重组人神经调节蛋白-1β在氧和葡萄糖剥夺条件下通过细胞外信号调节激酶信号通路对小鼠神经干细胞增殖的影响

摘要

目的 观察重组人神经调节蛋白(recombinant neuregulin-1beta,rhNRG)-1β在氧和葡萄糖剥夺(oxygen-glucose deprivation,OGD)条件下通过细胞外信号调节激酶(ERK)信号通路对小鼠神经干细胞增殖能力的影响.方法 将怀孕14～ 17 d的小鼠处死,分离脑室管膜下区的脑组织,提取神经干细胞.采用体外细胞培养方法进行细胞传代和鉴定.神经干细胞鉴定采用SABC-FITC (POD)双标试剂盒,标记免疫荧光细胞化学法镜下检测巢蛋白(Nestin)表达.空白对照组:取鉴定后的神经干细胞,细胞数为2×107个,放入24孔培养板中,DMEM/F12完全培养液培养3 h;OGD组:取鉴定后神经干细胞,细胞数为2×107个,放入24孔培养板中,加入不含葡萄糖的DMEM/F12培养液,将培养板移入潮湿密闭容器内(37℃恒温培养),容器内含有氮气(950ml/L)和氧气(50 ml/L)的混合气体,缺糖缺氧培养1h后,再更换完全培养液培养3 h;OGD+ rhNRG-1β组:在OGD干预前先加入100 μg/L rhNRG-1β,再培养3h.神经球形成:分别取3组干细胞,反复吹打成单细胞,按细胞个数1×106/ml将神经干细胞接种到含多聚赖氨酸盖玻片上的培养板中,培养7d后取出盖玻片,相差镜下观察3组于细胞神经球形成,计算神经干细胞增殖变化;克隆形成:分别取3组于细胞,接种于60 mm培养皿中,细胞个数2×107个,完全培养液培养24 h,相差镜下观察3组细胞克隆形成,计算神经干细胞增殖变化;蛋白免疫印迹法(Western blot):测定3组干细胞磷酸化ERK(pERK)蛋白的变化,观察rbNRG-1β通过ERK信号通路上调pERK蛋白表达对小鼠神经干细胞增殖能力的影响.结果 镜下原代培养的神经干细胞呈单个或成对生长,形态呈圆形;神经干细胞增殖呈团状集落;可见带有黄绿色荧光的特异性Nestin蛋白标记的神经干细胞.神经干细胞神经球数量、平均直径组间比较差异有统计学意义(F=693.66、1 002.09,P均＜0.01),其中OGD组神经干细胞神经球形成受到显著抑制,形成数量、平均直径[(88.78±7.14)个、(62.12±2.52)μm]明显低于对照组[(246.34土8.67)个、(128.45±2.33)μm]和OGD+rh-NRG-1β组[(237.87±6.61)个、(118.37±2.71) μm,P均＜0.01].神经干细胞克隆形成率组间比较差异有统计学意义(F=132.03,P＜0.01),其中OGD组神经干细胞克隆形成受到显著抑制,形成率[(11.65±0.94)％]明显低于对照组[(33.23士2.93)％]和OGD+ rh-NRG-1β组[(31.42土2.61)％,P均＜0.01].Western blot显示,神经干细胞pERK蛋白表达组间比较差异有统计学意义(F=63.76,P＜0.01),其中OGD组pERK蛋白的相对表达量(0.487±0.072)明显低于对照组(1.013±0.112)和OGD+ rh-NRG-1β组(1.752±0.278,P均＜0.01).结论 在OGD条件下,pERK蛋白表达下降,小鼠神经干细胞增殖受到抑制;rhNRG-1β能上调pERK蛋白表达,促进小鼠神经干细胞的增殖.小鼠神经干细胞的增殖能力受ERK信号通路的影响.%Objective To investigate the influence of recombinant neuregulin-1 beta (rhNRG-1β) on neural stem cell proliferation through extracellular regulated protein kinases (ERK) signaling pathway in oxygen and glucose deprivation (OGD) environment.Methods Neural stem cells were obtained from embryonic brain of mice pregnant for 14-17 d,cultured and identified by immunochemical staining through detection of the indicator nestin using the SABC-FITC (POD)double standard kit.Neural stem cells were divided into three experiment groups (OGD group,control group and OGD + rhNRG-1β group).Control group:identified neural stem cells,2 × 107,were cultured for 3 h in the 24-hole culture plate with DMEM/F12 complete culture medium;OGD group:neural stem cells,2 × 107,were cultured in the 24-hole culture plate deprived glucose DMEM/F12 in a wet airtight container (37 ℃ constant temperature),cells were cultured with mixed gas of nitrogen (950 ml/L) and oxygen (50 ml/L) for 1 h,and then the culture medium was replaced with complete culture medium and cultured for 3 h;OGD + rhNRG-1β group:before OGD intervention,100 μg/L rhNRG-1β was given for 3 h.Neurospheres formation:the three groups of stem cells were dispersed into single cells,1 × 106/ml cells were inoculated to culture plates containing cover slips coated with poly lysine,and cultivated for 7 d,and neurospheres formation of the 3 groups of neural stem cells was observed under microscope,which was aimed to record neural stem cells proliferation changes.Colony formation:the three groups of stem cells,vaccinated in 60 mm in a petri dish,2 × 107 in number,were cultivated in complete culture medium for 24 h.The colony formation of the three groups of cells was observed under microscope,and neural stem cells proliferation changes were observed.Western blotting:the change of phosphorylation ERK (pERK) protein of the three groups of stem cells was determined,and the effect of pERK protein expression regulated by rhNRG-1β in mice neural stem cells proliferation through ERK signaling pathway was observed.Results Microscopically the primary cultured stem cells grew in single or in pairs,in a round shape;neural stem cell proliferated in clumps or colony;neural stem cells expressed the specificity of nestin protein markers with fluorescent yellow-green color.The differences in the aspects of the average diameter of neurospheres and neurospheres quantity in the neural stem cells between groups were statistically significant (F =693.66,1 002.09,all P ＜ 0.01),and among them the neuropheres formation of OGD group was significantly suppressed.Formation quantity and average diameter in OGD group [(88.78 ± 7.14) numbers,(62.12 ± 2.52) μm] were significantly lower than those in the control group [(246.34 ± 8.67) numbers,(128.45 ± 2.33) μm] and those in OGD + rhNRG-1β group [(237.87 ± 6.61) numbers,(118.37 ± 2.71) μm,all P ＜ 0.01].The difference of colony formation rate of neural stem cell was statistically significant (F =132.03,P ＜ 0.01),and among them colony formation of OGD group significantly suppressed.Formation rate in OGD group [(11.65 ± 0.94)％] was significantly lower than that in the control group [(33.23 ± 2.93)％] and that in OGD + rhNRG-1β group [(31.42 ± 2.61)％,all P ＜ 0.01].Western blotting showed that the difference of pERK protein expression of neural stem cells between groups was statistically significant (F =63.76,P ＜ 0.01).Relative expression of the pERK protein in OGD group (0.487 ± 0.072) was significantly lower than that in the control group (1.013 ± 0.112) and that in OGD + rh-NRG-1β group (1.752 ± 0.278,all P ＜ 0.01).Conclusion rhNRG-1β preserves neural stem cell proliferation with phosphorylation ERK protein expression up-regulated in oxygen and glucose deprivation environment.