-Tubulin+/DAPI+阳性细胞数,10、50 mg·L^(-1)的TMP可延长放射性迁移距离,50 mg·L^(-1)的TMP还可促进NSCs的划痕修复并减少GFAP+/DAPI+阳性细胞数。但3个浓度对跨膜电阻均无明显效果。结论 TMP对体外NSCs微环境中NSCs的活性、增殖、迁移均有较好的促进作用,此外,在减少ACs方向化生的同时,还促进了神经元新生。Aim To study the ability of tetramethylpyrazine(TMP)on promoting neurogenesis in neural stem cell microenvironment after oxygen-glucose deprivation(OGD)injury in vitro.Methods Neural stem cells(NSCs),astrocytes(ACs)and cerebral microvascular endothelial cells(BMECs)were respectively extracted and separated to establish a co-culture system.The OGD modeling conditions were optimized by NSCs activity,and the concentration of TMP was optimized by Nissl staining.Then CCK-8 and Nestin+/DAPI+immunofluorescence assays were used to detect the proliferation of NSCs.The migration of NSCs was evaluated by radioactive migration and scratch repair assays.β3-tubulin+/DAPI+and GFAP+/DAPI+were employed to detect NSCs differentiation and neuronal axon growth.Transmembrane resistance(TEER)was measured to detect the integrity of the blood-brain barrier(BBB)in vitro.Results The activities of NSCs decreased by OGD injury in a time-dependent manner.The cell activity decreased to 60.72%when the duration of OGD was 6 h,which was used in the following studies.In the optimization of TMP concentration,5~50 mg·L^(-1) of TMP showed a significant dose-response relationship,and was employed for the subsequent experiment.Compared with the model group,5,10 and 50 mg·L^(-1) of TMP significantly increased the activities of NSCs,the number of Nestin+/DAPI+andβ3-tubulin+/DAPI+positive cells.Moreover,10 and 50 mg·L^(-1) of TMP enhanced the radioactive migration length.Additionally,50 mg·L^(-1)1 of TMP shortened the NSCs scratch distance and reduced GFAP+/DAPI+positive cells.However,there was no significant effect on the transmembrane resistance among the three doses of TMP.Conclusions TMP has the ability of promoting the activity,proliferation and migration of NSCs.Furthermore,it increased neurogenesis with the reduction of ACs formation in the NSCs microenvironment in vitro.

关 键 词：川芎嗪 神经元新生 神经干细胞微环境 神经干细胞 星形胶质细胞 脑微血管内皮细胞 

分 类 号：R-332[医药卫生] R284.1R322.8R329.24R338.1