【摘要】：目的:研究神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y)α7神經(jīng)型尼古丁受體基因(α7 n ACh R)表達(dá)增強(qiáng)及沉默后對(duì)細(xì)胞突觸相關(guān)蛋白的影響,以及對(duì)Aβ引起的突觸損傷的作用,探討α7 n ACh R神經(jīng)保護(hù)作用機(jī)制及在阿爾茨海默病(Alzheimer disease,AD)的發(fā)病機(jī)制中的作用。方法:(1)采用逆轉(zhuǎn)錄PCR的方法獲取人α7 n ACh R基因,用T4 DNA連接酶將其連接到pc DNA 3.1質(zhì)粒,構(gòu)建重組體質(zhì)粒pc DNA3.1-?7 n ACh R,α7 n ACh R sh RNA Plasmid購(gòu)自Santa Curz公司;(2)通過穩(wěn)定轉(zhuǎn)染方法,篩選出α7 n ACh R基因高表達(dá)及沉默的SH-SY5Y細(xì)胞株;(3)用Real-time PCR法和蛋白免疫印跡(Western Blot)法分別測(cè)定細(xì)胞中囊泡相關(guān)蛋白(synaptophysin),突觸前膜蛋白(SNAP-25)和突觸后膜蛋白(PSD-95)m RNA和蛋白表達(dá)水平的變化;(4)用終濃度為1μmol/L的Aβ1-42寡聚體處理培養(yǎng)的SH-SY5Y細(xì)胞、空質(zhì)粒組細(xì)胞、α7 n ACh R水平上調(diào)組和沉默組的細(xì)胞24 h,再用Real-time PCR法和蛋白免疫印跡(Western Blot)法分別測(cè)定細(xì)胞中囊泡相關(guān)蛋白synaptophysin,SNAP-25和PSD-95m RNA和蛋白表達(dá)水平的變化。結(jié)果:(1)成功構(gòu)建pc DNA3.1-?7 n ACh R過表達(dá)質(zhì)粒,轉(zhuǎn)入SH-SY5Y細(xì)胞后,經(jīng)G418篩選獲得穩(wěn)定轉(zhuǎn)染細(xì)胞株,與對(duì)照組相比,α7 n ACh R m RNA及蛋白表達(dá)量分別增加了533%和110%;SH-SY5Y細(xì)胞轉(zhuǎn)染α7 n ACh R sh RNA Plasmid之后經(jīng)嘌呤霉素篩選,獲得穩(wěn)定轉(zhuǎn)染α7 n ACh R sh RNA重組質(zhì)粒的細(xì)胞株,與對(duì)照組相比,α7 n ACh R m RNA及蛋白表達(dá)量分別減少了95%和80%。(2)α7 n ACh R上調(diào)組的突觸相關(guān)蛋白PSD-95、SNAP-25、SYP m RNA及蛋白表達(dá)都有明顯的增加,而α7 n ACh R沉默組的突觸相關(guān)蛋白PSD-95、SNAP-25、SYP的m RNA及蛋白表達(dá)明顯的降低。(3)Aβ1-42寡聚體處理SH-SY5Y細(xì)胞和空質(zhì)粒組細(xì)胞24小時(shí)后,PSD-95、SNAP-25、SYP m RNA及蛋白表達(dá)水平較正常對(duì)照組都有明顯的降低(P0.01)。α7 n ACh R上調(diào)組經(jīng)Aβ處理后,PSD-95、SNAP-25、SYP與Aβ處理組相比,m RNA及蛋白表達(dá)水平有明顯的升高,而α7 n ACh R沉默組經(jīng)Aβ處理后,與Aβ處理組相比,PSD-95、SNAP-25、SYP m RNA及蛋白表達(dá)水平有明顯的降低。結(jié)論:上調(diào)SH-SY5Y細(xì)胞α7 n ACh R水平能夠使細(xì)胞突觸相關(guān)蛋白水平升高,而降低SH-SY5Y細(xì)胞α7 n ACh R水平會(huì)抑制細(xì)胞突觸蛋白水平;同時(shí)α7 n ACh R受體水平的增加能夠?qū)笰β對(duì)突觸的損傷,降低α7 n ACh R水平使Aβ對(duì)細(xì)胞突觸的毒性作用增強(qiáng)及突觸損傷情況更嚴(yán)重。這可能提示了α7 n ACh R與細(xì)胞突觸密切相關(guān),并且α7 n ACh R的增加可對(duì)Aβ誘導(dǎo)神經(jīng)細(xì)胞的突觸損傷有一定的保護(hù)作用,進(jìn)一步說(shuō)明α7n ACh R在阿爾茨海默病的發(fā)病中起著重要作用。
[Abstract]:Aim: to study the effect of 偽 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 偽 7 n ACh R and its role in the pathogenesis of Alzheimer's disease (Alzheimer disease,AD). Methods: (1) Human 偽 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, 偽 7 n ACh R sh RNA Plasmid was obtained from Santa Curz Company. (2) SH-SY5Y cell lines with high expression and silencing of 偽 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, 偽 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 偽 7 n ACh R m RNA and protein increased by 53 3% and 110%, respectively. SH-SY5Y cells transfected with 偽 7 n ACh R sh RNA Plasmid were screened by purine mycin, and then stable transfected with 偽 7 n ACh R sh RNA recombinant plasmid was obtained, which was compared with the control group. The expression of 偽 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 偽 7 n ACh R upregulated group was significantly increased. In 偽 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 偽 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 偽 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 偽 7 n ACh R silencing group was significantly lower than that in A 尾 treated group. Conclusion: upregulation of 偽 7 n ACh R level in SH-SY5Y cells can increase the level of synaptophysin, while decreasing 偽 7 n ACh R level in SH-SY5Y cells can inhibit the level of synaptic protein. At the same time, the increase of 偽 7 n ACh R receptor level could antagonize the synaptic damage caused by A 尾, and the decrease of 偽 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 偽 7 n ACh R is closely related to the synapse of the cells, and the increase of 偽 7 n ACh R may protect the synaptic injury induced by A 尾. It is further demonstrated that 偽 7n ACh R plays an important role in the pathogenesis of Alzheimer's disease.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R749.16

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