本文選題：阿爾茨海默氏病 + β-淀粉樣蛋白��； 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討激活大鼠腦組織原代星形膠質(zhì)細(xì)胞的α7尼古丁乙酰膽堿能受體(nicotinic acetylcholine receptor,nAChR)調(diào)控β-淀粉樣蛋白(β-amyloid peptide,Aβ)聚集的研究。方法:分離新生大鼠大腦皮質(zhì),培養(yǎng)原代星形膠質(zhì)細(xì)胞并純化、傳代,使用細(xì)胞免疫熒光的方法鑒定細(xì)胞及純度;體外制備Aβ1-42寡聚體;將培養(yǎng)細(xì)胞分為對(duì)照組、α7 nAChR激動(dòng)劑組、α7 nAChR阻斷劑組、PI3K/Akt信號(hào)通路抑制劑組以及Aβ組、激動(dòng)劑+Aβ組、阻斷劑+Aβ組。激動(dòng)劑有尼古丁、PNU、s24795,阻斷劑為甲基牛扁亭(methyllycaconitine,MLA)、LY294002。用蛋白印跡法(Western blotting)檢測(cè)星形膠質(zhì)細(xì)胞內(nèi)源性B-晶狀體蛋白(αB-crystallin,Cryab)、磷酸化Akt、Aβ的表達(dá)。結(jié)果:采用免疫熒光法鑒定所培養(yǎng)的細(xì)胞,結(jié)果顯示星形膠質(zhì)細(xì)胞占所有細(xì)胞的95%以上。檢測(cè)內(nèi)源性Cryab蛋白表達(dá)結(jié)果顯示:(1)尼古丁組與對(duì)照組比較,尼古丁組的內(nèi)源性Cryab表達(dá)明顯增高(P0.05),MLA阻斷劑組與尼古丁組比較,阻斷劑組的內(nèi)源性Cryab表達(dá)降低(P0.01),LY294002抑制劑組與尼古丁組比較,抑制劑組的內(nèi)源性Cryab表達(dá)降低(P0.01);(2)PNU組與對(duì)照組比較,PNU組的內(nèi)源性Cryab的表達(dá)增加(P0.05),MLA阻斷劑組與PNU組相比,內(nèi)源性Cryab表達(dá)降低(P0.01),LY294002抑制劑組與PNU組比較,抑制劑組的內(nèi)源性Cryab表達(dá)降低(P0.01);(3)s24795組與對(duì)照比較,s24795組的內(nèi)源性Cryab表達(dá)明顯升高(P0.01),MLA阻斷劑組與s24795組比較,阻斷劑組的內(nèi)源性Cryab表達(dá)明顯降低(P0.01),LY294002抑制劑組與s24795組比較,抑制劑組的內(nèi)源性cryab表達(dá)降低(p0.01)。檢測(cè)磷酸化akt蛋白的表達(dá)結(jié)果為:(1)尼古丁組與對(duì)照組比較,尼古丁組的磷酸化akt表達(dá)明顯增高(p0.05),mla阻斷劑組與尼古丁組比較,阻斷劑組的磷酸化akt表達(dá)降低(p0.01),ly294002抑制劑組與尼古丁組比較,抑制劑組的磷酸化akt表達(dá)降低(p0.01);(2)pnu組與對(duì)照組比較,pnu組的磷酸化akt的表達(dá)增加(p0.05),mla阻斷劑組與pnu組相比,磷酸化akt表達(dá)降低(p0.01),ly294002抑制劑組與pnu組比較,抑制劑組的磷酸化akt表達(dá)降低(p0.01);(3)s24795組與對(duì)照比較,s24795組的磷酸化akt表達(dá)明顯升高(p0.05),mla阻斷劑組與s24795組比較,阻斷劑組的磷酸化akt表達(dá)明顯降低(p0.01),ly294002抑制劑組與s24795組比較,抑制劑組的磷酸化akt表達(dá)降低(p0.01)。檢測(cè)aβ蛋白的表達(dá)結(jié)果為:(1)對(duì)照組正常的細(xì)胞內(nèi)沒(méi)有aβ蛋白的表達(dá),尼古丁組與aβ組相比,尼古丁組的aβ蛋白表達(dá)均明顯降低(p0.01),ly294002抑制劑組尼古丁組比較,抑制劑組的aβ蛋白的表達(dá)均明顯升高(p0.01);(2)對(duì)照組正常的細(xì)胞內(nèi)沒(méi)有aβ蛋白的表達(dá),pnu組與aβ組相比,pnu組的aβ蛋白表達(dá)均明顯降低(p0.01),ly294002抑制劑組與pnu組比較,抑制劑組的aβ蛋白的表達(dá)均升高(p0.01);(3)對(duì)照組正常的細(xì)胞內(nèi)沒(méi)有aβ蛋白的表達(dá),細(xì)胞總蛋白中,對(duì)照組aβ蛋白無(wú)表達(dá),s24795組與aβ組相比,s24795組的aβ蛋白表達(dá)降低(p0.05),ly294002抑制劑組與s24795組比較,抑制劑組的aβ蛋白的表達(dá)明顯升高(p0.01)。結(jié)論:尼古丁、pnu以及s24795通過(guò)激活星形膠質(zhì)細(xì)胞α7nachrs上調(diào)內(nèi)源性cryab從而抑制aβ集聚;pi3k/akt信號(hào)通路很可能是這3種激動(dòng)劑通過(guò)星形膠質(zhì)細(xì)胞α7nachrs上調(diào)內(nèi)源性cryab從而抑制aβ集聚過(guò)程的重要參與因素。為進(jìn)一步研究cryab蛋白可能是治療ad的一個(gè)潛在靶點(diǎn)提供了一定的基礎(chǔ)。
[Abstract]:Objective: To investigate the effect of nicotinic acetylcholine receptor (nAChR) on the aggregation of beta amyloid (beta -amyloid peptide, A beta) protein (beta -amyloid peptide, A beta) that activates primary astrocytes in the brain tissue of rats. Methods: to isolate the cerebral cortex of neonatal rats and to cultivate primary astrocytes and to purify, subculture, and use cell free cells. A beta 1-42 oligomer was prepared by the method of immunofluorescence, and the cultured cells were divided into control group, alpha 7 nAChR agonist group, alpha 7 nAChR blocker group, PI3K/Akt signaling pathway inhibitor group and A beta group, agonist +A beta group, blocker +A beta group. The agonist was niodein, PNU, s24795, and blocker was methyl bull kiosk (methyllycaconit) Ine, MLA), LY294002. was used to detect the endogenous B- lens protein (alpha B-crystallin, Cryab) and the expression of phosphorylated Akt and A beta in astrocytes by Western blotting. Results: the immunofluorescence method was used to identify the cultured cells. The results showed that astrocytes were more than 95% of all cells. The expression of endogenous Cryab protein was detected. The results showed: (1) the endogenous Cryab expression in nicotine group was significantly higher than that of the control group (P0.05). The endogenous Cryab expression in the blocker group was lower than the nicotine group in the MLA blocker group (P0.01), the LY294002 inhibitor group was compared with the nicotine group, and the endogenous Cryab expression was reduced (P0.01) in the inhibitor group (P0.01); (2) the PNU group and the control group were (P0.01). The endogenous Cryab expression in the PNU group was increased (P0.05). The endogenous Cryab expression in the MLA blocker group was lower than that in the PNU group (P0.01). The endogenous Cryab expression of the inhibitor group decreased (P0.01) compared with the PNU group. (3) the endogenous expression of the inhibitor group was significantly higher than that of the control group. Compared with the s24795 group, the endogenous Cryab expression in the blocker group was significantly decreased (P0.01). The endogenous CRYAB expression in the inhibitor group was lower than that in the s24795 group (P0.01). The results of the expression of the phosphorylated Akt protein were: (1) the nicotine group was significantly higher than the control group, and the expression of Akt in the nicotine group was significantly higher (P0.05), MLA resistance. Compared with nicotine group, the expression of phosphorylated Akt in the blocker group decreased (P0.01), the LY294002 inhibitor group was compared with the nicotine group, and the phosphorylated Akt expression of the inhibitor group decreased (P0.01). (2) the expression of phosphorylated Akt in the PNU group increased (P0.05) compared with the control group (P0.05), and the MLA blocker group was lower than the PNU group, and the phosphorylated Akt expression decreased. 1) the LY294002 inhibitor group was compared with the PNU group, and the phosphorylated Akt expression of the inhibitor group decreased (P0.01); (3) the expression of phosphorylated Akt in the group s24795 group was significantly higher than that of the control group (P0.05). The MLA blocker group was compared with the s24795 group, and the Akt phosphorylation of the blocker group was significantly reduced (P0.01). The inhibitor group was compared with the inhibitor group. The inhibitor group was compared with the group of inhibitors. The expression of phosphorylated Akt was reduced (P0.01). The results of a beta protein expression were as follows: (1) there was no a beta protein expression in the normal cells of the control group, and the nicotine group was significantly lower than the A beta group (P0.01). The expression of a beta protein in the inhibitor group was significantly higher than that of the LY294002 inhibitor group nikolin group (p0.). 01) (2) there was no expression of a beta protein in normal cells in the control group, and the expression of a beta protein in group PNU was significantly lower than that in group A beta (P0.01). The expression of a beta protein in the inhibitor group increased (P0.01) in the group of LY294002 inhibitors and PNU (P0.01), and (3) there was no expression of a beta protein in the normal cells of the control group, and the control group A was in the total cell protein. The expression of a beta protein in group s24795 was lower than that in group A beta (P0.05). The expression of a beta protein in the inhibitor group was significantly higher than that in the s24795 group (P0.01). Conclusion: nicotine, PNU and s24795 inhibit the accumulation of endogenous s24795 by activating astrocyte alpha 7nachrs. Signaling pathway is probably an important participant in the inhibition of a beta aggregation by the 3 agonists up regulation of endogenous CRYAB through astrocyte alpha 7nachrs, which may be a potential target for the further study of CRYAB protein as a potential target for the treatment of ad.

【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】：2017
【分類號(hào)】：R749.16

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