摘要
目的:观察不同浓度BDNF对Aβ致伤神经元突触形态及突触蛋白Ng、Nm表达的影响。方法:大鼠海马神经元体外常规培养、鉴定,建立Aβ1-42寡聚体致伤神经元模型。应用免疫荧光细胞化学法,检测不同浓度BDNF与体外培养的Aβ1-42致伤神经元共孵育,对突触蛋白Ng、Nm表达水平的影响。结果:Aβ1-42与神经元共孵育24h后,10μmol/L、20μmol/L Aβ1-42神经细胞生存率较对照组明显降低,凋亡率较对照组明显增高(P<0.05)。Aβ致伤后神经细胞明显减少,胞体小,突起变短、消失。与0ng/mL组比较,BDNF5ng/mL、20ng/mL、100ng/mL浓度组随着浓度增高,神经细胞存活状态有所恢复,有较多的圆形细胞和悬浮细胞,多数细胞突起数量较少、长度变短。正常对照组Ng、Nm免疫荧光染色表达强阳性,BDNF、10μmol/L Aβ1-42与神经元共孵育24h,海马神经细胞Ng、Nm平均荧光强度较正常对照组明显降低(P<0.01);与0ng/mL浓度组相比,其他各组(5ng/mL、20ng/mL、100ng/mL浓度)Ng、Nm荧光强度明显增强(P<0.01),但与正常对照组相比Ng、Nm荧光强度仍较弱(P<0.05)。结论:10μmol/L是Aβ1-42致伤海马神经元的较佳有效浓度;Aβ1-42可导致突触蛋白Ng、Nm的表达减少;BDNF可以通过抑制Aβ1-42寡聚体所致突触蛋白Ng、Nm减少,发挥突触修复及神经保护作用。
Objective：Study the effects of various concentrations of BDNF against the synapses damage introduced by Aβ and the expressions of synaptic proteins Ng and Nm.Methods：We cultured and identified the hippocampal neurons in vitro rat; established the Aβ1-42 oligomer injured neuron model; detected the cell metabolic activity through MTT; explored the apoptosis rate of neuronal cells by TUNEL method; cultured in vitro with different concentrations of BDNF（0,5,20,100ng/mL） and neurons injured by Aβ1-42 oligomer; detected the effects of different concentrations of BDNF against the expressions of synaptic proteins Ng and Nm by the immunofluorescence cytochemistry method.Results：Neurons were incubated with Aβ1-42 oligomer for 24h,apoptotic neurons was significantly reduced,the refractive index decreased or disappeared,protrusions began to retract,breaking branches connected to a large area of retraction,the cells surrounding the halo was gone,cells were punctuate scattered.With 2μmol/L and 5μmol/L Aβ1-42 oligomer,there is no significant difference for the survival and apoptosis rates of nerve cells,compared with the control group（P〈0.05）.Regarding as 10 and 20μmol/L,the survivals of nerve cells were significantly reduced compared with the control group,with apoptosis rate increased（P〈0.05 and P〈0.01）.In control group,differentiated cells protrusions gradually extended,intensive,woven into a network with neighboring differentiated cells.With the treatment of Aβ1-42 oligomer and 0ng/mL of BDNF,the nerve cells were significantly reduced and their bodies are small,protruding shorter disappears.Compared with 0ng/mL,5,20,100ng/mL concentrations BDNF introduced increase of nerve cell survivals.There are more round and floating cells,the majority of cell processes shorter,the projection of the number of cells and the number of projection neurons were reduced.Within the normal control group,Ng and Nm immun of luorescence staining strongly positive expression.When neurons were incubated with BDNF and 10μmol/L Aβ1-42 oligomer for 24h,the expressions of Ng and Nm in injured primary hippocampal nerve cells were significantly decreased,with the value being lower than that of control group significantly（P〈0.01）.With the increased concentrations of BDNF（5,20,100ng/mL concentration）,the expressions of Ng and Nm were increased with significantly increased fluorescence intensity（P〈0.01）.In addition,the fluorescence intensity in the highdose group has not significant difference（P〈0.05）,but compared with the normal control group,the expressions of Ng and Nm were still weak,the difference was significant（P〈0.05）.Conclusion：The preferred effective concentration of Aβ1-42 oligomer was 10μmol/L,to introduced the injured hippocampal neurons.With the treatment of Aβ1-42 oligomer,the expressions of Ng and Nm were significantly decreased.The action mechanism of BDNF can reduce the number of Ng and Nm by inhibiting Aβ1-42 oligomer and gave play to synapse repair and neurons protection.
出处
《黑龙江医药科学》
2013年第6期7-10,共4页
Heilongjiang Medicine and Pharmacy
基金
黑龙江省自然科学基金面上项目
编号:D201017
黑龙江省教育厅科研项目
编号:12531666和12521543
