海马胆碱能神经元刺激肽促进海马胆碱能神经元再生的研究

The effect of hippocampal cholinergic neurostimulating peptide in promoting hippocampal cholinergic neural regeneration

目的:探讨海马胆碱能神经元刺激肽(HCNP)对切割穹窿海马伞后海马胆碱能神经元的促进作用。方法:36只SD大鼠随机分成两组,切割右侧穹窿海马伞后,术后连续5d腹腔注射BrdU,经侧脑室分别注射HCNP和人工脑脊液(ACSF),每3天1次,共6次。切割术前、术后及治疗术后行Morris水迷宫行为学测试;术后第35天,灌注取脑行溴脱氧尿苷(5-BrdU)、胆碱乙酰转移酶(ChAT)免疫荧光检测;提取总RNA,RT-PCR检测ChAT mRNA表达量;提取总蛋白,Western blot检测ChAT蛋白表达。结果:切割穹窿海马伞后,两组大鼠平均逃避潜伏期(AEL)均明显增加,记忆力下降,经过治疗后,实验组大鼠AEL显著缩短,对照组无改善,两组AEL有显著性差异(P<0.05);HCNP组海马齿状回颗粒下层可见较多BrdU阳性细胞及海马ChAT阳性神经元,ACSF组仅见少量BrdU阳性细胞,未见ChAT阳性神经元;RT-PCR结果显示,HCNP组ChAT mRNA约为ACSF组2倍(P<0.01);Western blot结果表明,实验组ChAT蛋白相对表达量为0.412±0.018、对照组为0.218±0.017,实验组明显高于对照组,二者存在显著性差异(P<0.01)。结论:HCNP可促进海马齿状回颗粒下层神经干细胞的增殖及其向胆碱能神经元分化。

Objective： To investigate the promuting effect of hippocampal cholinergic neurostimulating peptide （HCNP） on hippocampal neural stem cells （NSCs） differentiation into neurons or cholinergic neurons. Method： Thirty six SD rats were randomly divided into experimental group and control group. The right timbria-fornix were transected by three-diamensional positioning techonology. BrdU was injected into the rats＇ peritoneal for 5 consecutive days to label hippocampal autologous NSCs after surgery. HCNP or ACSF was injected into intracerebroventricular on the 9th day after surgery and supplemented every 3 days for a total of 6 times. The cognitive function of rats were detected by Morris water maze before and after right fimbria-fornix transection, also after treatment; The cholinergic neurons derived from hippocampal NSCs were detected by BrdU/ChAT immunofluorescence on the postoperative 35th day. Total RNA was extracted to detect the expression of ChAT mRNA by Real-time PCR; Total protein was extracted to detect the expression of ChAT protein by Western blot. Result： Morris water maze results showed that the average escape latency （AEL） in both of two groups increased clearly, memory lost after surgery. The AEL of experimental group was significantly shorter in the group treated by HCNP than that in the control group treated by ACSF compared with that after the fight timbria fomix transected （P〈0.05）; The immunofluorescence results indicated that more BrdU positive ceils, ChAT positive neurons and ChAT/BrdU double-labeled cells in the subgranular layer of hippocampal dentate gyrus and hilus area in experimental group; while in control group, only a few BrdU positive cells, ChAT positive neurons and ChAT/BrdU double-labeled ceils could be detected; Real-time PCR results showed that ChAT mRNA in experimental group was two-folds higher than that in the control group （P〈0.01）; Western blot results showed that the expression of ChAT protein in experimental group was significantly higher than that in control group （P〈0.01）.Conclusion： This study demonstrated that HCNP could promote the proliferation and cholinergic differentiation of NSCs in hippocampal dentate gyrus.