激活原代星形胶质细胞α7尼古丁型乙酰胆碱能受体调控内源性Cryab抑制β-淀粉样蛋白聚集的机制研究

发布时间：2018-05-04 13:49

本文选题：阿尔茨海默氏病 + β-淀粉样蛋白　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:探讨激活大鼠脑组织原代星形胶质细胞的α7尼古丁乙酰胆碱能受体(nicotinic acetylcholine receptor,nAChR)调控β-淀粉样蛋白(β-amyloid peptide,Aβ)聚集的研究。方法:分离新生大鼠大脑皮质,培养原代星形胶质细胞并纯化、传代,使用细胞免疫荧光的方法鉴定细胞及纯度;体外制备Aβ1-42寡聚体;将培养细胞分为对照组、α7 nAChR激动剂组、α7 nAChR阻断剂组、PI3K/Akt信号通路抑制剂组以及Aβ组、激动剂+Aβ组、阻断剂+Aβ组。激动剂有尼古丁、PNU、s24795,阻断剂为甲基牛扁亭(methyllycaconitine,MLA)、LY294002。用蛋白印迹法(Western blotting)检测星形胶质细胞内源性B-晶状体蛋白(αB-crystallin,Cryab)、磷酸化Akt、Aβ的表达。结果:采用免疫荧光法鉴定所培养的细胞,结果显示星形胶质细胞占所有细胞的95%以上。检测内源性Cryab蛋白表达结果显示:(1)尼古丁组与对照组比较,尼古丁组的内源性Cryab表达明显增高(P0.05),MLA阻断剂组与尼古丁组比较,阻断剂组的内源性Cryab表达降低(P0.01),LY294002抑制剂组与尼古丁组比较,抑制剂组的内源性Cryab表达降低(P0.01);(2)PNU组与对照组比较,PNU组的内源性Cryab的表达增加(P0.05),MLA阻断剂组与PNU组相比,内源性Cryab表达降低(P0.01),LY294002抑制剂组与PNU组比较,抑制剂组的内源性Cryab表达降低(P0.01);(3)s24795组与对照比较,s24795组的内源性Cryab表达明显升高(P0.01),MLA阻断剂组与s24795组比较,阻断剂组的内源性Cryab表达明显降低(P0.01),LY294002抑制剂组与s24795组比较,抑制剂组的内源性cryab表达降低(p0.01)。检测磷酸化akt蛋白的表达结果为:(1)尼古丁组与对照组比较,尼古丁组的磷酸化akt表达明显增高(p0.05),mla阻断剂组与尼古丁组比较,阻断剂组的磷酸化akt表达降低(p0.01),ly294002抑制剂组与尼古丁组比较,抑制剂组的磷酸化akt表达降低(p0.01);(2)pnu组与对照组比较,pnu组的磷酸化akt的表达增加(p0.05),mla阻断剂组与pnu组相比,磷酸化akt表达降低(p0.01),ly294002抑制剂组与pnu组比较,抑制剂组的磷酸化akt表达降低(p0.01);(3)s24795组与对照比较,s24795组的磷酸化akt表达明显升高(p0.05),mla阻断剂组与s24795组比较,阻断剂组的磷酸化akt表达明显降低(p0.01),ly294002抑制剂组与s24795组比较,抑制剂组的磷酸化akt表达降低(p0.01)。检测aβ蛋白的表达结果为:(1)对照组正常的细胞内没有aβ蛋白的表达,尼古丁组与aβ组相比,尼古丁组的aβ蛋白表达均明显降低(p0.01),ly294002抑制剂组尼古丁组比较,抑制剂组的aβ蛋白的表达均明显升高(p0.01);(2)对照组正常的细胞内没有aβ蛋白的表达,pnu组与aβ组相比,pnu组的aβ蛋白表达均明显降低(p0.01),ly294002抑制剂组与pnu组比较,抑制剂组的aβ蛋白的表达均升高(p0.01);(3)对照组正常的细胞内没有aβ蛋白的表达,细胞总蛋白中,对照组aβ蛋白无表达,s24795组与aβ组相比,s24795组的aβ蛋白表达降低(p0.05),ly294002抑制剂组与s24795组比较,抑制剂组的aβ蛋白的表达明显升高(p0.01)。结论:尼古丁、pnu以及s24795通过激活星形胶质细胞α7nachrs上调内源性cryab从而抑制aβ集聚;pi3k/akt信号通路很可能是这3种激动剂通过星形胶质细胞α7nachrs上调内源性cryab从而抑制aβ集聚过程的重要参与因素。为进一步研究cryab蛋白可能是治疗ad的一个潜在靶点提供了一定的基础。
[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.

【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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