不同临床有效浓度异丙酚对新生大鼠海马区来源神经干细胞增殖分化和凋亡的影响

Effects of Different Clinical Effective Concentrations of Propofol on Proliferation,Differentiation and Apoptosis of Neural Stem Cells Isolated from Newborn Rat Hippocampus

目的:探讨不同临床有效浓度异丙酚对新生大鼠海马区来源神经干细胞增殖分化及凋亡的影响。方法:采用已建立的新生SD大鼠海马区来源神经干细胞单细胞克隆系细胞株,将其均匀地接种到24或48孔培养板中(含血清培养基培养孔用于分化和凋亡检测,无血清培养基培养孔用于增殖周期和凋亡检测)。将细胞随机分为五组(每组5个培养孔):⑴低异丙酚浓度组(P1组):终浓度2.5μg/mL;⑵中异丙酚浓度组(P2组):终浓度5.0μg/mL;⑶高异丙酚浓度组(P3组):终浓度10.0μg/mL;⑷C组:正常(control)对照组;⑸L组:脂肪乳(introlipid)对照组。培养8 h后,5-溴脱氧尿(Brdu)检测细胞增殖周期,培养48 h后,流式细胞仪检测各组神经干细胞增殖分化过程中的增殖和凋亡情况;72 h免疫荧光技术检测神经元特异性微管蛋白抗体(β-tubulin)和星形胶质细胞特异性胶质酸性蛋白(GFAP),观察神经干细胞分化的各类神经细胞形态学变化、分化比率及分化细胞的凋亡情况。结果:与C组比较,L组细胞增殖周期和分化比率无明显差异(P>0.05),分化细胞无明显凋亡且细胞形态正常;P2和P3组细胞增殖周期显著抑制,增殖和分化过程中凋亡比率明显升高(其中P2组P<0.05;P3组P<0.01),分化细胞的突触或轴突发育差且发生大量凋亡,未凋亡的星形胶质细胞增生肥大;P1组神经元分化比率高(P<0.05)。结论:中高浓度异丙酚均明显抑制新生大鼠海马区来源神经干细胞的增殖和分化,并诱发神经细胞的大量凋亡,影响到分化细胞的神经突起分支、树突棘的生长发育;低浓度异丙酚能诱导神经干细胞向神经元细胞分化。

Objective To investigate the effects of different clinical effective concentrations of propofol on the proliferation,differentiation and apoptosis of neural stem cells( NSCs) isolated from newborn rat hippocampus. MethodsThe hippocampal derived monoclonal NSCs from newborn SD rat were cultured with serum medium or serum-free medium in 24 or 48-well culture plates,so as to determine the proliferation,apoptosis or cell cycle. Then all the cells were randomly divided into 5groups( n = 5),including control group( group C),introlipid group( group L) and propofol treated groups( group P1,P2,P3were treated with propofol with the doses of 2. 5,5. 0,10. 0 μg / mL,respectively). The proliferative cycle was determined with 5-Brdu after cultured for 8 h,the proliferation and apoptosis of NSCs was detected by flow cytometry after cultured for48 h,the contents of protein β-tubulin and glial fibrillary acidic protein( GFAP) were determined with fluorescence staining after cultured for 72 h. The morphological changes,differentiaation ratio and apoptosis of NSCs were observed. Results The proliferative cycle and differentiation ratio had no significant difference between group C and group L( P > 0. 05),and thedifferentiated NSCs had no apoptosis and abnormal morphology in this two groups. The proliferative cycle of group P 2 and P 3 were significantly inhibited,and the differentiation ratio was increased in the progress of proliferation and differention( group P 2,P <0. 05; group P 3,P <0. 01). The synapses or axons of differentiated cells were poor development,most cells were apoptosis and the non-apoptotic astrocytes were hyperplasia or hypertrophy. The differentiation ratio of neuron in group P 1 was higher than that of group C( P < 0. 05). Conclusion Low concentration of propofol could induce NSCs differentiation to neurons. Median and high concentrations of propofol could inhibit proliferation and differentiation,induce apoptosis of NSCs isolated from newborn rat hippocampus,and even affect the development of nervous apophysis branch and dendritic spine.