SH-SY5Y细胞α7神经型尼古丁受体基因表达水平的改变对突触相关蛋白的影响

发布时间：2019-01-07 10:09

【摘要】：目的:研究神经母细胞瘤细胞(SH-SY5Y)α7神经型尼古丁受体基因(α7 n ACh R)表达增强及沉默后对细胞突触相关蛋白的影响,以及对Aβ引起的突触损伤的作用,探讨α7 n ACh R神经保护作用机制及在阿尔茨海默病(Alzheimer disease,AD)的发病机制中的作用。方法:(1)采用逆转录PCR的方法获取人α7 n ACh R基因,用T4 DNA连接酶将其连接到pc DNA 3.1质粒,构建重组体质粒pc DNA3.1-?7 n ACh R,α7 n ACh R sh RNA Plasmid购自Santa Curz公司;(2)通过稳定转染方法,筛选出α7 n ACh R基因高表达及沉默的SH-SY5Y细胞株;(3)用Real-time PCR法和蛋白免疫印迹(Western Blot)法分别测定细胞中囊泡相关蛋白(synaptophysin),突触前膜蛋白(SNAP-25)和突触后膜蛋白(PSD-95)m RNA和蛋白表达水平的变化;(4)用终浓度为1μmol/L的Aβ1-42寡聚体处理培养的SH-SY5Y细胞、空质粒组细胞、α7 n ACh R水平上调组和沉默组的细胞24 h,再用Real-time PCR法和蛋白免疫印迹(Western Blot)法分别测定细胞中囊泡相关蛋白synaptophysin,SNAP-25和PSD-95m RNA和蛋白表达水平的变化。结果:(1)成功构建pc DNA3.1-?7 n ACh R过表达质粒,转入SH-SY5Y细胞后,经G418筛选获得稳定转染细胞株,与对照组相比,α7 n ACh R m RNA及蛋白表达量分别增加了533%和110%;SH-SY5Y细胞转染α7 n ACh R sh RNA Plasmid之后经嘌呤霉素筛选,获得稳定转染α7 n ACh R sh RNA重组质粒的细胞株,与对照组相比,α7 n ACh R m RNA及蛋白表达量分别减少了95%和80%。(2)α7 n ACh R上调组的突触相关蛋白PSD-95、SNAP-25、SYP m RNA及蛋白表达都有明显的增加,而α7 n ACh R沉默组的突触相关蛋白PSD-95、SNAP-25、SYP的m RNA及蛋白表达明显的降低。(3)Aβ1-42寡聚体处理SH-SY5Y细胞和空质粒组细胞24小时后,PSD-95、SNAP-25、SYP m RNA及蛋白表达水平较正常对照组都有明显的降低(P0.01)。α7 n ACh R上调组经Aβ处理后,PSD-95、SNAP-25、SYP与Aβ处理组相比,m RNA及蛋白表达水平有明显的升高,而α7 n ACh R沉默组经Aβ处理后,与Aβ处理组相比,PSD-95、SNAP-25、SYP m RNA及蛋白表达水平有明显的降低。结论:上调SH-SY5Y细胞α7 n ACh R水平能够使细胞突触相关蛋白水平升高,而降低SH-SY5Y细胞α7 n ACh R水平会抑制细胞突触蛋白水平;同时α7 n ACh R受体水平的增加能够对抗Aβ对突触的损伤,降低α7 n ACh R水平使Aβ对细胞突触的毒性作用增强及突触损伤情况更严重。这可能提示了α7 n ACh R与细胞突触密切相关,并且α7 n ACh R的增加可对Aβ诱导神经细胞的突触损伤有一定的保护作用,进一步说明α7n ACh R在阿尔茨海默病的发病中起着重要作用。
[Abstract]:Aim: to study the effect of 伪 7 n ACh R) expression on synaptophysin in neuroblastoma cells (SH-SY5Y) and the synaptic injury induced by A 尾. To explore the neuroprotective mechanism of 伪 7 n ACh R and its role in the pathogenesis of Alzheimer's disease (Alzheimer disease,AD). Methods: (1) Human 伪 7 n ACh R gene was obtained by reverse transcription PCR and ligated to pc DNA 3.1 plasmid by T4 DNA ligase. The recombinant pc DNA3.1-?7 n ACh R, 伪 7 n ACh R sh RNA Plasmid was obtained from Santa Curz Company. (2) SH-SY5Y cell lines with high expression and silencing of 伪 7 n ACh R gene were screened by stable transfection. (3) the expression of (synaptophysin), presynaptic membrane protein (SNAP-25), postsynaptic membrane protein (PSD-95) m RNA) and postsynaptic membrane protein (PSD-95) m RNA) were measured by Real-time PCR and Western blot (Western Blot). (4) the cultured SH-SY5Y cells were treated with A 尾 1-42 oligodeoxynucleotides (A 尾 1-42) at the final concentration of 1 渭 mol/L for 24 h. The cells in the empty plasmid group, 伪 7 n ACh R upregulation group and silencing group were treated for 24 h. The expression levels of vesicular associated protein (synaptophysin,SNAP-25), PSD-95m RNA and protein were determined by Real-time PCR and Western blot (Western Blot) respectively. Results: (1) pc DNA3.1-?7 n ACh R overexpression plasmid was successfully constructed and transfected into SH-SY5Y cells. The expression of 伪 7 n ACh R m RNA and protein increased by 53 3% and 110%, respectively. SH-SY5Y cells transfected with 伪 7 n ACh R sh RNA Plasmid were screened by purine mycin, and then stable transfected with 伪 7 n ACh R sh RNA recombinant plasmid was obtained, which was compared with the control group. The expression of 伪 7 n ACh R m RNA and protein decreased by 95% and 80%, respectively. (2) the expression of PSD-95,SNAP-25,SYP m RNA and protein in 伪 7 n ACh R upregulated group was significantly increased. In 伪 7 n ACh R silencing group, m RNA and protein expression of PSD-95,SNAP-25,SYP were significantly decreased. (3) A 尾 1-42 oligomer treated SH-SY5Y cells and empty plasmid group for 24 hours, PSD-95,SNAP-25,. The expression of SYP m RNA and protein in 伪 7 n ACh R upregulated group was significantly lower than that in normal control group (P0.01). After treatment with A 尾, the expression of, m RNA and protein in 伪 7 n ACh R upregulated group was significantly higher than that in A 尾 treated group. The expression of PSD-95,SNAP-25,SYP m RNA and protein in 伪 7 n ACh R silencing group was significantly lower than that in A 尾 treated group. Conclusion: upregulation of 伪 7 n ACh R level in SH-SY5Y cells can increase the level of synaptophysin, while decreasing 伪 7 n ACh R level in SH-SY5Y cells can inhibit the level of synaptic protein. At the same time, the increase of 伪 7 n ACh R receptor level could antagonize the synaptic damage caused by A 尾, and the decrease of 伪 7 n ACh R level increased the toxicity of A 尾 to the synapse and made the synaptic injury more serious. This may suggest that 伪 7 n ACh R is closely related to the synapse of the cells, and the increase of 伪 7 n ACh R may protect the synaptic injury induced by A 尾. It is further demonstrated that 伪 7n ACh R plays an important role in the pathogenesis of Alzheimer's disease.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.16

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