本文選題：外源性硫化氫 + 多奈哌齊��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：第一部分外源性硫化氫和多奈哌齊對Aβ25-35致PC12細(xì)胞損傷的保護(hù)作用 目的：觀察外源性硫化氫(H2S)及多奈哌齊(donepezil)對β淀粉樣蛋白25-35(β-amyloid peptide25-35,Aβ25-35)致大鼠嗜鉻細(xì)胞瘤(PC12)細(xì)胞損傷的細(xì)胞生存保護(hù)作用。方法：以體外培養(yǎng)的PC12細(xì)胞為研究對象,用硫氫化鈉(NaHS)作為外源性H2S供體。實(shí)驗(yàn)設(shè)空白對照組、Aβ25-35組、NaHS組、NaHS+Aβ25-35組、多奈哌齊組、多奈哌齊+Aβ25-35組共6組。NaHS的濃度為10、25、50、100、200、500、1000μmol.L,而Aβ25-35和多奈哌齊的濃度都為20μmol/L.按分組濃度分別處理PC12細(xì)胞24h后,用甲氮甲唑藍(lán)法(MTT)檢測細(xì)胞成活率。結(jié)果：①Aβ25-35能顯著降低PC12細(xì)胞的存活率(P0.05)；②用不同濃度NaHS預(yù)處理后,10μmol/L的NaHS本身不影響PC12細(xì)胞的存活率(P0.05),但25μmol/L～200μmol.LNaHS能提高PC12細(xì)胞的存活率(P0.05),也能抑制20μmol/L Aβ25-35誘導(dǎo)的細(xì)胞毒性,可使PC12細(xì)胞存活率明顯提高(P0.05),然而100μmol/LNaHS和200μmol/LNaHS相比,100μmol/LNaHS具有更好的保護(hù)細(xì)胞的作用(P0.05)。但500和1000μmol/L的NaHS對PC12細(xì)胞本身具有毒性作用(P0.05)；③多奈哌齊能降低Aβ25-35引起的細(xì)胞毒性作用,提高PC12細(xì)胞存活率(P0.05)。④20μmol/L多奈哌齊與200μmol/LNaHS比較,無明顯差異,都能保護(hù)Aβ25-35所致的PC12損傷。對培養(yǎng)PC12細(xì)胞存活的最佳NaHS濃度是100μmol/L。結(jié)論：H2S及多奈哌齊都對Aβ25-35致PC12細(xì)胞損傷具有保護(hù)作用。 第二部分外源性硫化氫和多奈哌齊對PC12細(xì)胞APP/Aβ代謝途徑的影響 目的：探討外源性H2S及多奈哌齊對PC12細(xì)胞淀粉樣前體蛋白/β-位淀粉樣蛋白(APP/Aβ)代謝的影響。方法：以PC12細(xì)胞為研究對象,用NaHS作外源性H2S供體,實(shí)驗(yàn)設(shè)空白對照組、NaHS10μmol/L組、25、50、100、200μmol/L組、多奈哌齊20μmol/L組,共7組。按分組濃度分別處理PC12細(xì)胞24h后,用Western blot檢測代謝過程中關(guān)鍵蛋白APP表達(dá)變化；ELISA檢測細(xì)胞培養(yǎng)上清液中Aβ40、Aβ42、sAPPα、 sAPPβ的蛋白水平。結(jié)果：①不同濃度NaHS和多奈哌齊對APP蛋白表達(dá)水平無明顯差異(P0.05)；②不同濃度NaHS和多奈哌齊均可降低Aβ40, Aβ42和sAPPβ蛋白表達(dá)水平,還可增高sAPPα蛋白表達(dá)水平,這種作用與H2S濃度有相關(guān)性。結(jié)論：外源性H2S和多奈哌齊可以誘導(dǎo)APP/Aβ代謝途徑向非淀粉樣途徑(α途徑)轉(zhuǎn)變,H2S的這種作用具有濃度依賴性。 第三部分外源性硫化氫和多奈哌齊對PC12細(xì)胞α分泌酶(ADAM10/ADAM17)的影響 目的：觀察H2S和多奈哌齊對PC12細(xì)胞α-分泌酶ADAM10和ADAM10蛋白的影響,探討二者對APP代謝途徑的轉(zhuǎn)變作用是否與ADAM10和ADAM17相關(guān)。方法：以PC12細(xì)胞為研究對象,用NaHS作外源性H2S供體,實(shí)驗(yàn)設(shè)空白對照組、NaHS10、25、50、100和200μmol/L組、多奈哌齊20μmol/L組,共7組。按分組濃度分別處理PC12細(xì)胞24h后,用Western blot檢測代謝過程中關(guān)鍵蛋白ADAM10和ADAM17的表達(dá)水平。結(jié)果：不同濃度NaHS和多奈哌齊組的ADAM10和ADAM17蛋白表達(dá)水平較對照組增高,且隨著NaHS濃度增加,蛋白表達(dá)水平呈遞增趨勢(P0.05)。100μmol/L和200μmol/LNaHS組的ADAM10及ADAM17表達(dá)水平無明顯差異(P0.05),但100μmol/LNaHS組的ADAM10及ADAM17蛋白表達(dá)水平最高(P0.01)。結(jié)論：①外源性H2S和多奈哌齊具有上調(diào)體外培養(yǎng)的PC12細(xì)胞α分泌酶(ADAM10、ADAM17)蛋白表達(dá)水平；②外源性H2S和多奈哌齊使APP/Aβ代謝途徑向非淀粉樣途徑轉(zhuǎn)變,可能與α分泌酶的核心蛋白ADAM10和ADAM17蛋白表達(dá)水平增高有關(guān)。 第四部分外源性硫化氫和多奈哌齊對調(diào)控APP的α代謝途徑三條細(xì)胞信號通路的研究 目的:觀察外源性H2S及多奈哌齊對APP的α代謝途徑中PI3-K、 MAPKs及JNK3條主要細(xì)胞信號通路的影響,探討二者的細(xì)胞信號機(jī)制。方法：以體外培養(yǎng)的PC12細(xì)胞為研究對象,用NaHS作外源性H2S供體。①實(shí)驗(yàn)先設(shè)為空白對照組,P13-K抑制劑LY組(LY294002,20μmol/L),p38MAPK抑制劑SB組(SB203580,30μmol/L)、 JNK抑制劑SP組(SP600125,20μmol/L),共4組。②實(shí)驗(yàn)再設(shè)置為空白對照組,NaHS100μmol/L組,NaHS100μmol/L+SP組,NaHS100μmol/L+LY組,NaHS100μmol/L+SB組,多奈哌齊20μmol/L組,多奈哌齊20μmol/L+SP組,多奈哌齊20μmol/L+LY組,多奈哌齊20μmol/L+SB組,共9組。PC12細(xì)胞在加入NaHS100μmol/L或多奈哌齊μmol/L前30min,先加入SP600125、LY294002、SB203580。干預(yù)24h后,用Western blot法檢測APP、ADAM10和ADAM17蛋白表達(dá)水平；ELISA檢測細(xì)胞培養(yǎng)上清液中Aβ40、Aβ42、sAPPα和sAPβ的蛋白水平。結(jié)果：①SP組、LY組、SB組APP蛋白以及SB組ADAM10和ADAM17蛋白表達(dá)水平無明顯影響(P0.05),而SP組、LY組ADAM10和ADAM17的蛋白表達(dá)降低。②SB組Aβ40、Aβ42、sAPPβ和sAPPα蛋白表達(dá)與對照組比較無顯著變化(P0.05)。LY組、SP組Aβ40、Aβ42和sAPPβ蛋白表達(dá)顯著增強(qiáng),并降低sAPPα蛋白的表達(dá),與對照組比較具有顯著差異性(P0.01)。③SP組、LY組H2S增強(qiáng)ADAM10蛋白表達(dá)的作用降低,H2S降低Aβ40、Aβ42和sAPPβ蛋白表達(dá)的作用減弱,與對照組比較具有顯著差異(P0.01)；SB組、SP組、LY組多奈哌齊增強(qiáng)ADAM10、ADAM17蛋白表達(dá)的作用均降低,多奈哌齊降低Aβ40、Aβ42和sAPPβ蛋白表達(dá)的作用減弱,與對照組比較具有顯著差異(P0.01)。④SP組、LY組可抑制H2S升高sAPPα蛋白的表達(dá),與對照組比較具有顯著差異(P0.01)；SB組、SP組、LY組可抑制多奈哌齊升高sAPPα蛋白的表達(dá),與對照組比較具有顯著差異(P0.01)。結(jié)論：①外源性H2S具有上調(diào)體外培養(yǎng)的PC12細(xì)胞APPα代謝途徑中關(guān)鍵蛋白ADAM10和ADAM17,能明顯提高sAPPα的表達(dá),有效的減少Aβ40、Aβ42和sAPPβ病理產(chǎn)物的生成,使APP/Aβ代謝途徑向非淀粉樣途徑(α途徑)轉(zhuǎn)變,其機(jī)制可能涉及P13-K或JNK信號通路。②多奈哌齊具有H2S相似的作用,其機(jī)制可能涉及PI3-K、JNK信號通路或p38MAPK通路。說明H2S與多奈哌齊在增強(qiáng)APP的非淀粉樣代謝途徑(α代謝途徑)調(diào)控中具有相似的細(xì)胞信號機(jī)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R749.16

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本文編號：1936293