外源性硫化氢和多奈哌齐对PC12细胞APP代谢α途径的影响与机制研究

发布时间：2018-05-26 06:32

本文选题：外源性硫化氢 + 多奈哌齐　；参考：《重庆医科大学》2012年硕士论文

【摘要】：第一部分外源性硫化氢和多奈哌齐对Aβ25-35致PC12细胞损伤的保护作用目的：观察外源性硫化氢(H2S)及多奈哌齐(donepezil)对β淀粉样蛋白25-35(β-amyloid peptide25-35,Aβ25-35)致大鼠嗜铬细胞瘤(PC12)细胞损伤的细胞生存保护作用。方法：以体外培养的PC12细胞为研究对象,用硫氢化钠(NaHS)作为外源性H2S供体。实验设空白对照组、Aβ25-35组、NaHS组、NaHS+Aβ25-35组、多奈哌齐组、多奈哌齐+Aβ25-35组共6组。NaHS的浓度为10、25、50、100、200、500、1000μmol.L,而Aβ25-35和多奈哌齐的浓度都为20μmol/L.按分组浓度分别处理PC12细胞24h后,用甲氮甲唑蓝法(MTT)检测细胞成活率。结果：①Aβ25-35能显著降低PC12细胞的存活率(P0.05)；②用不同浓度NaHS预处理后,10μmol/L的NaHS本身不影响PC12细胞的存活率(P0.05),但25μmol/L～200μmol.LNaHS能提高PC12细胞的存活率(P0.05),也能抑制20μmol/L Aβ25-35诱导的细胞毒性,可使PC12细胞存活率明显提高(P0.05),然而100μmol/LNaHS和200μmol/LNaHS相比,100μmol/LNaHS具有更好的保护细胞的作用(P0.05)。但500和1000μmol/L的NaHS对PC12细胞本身具有毒性作用(P0.05)；③多奈哌齐能降低Aβ25-35引起的细胞毒性作用,提高PC12细胞存活率(P0.05)。④20μmol/L多奈哌齐与200μmol/LNaHS比较,无明显差异,都能保护Aβ25-35所致的PC12损伤。对培养PC12细胞存活的最佳NaHS浓度是100μmol/L。结论：H2S及多奈哌齐都对Aβ25-35致PC12细胞损伤具有保护作用。第二部分外源性硫化氢和多奈哌齐对PC12细胞APP/Aβ代谢途径的影响目的：探讨外源性H2S及多奈哌齐对PC12细胞淀粉样前体蛋白/β-位淀粉样蛋白(APP/Aβ)代谢的影响。方法：以PC12细胞为研究对象,用NaHS作外源性H2S供体,实验设空白对照组、NaHS10μmol/L组、25、50、100、200μmol/L组、多奈哌齐20μmol/L组,共7组。按分组浓度分别处理PC12细胞24h后,用Western blot检测代谢过程中关键蛋白APP表达变化；ELISA检测细胞培养上清液中Aβ40、Aβ42、sAPPα、 sAPPβ的蛋白水平。结果：①不同浓度NaHS和多奈哌齐对APP蛋白表达水平无明显差异(P0.05)；②不同浓度NaHS和多奈哌齐均可降低Aβ40, Aβ42和sAPPβ蛋白表达水平,还可增高sAPPα蛋白表达水平,这种作用与H2S浓度有相关性。结论：外源性H2S和多奈哌齐可以诱导APP/Aβ代谢途径向非淀粉样途径(α途径)转变,H2S的这种作用具有浓度依赖性。第三部分外源性硫化氢和多奈哌齐对PC12细胞α分泌酶(ADAM10/ADAM17)的影响目的：观察H2S和多奈哌齐对PC12细胞α-分泌酶ADAM10和ADAM10蛋白的影响,探讨二者对APP代谢途径的转变作用是否与ADAM10和ADAM17相关。方法：以PC12细胞为研究对象,用NaHS作外源性H2S供体,实验设空白对照组、NaHS10、25、50、100和200μmol/L组、多奈哌齐20μmol/L组,共7组。按分组浓度分别处理PC12细胞24h后,用Western blot检测代谢过程中关键蛋白ADAM10和ADAM17的表达水平。结果：不同浓度NaHS和多奈哌齐组的ADAM10和ADAM17蛋白表达水平较对照组增高,且随着NaHS浓度增加,蛋白表达水平呈递增趋势(P0.05)。100μmol/L和200μmol/LNaHS组的ADAM10及ADAM17表达水平无明显差异(P0.05),但100μmol/LNaHS组的ADAM10及ADAM17蛋白表达水平最高(P0.01)。结论：①外源性H2S和多奈哌齐具有上调体外培养的PC12细胞α分泌酶(ADAM10、ADAM17)蛋白表达水平；②外源性H2S和多奈哌齐使APP/Aβ代谢途径向非淀粉样途径转变,可能与α分泌酶的核心蛋白ADAM10和ADAM17蛋白表达水平增高有关。第四部分外源性硫化氢和多奈哌齐对调控APP的α代谢途径三条细胞信号通路的研究目的:观察外源性H2S及多奈哌齐对APP的α代谢途径中PI3-K、 MAPKs及JNK3条主要细胞信号通路的影响,探讨二者的细胞信号机制。方法：以体外培养的PC12细胞为研究对象,用NaHS作外源性H2S供体。①实验先设为空白对照组,P13-K抑制剂LY组(LY294002,20μmol/L),p38MAPK抑制剂SB组(SB203580,30μmol/L)、 JNK抑制剂SP组(SP600125,20μmol/L),共4组。②实验再设置为空白对照组,NaHS100μmol/L组,NaHS100μmol/L+SP组,NaHS100μmol/L+LY组,NaHS100μmol/L+SB组,多奈哌齐20μmol/L组,多奈哌齐20μmol/L+SP组,多奈哌齐20μmol/L+LY组,多奈哌齐20μmol/L+SB组,共9组。PC12细胞在加入NaHS100μmol/L或多奈哌齐μmol/L前30min,先加入SP600125、LY294002、SB203580。干预24h后,用Western blot法检测APP、ADAM10和ADAM17蛋白表达水平；ELISA检测细胞培养上清液中Aβ40、Aβ42、sAPPα和sAPβ的蛋白水平。结果：①SP组、LY组、SB组APP蛋白以及SB组ADAM10和ADAM17蛋白表达水平无明显影响(P0.05),而SP组、LY组ADAM10和ADAM17的蛋白表达降低。②SB组Aβ40、Aβ42、sAPPβ和sAPPα蛋白表达与对照组比较无显著变化(P0.05)。LY组、SP组Aβ40、Aβ42和sAPPβ蛋白表达显著增强,并降低sAPPα蛋白的表达,与对照组比较具有显著差异性(P0.01)。③SP组、LY组H2S增强ADAM10蛋白表达的作用降低,H2S降低Aβ40、Aβ42和sAPPβ蛋白表达的作用减弱,与对照组比较具有显著差异(P0.01)；SB组、SP组、LY组多奈哌齐增强ADAM10、ADAM17蛋白表达的作用均降低,多奈哌齐降低Aβ40、Aβ42和sAPPβ蛋白表达的作用减弱,与对照组比较具有显著差异(P0.01)。④SP组、LY组可抑制H2S升高sAPPα蛋白的表达,与对照组比较具有显著差异(P0.01)；SB组、SP组、LY组可抑制多奈哌齐升高sAPPα蛋白的表达,与对照组比较具有显著差异(P0.01)。结论：①外源性H2S具有上调体外培养的PC12细胞APPα代谢途径中关键蛋白ADAM10和ADAM17,能明显提高sAPPα的表达,有效的减少Aβ40、Aβ42和sAPPβ病理产物的生成,使APP/Aβ代谢途径向非淀粉样途径(α途径)转变,其机制可能涉及P13-K或JNK信号通路。②多奈哌齐具有H2S相似的作用,其机制可能涉及PI3-K、JNK信号通路或p38MAPK通路。说明H2S与多奈哌齐在增强APP的非淀粉样代谢途径(α代谢途径)调控中具有相似的细胞信号机制。
[Abstract]:Part one: protective effects of exogenous hydrogen sulfide and donepezil on PC12 cell injury induced by A beta 25-35
Objective: To observe the survival and protective effects of exogenous hydrogen sulfide (H2S) and donepezil (donepezil) on the cell damage of rat pheochromocytoma (PC12) cells induced by beta amyloid 25-35 (beta -amyloid peptide25-35, A beta 25-35). Methods: PC12 cells cultured in vitro were used as research subjects and sodium hydrogen sulfide (NaHS) was used as a exogenous H2S donor. In the blank control group, A beta 25-35, group NaHS, NaHS+A beta 25-35, donepezil group, and donepezil +A beta 25-35 group were 10,25,501002005001000 mol.L, while A beta 25-35 and donepezil concentration were 20 u mol/L. respectively for PC12 cell 24h, and methazolate blue method (MTT) was used to detect the survival rate. Fruit: (1) A beta 25-35 can significantly reduce the survival rate of PC12 cells (P0.05). After preconditioning with different concentrations of NaHS, NaHS itself does not affect the survival rate of PC12 cells (P0.05), but 25 u mol/L to 200 micron mol.LNaHS can increase the survival rate of PC12 cells (P0.05), and also inhibit the cytotoxicity induced by 20 mu 25-35. The survival rate was significantly increased (P0.05), however, compared with 100 mu mol/LNaHS and 200 mu mol/LNaHS, 100 mu mol/LNaHS had better protective effect (P0.05). But 500 and 1000 micron mol/L NaHS had toxic effects on PC12 cells (P0.05); 3. Donepezil could reduce the cytotoxicity of A beta 25-35 and increase the survival rate of PC12 cells (P0.05). (4) 2 0 mu mol/L donepezil, compared with 200 mol/LNaHS, has no obvious difference, which can protect the PC12 damage caused by A beta 25-35. The best NaHS concentration for the survival of PC12 cells is 100 mu mol/L. conclusion: H2S and donepezil have protective effects on A beta 25-35 induced PC12 cell damage.
The second part is the effect of exogenous hydrogen sulfide and donepezil on APP/A beta metabolism pathway in PC12 cells.
Objective: To investigate the effects of exogenous H2S and donepezil on the metabolism of amyloid precursor protein / beta amyloid (APP/A beta) protein (APP/A beta) in PC12 cells. Methods: PC12 cells were used as the research object and NaHS was used as a exogenous H2S donor. The experiment set up a blank control group, NaHS10 Mu mol/L group, 25,50100200 mu mol/L group, and donepezil 20 mu mol/L group, 7 groups. Groups were grouped. After the concentration of PC12 cells was treated with 24h, the expression of the key protein APP in the metabolic process was detected by Western blot, and ELISA was used to detect the protein level of A beta 40, A beta 42, sAPP alpha and sAPP beta in the culture supernatant. Piperazine can reduce the expression level of A beta 40, A beta 42 and sAPP beta protein, and also increase the expression level of sAPP alpha protein. This effect is related to the concentration of H2S. Conclusion: exogenous H2S and donepezil can induce the transition of APP/A beta metabolic pathway to the non amyloid pathway (alpha pathway). This effect of H2S is dependent on the concentration of H2S.
The third part is the effect of exogenous hydrogen sulfide and donepezil on PC12 secreting enzyme (ADAM10/ADAM17).
Objective: To observe the effect of H2S and donepezil on the alpha secretase ADAM10 and ADAM10 protein of PC12 cells, and to explore whether the changes in the metabolic pathway of APP were related to ADAM10 and ADAM17. Methods: PC12 cells were used as the research object and NaHS was used as a exogenous H2S donor. The experimental group was set up in the blank control group, NaHS10,25,50100 and 200 micron mol/L groups, donepep. The expression level of the key protein ADAM10 and ADAM17 in the metabolic process was detected by Western blot. The expression level of ADAM10 and ADAM17 proteins in the group of NaHS and donepezil at different concentrations was higher than that of the control group. The protein expression level increased with the increase of NaHS concentration, and the expression level of protein expression increased with the concentration of NaHS and donepezil at different concentrations. The expression level of protein expression was increased with the concentration of NaHS and donepezil at different concentrations. The expression level of protein expression was increased with the increase of NaHS concentration. The expression level of the protein expression level was increased with the increase of NaHS concentration in the group of mol/L and donepezil. There was no significant difference in the expression level of ADAM10 and ADAM17 in the trend (P0.05).100 and 200 mol/LNaHS groups (P0.05), but the expression level of ADAM10 and ADAM17 proteins in the 100 mu mol/LNaHS group was the highest (P0.01). Sex H2S and donepezil change the APP/A beta pathway to the non amyloid pathway, which may be related to the increase in the expression level of the core protein ADAM10 and ADAM17 protein of the alpha secretase.
The fourth part is exogenous hydrogen sulfide and donepezil to regulate the signal pathway of three cell pathways of APP alpha metabolism pathway.
Objective: To observe the effect of exogenous H2S and donepezil on the main cell signaling pathways of PI3-K, MAPKs and JNK3 in the alpha metabolic pathway of APP, and to explore the cell signaling mechanism of the two groups. Methods: PC12 cells cultured in vitro as the research object and NaHS as the exogenous H2S donor. (1) the experiment was set as a blank control group, P13-K inhibitor LY group (LY29400). 2,20 mu mol/L), p38MAPK inhibitor SB group (SB203580,30 mu mol/L), JNK inhibitor SP group (SP600125,20 micron mol/L), a total of 4 groups. Group, donepezil 20 mol/L+SB group, a total of 9 groups of.PC12 cells were added to NaHS100 mu mol/L or donepezil mu mol/L 30min before adding SP600125, LY294002, SB203580. interfered 24h, and the expression level of Western blot was detected by Western blot, and the protein levels of beta 40, beta 42, alpha and beta were detected in cell culture supernatant. Fruit: (1) SP group, LY group, SB group APP protein and SB group ADAM10 and ADAM17 protein expression level had no obvious effect (P0.05), while SP group, LY group ADAM10 and ADAM17 protein expression decreased. The expression of sAPP alpha protein was significantly different from that of the control group (P0.01). (3) group SP, group LY, H2S enhanced the expression of ADAM10 protein, H2S decreased A beta 40, A beta 42 and sAPP beta protein expression decreased, and there was a significant difference compared with the control group (P0.01). The effect of donepezil decreased A beta 40, A beta 42 and sAPP beta protein expression decreased, compared with the control group (P0.01). (4) group SP, LY group inhibited the expression of sAPP alpha protein in H2S, and was significantly different from the control group (P0.01); SB, SP group, LY group could inhibit the expression of donepezil to increase the expression of alpha protein. There were significant differences in the control group (P0.01). Conclusion: (1) exogenous H2S can increase the expression of sAPP a, the key protein ADAM10 and ADAM17 in the APP alpha metabolism pathway of PC12 cells in vitro, which can effectively reduce the formation of A beta 40, A beta 42 and sAPP beta, and make the APP/A beta metabolic pathway transition to the non amyloid pathway (alpha pathway). The mechanism may involve P13-K or JNK signaling pathways. (2) donepezil has a similar role in H2S, and its mechanism may involve PI3-K, JNK signaling pathway or p38MAPK pathway. It shows that H2S and donepezil have a similar cellular signaling mechanism in the regulation of non amyloid metabolic pathway (alpha metabolic pathway) in the enhancement of APP.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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