不同浓度瑞芬太尼对新生大鼠海马区神经干细胞内钙浓度及细胞凋亡的影响

摘要

Objective To investigate the effect of different clinical doses of remifentanil on the apoptosis and the related intracellular Ca2 + concentration([Ca2 +]i )in newborn rat hippocampal neural stem cells(NSCs).Methods The hippocampal monoclonal NSCs strains of newborn SD rat were seeded into 12-well plates at the density of 5 ×105 cells/well,and cultured with 1 mL of DMEM/F12 medium containing 10% fetal bovine serum.NSCs were incubated in humidified incubator at 37 °C with 5% CO2 for 24 hours.Cells were assigned into four groups by different treatments as follows:(1)control group:NSCs were treated by saline;(2)low dose group (R5 group):NSCs were stimulated with 5 μg·L -1 of remifentanil;(3)middle dose group(R10 group):NSCs were incubated in 10 μg ·L -1 of remifentanil;(4)high dose group(R20 group):NSCs were stimulated with 20 μg·L -1 of remifentanil.In every group the treatment was repeated for four times.Laser scanning confocal microscope(LSCM)scanning was used after 300 s,600 s and 900 s of remifentanil treatment for detection of [Ca2 +]i .NSCs apoptosis was assayed by flow cytometry 24 hours after the treatment of remifen-tanil.Results Compared with control group,R5 group showed no significant changes in [Ca2 +]i (P >0.05)and the concentrations of [Ca2 +]i of NSCs in R10 and R20 group were increased markedly (P <0.05 and P <0.01,respectively).Compared with control group, NSCs in R5 group were almost not impaired,while the apoptosis of NSCs was significantly increased in R10 and R20 group(P <0.05 and P <0.01,respectively).Conclusions Low clinical dose of remifentanil has little effect on NSC apoptosis and the related [Ca2 +]i , while the middle and high doses of clinical remifentanil notably increase the level of [Ca2 +]i ,resulting in significant apoptosis in NSCs.%目的：探讨不同浓度瑞芬太尼对新生大鼠海马区神经干细胞凋亡及细胞内钙浓度的影响。方法采用已建立的新生SD 大鼠海马区神经干细胞单细胞克隆系细胞株，将5×108个·L －1活细胞的密度均匀地接种到的12孔培养板中，每孔加入1 mL 含有10％胎牛血清的 DMEM／F12的培养基，放入温度37℃、5％浓度 CO2培养箱中孵育24 h，镜下观察细胞球贴壁后，每孔分别加入生理盐水或不同浓度的瑞芬太尼（R），分为以下4组：（1）对照组，每孔加入生理盐水；（2）低浓度组，每孔加入瑞芬太尼5μg·L －1，即 R5组；（3）中浓度组，每孔加入瑞芬太尼10μg·L －1，即 R10组；（4）高浓度组，每孔加入瑞芬太尼20μg ·L －1，即 R20组。每组重复4次。加药后培养300、600、900 s 用激光扫描共聚焦显微镜（LSCM）检测各组神经干细胞内游离钙浓度的动态变化，24 h 后流式细胞仪检测各组神经干细胞的凋亡情况。结果（1）各组细胞内钙浓度变化情况：与对照组比较，R5组细胞内钙浓度差异无统计学意义（P ＞0．05）；R10、R20组细胞内钙浓度明显升高（其中 R10组 P ＜0．05，R20组 P ＜0．01）。（2）各组神经干细胞凋亡变化情况：与对照组比较，R5组神经干细胞凋亡差异无统计学意义（P ＞0．05）；R10、R20组神经干细胞凋亡明显升高（其中 R10组 P ＜0．05，R20组 P ＜0．01）。结论低浓度的临床有效血药浓度的瑞芬太尼对新生大鼠海马区神经干细胞内钙浓度、细胞凋亡没有影响，中高浓度的临床有效血药浓度的瑞芬太尼应用使细胞内钙浓度显著升高，从而诱发细胞大量凋亡。