【摘要】：目的：Wnt信號(hào)通路在多巴胺能神經(jīng)元的發(fā)育及帕金森病多巴胺能神經(jīng)元的損傷中發(fā)揮著重要作用。本研究旨在觀察經(jīng)典Wnt信號(hào)通路的相關(guān)蛋白及其抑制劑Dkk1是否在MPP+誘導(dǎo)的PC12細(xì)胞中發(fā)生改變,并初步探討其可能機(jī)制。 方法：用不同濃度的MPP+作用于PC12細(xì)胞,MTT法檢測(cè)在不同時(shí)間細(xì)胞的存活率及Western Blot檢測(cè)不同時(shí)間點(diǎn)Dkk、β-catenin和p-Ser9-GSK-3β表達(dá)的變化情況。LiCl預(yù)處理1h后,再用MPP+作用PC12細(xì)胞,同時(shí)也檢測(cè)細(xì)胞的存活率及凋亡的情況,檢測(cè)不同時(shí)間點(diǎn)Dkk1. β-cateninn和p-Ser9-GSK-3p表達(dá)的變化情況。 結(jié)果：①不同濃度的MPP+對(duì)PC12細(xì)胞作用24h,細(xì)胞存活率沒(méi)有顯著變化,高濃度的MPP+對(duì)PC12細(xì)胞作用48h和72h后細(xì)胞存活率有顯著變化,并且在1000μMMPP+作用72h后,細(xì)胞存活率降至最低45.0%,而1000μMMPP+作用48h和72h對(duì)細(xì)胞存活率的影響并沒(méi)有顯著性差異。②Western Blot結(jié)果顯示,在6-24h,MPP+誘導(dǎo)Dkk1的表達(dá)顯著增加,其表達(dá)高峰時(shí)間先于細(xì)胞的死亡時(shí)間,但是Dkk1的水平在48h恢復(fù)至正常。MPP+作用8h后,β-catenin的水平開(kāi)始有降低的趨勢(shì),在24h至48h其水平則顯著降低,p-Ser9-GSK-3β(GSK-3β的非活化形式)在MPP+作用6-48h減少。③LiCl預(yù)處理可顯著增加PC12細(xì)胞中β-catenin和p-Ser9-GSK-3β的表達(dá)。④LiCl預(yù)處理減輕MPP+誘導(dǎo)的PC12細(xì)胞存活率的減少,Annexin Ⅴ-FITC/PI流式細(xì)胞術(shù)及TUNEL法顯示MPP+誘導(dǎo)的PC12細(xì)胞凋亡率的增加也可被LiCl(1mM)減少。 結(jié)論：經(jīng)典Wnt信號(hào)通路蛋白和其抑制劑Dkk1可能參與了MPP+誘導(dǎo)的PC12細(xì)胞的減少,Dkk1可能通過(guò)抑制經(jīng)典Wnt信號(hào)通路蛋白發(fā)揮作用。 目的：第一部分實(shí)驗(yàn)表明Dkk1在MPP+致PC12細(xì)胞損傷模型中的誘導(dǎo)表達(dá)先于細(xì)胞死亡,本實(shí)驗(yàn)旨在探討Dkk1在MPP+致PC12細(xì)胞損傷模型中的機(jī)制。 方法：通過(guò)對(duì)外源性的給予·rhDkkl聯(lián)合MPP+處理PC12細(xì)胞48h后,檢測(cè)細(xì)胞存活率及Wnt信號(hào)通路相關(guān)蛋白表達(dá)的變化情況。通過(guò)siRNA技術(shù)下調(diào)Dkkl在PC12細(xì)胞的表達(dá),觀察細(xì)胞凋亡情況及Wnt信號(hào)通路相關(guān)蛋白表達(dá)的變化情況。 結(jié)果：①hDkk1(100ng/ml)與MPP+聯(lián)合作用細(xì)胞48h后檢測(cè)細(xì)胞存活率,結(jié)果顯示：rhDkkl加重MPP+對(duì)PC12細(xì)胞的損傷作用,而LiCl預(yù)處理1h并不能顯著改善兩者聯(lián)合應(yīng)用引起的細(xì)胞損傷作用。②hDkk1促進(jìn)MPP+誘導(dǎo)的PC12細(xì)胞中TH的減少。與MPP+單獨(dú)處理組相比,兩者聯(lián)合應(yīng)用β-catenin減少的更多,但p-Ser9-GSK-33β輕微減少。③Dkk1-siRNA可以顯著減輕MPP+誘導(dǎo)的PC12細(xì)胞的凋亡。④Dkk1-siRNA有效的增加β-catenin和p-Ser9-GSK-3β的表達(dá),同時(shí)也顯著提高TH的水平。 結(jié)論：Dkkl參與了MPP+誘導(dǎo)的PC12細(xì)胞的凋亡,其機(jī)制可能與抑制經(jīng)典Wnt信號(hào)通路相關(guān)。 目的：本實(shí)驗(yàn)旨在觀察Dkkl在6-OHDA損傷大鼠的帕金森病模型中的作用。 方法：通過(guò)腦立體定位儀經(jīng)MFB給予6-OHDA損傷大鼠,制備帕金森病模型。另有LiCl預(yù)處理7d后再給予6-OHDA損傷大鼠組,及通過(guò)黑質(zhì)給予rhDkk1與MFB給予6-OHDA聯(lián)合損傷大鼠組。術(shù)后35d多聚甲醛灌注免疫組化檢測(cè)各組黑質(zhì)多巴胺能神經(jīng)元的損傷情況,并取大鼠腹側(cè)中腦Western Blot檢測(cè)TH、Dkk1及β-catenin、 p-Ser9-GSK-3β的變化情況。 結(jié)果：①6-OHDA損傷組TH陽(yáng)性的神經(jīng)元數(shù)量明顯減少,LiC1預(yù)處理組的TH陽(yáng)性的神經(jīng)元數(shù)量則明顯增加；rhDkk1與6-OHDA聯(lián)合損傷組,與6-OHDA單獨(dú)損傷組相比,TH陽(yáng)性的神經(jīng)元數(shù)量則降低的更明顯。②Dkk1在6-OHDA損傷的大鼠術(shù)后35d腹側(cè)中腦的表達(dá)明顯增加。③經(jīng)典Wnt信號(hào)通路的抑制在6-OHDA損傷的大鼠腹側(cè)中腦中也被檢測(cè)到,包括β-catenin和p-Ser9-GSK-3β的降低,而兩者表達(dá)水平均可被LiCl升高；rhDkkl與6-OHDA聯(lián)合應(yīng)用可進(jìn)一步加劇β-catenin和p-Ser9-GSK-3β的減少。 結(jié)論：Dkkl在6-OHDA損傷大鼠的腹側(cè)中腦被誘導(dǎo)表達(dá),其可能通過(guò)抑制經(jīng)典Wnt信號(hào)通路而促進(jìn)6-OHDA損傷大鼠黑質(zhì)多巴胺能神經(jīng)元的損傷。
[Abstract]:AIM: Wnt signaling pathway plays an important role in the development of dopaminergic neurons and the damage of dopaminergic neurons in Parkinson's disease.
METHODS: PC12 cells were treated with different concentrations of MPP +. MTT assay was used to detect the survival rate of PC12 cells at different time points and Western Blot to detect the expression of Dkk, beta-catenin and p-Ser9-GSK-3 beta at different time points. Changes in the expression of Dkk1. -cateninn and p-Ser9-GSK-3p were observed.
RESULTS: The survival rate of PC12 cells treated with different concentrations of MPP + for 24 hours had no significant change. The survival rate of PC12 cells treated with high concentrations of MPP + for 48 hours and 72 hours had significant change. After 72 hours of treatment with 1000 mu MMPP + for 72 hours, the cell survival rate decreased to the lowest level of 45.0%, while the effect of 1000 mu mu MMPP + for 48 hours and 72 hours had no effect on the cell survival rate. The results of Western Blot showed that the expression of Dkk1 was significantly increased at 6-24 h, and the peak time of Dkk1 expression was earlier than the time of cell death, but the level of Dkk1 returned to normal at 48 h. After 8 h of MPP + treatment, the level of beta-catenin began to decrease, but decreased significantly at 24 h to 48 h, and p-Ser9-GSK-3 beta (GSK-3 beta) was decreased. LiCl pretreatment significantly increased the expression of beta-catenin and p-Ser9-GSK-3 beta in PC12 cells. LiCl pretreatment reduced the decrease of MPP+induced survival rate of PC12 cells. Annexin V-FITC/PI flow cytometry and TUNEL assay showed that the increase of apoptosis rate of PC12 cells induced by MPP+ could also be induced by LiCl (1mM). Reduce.
CONCLUSION: The classical Wnt signaling pathway protein and its inhibitor Dkk1 may be involved in the decrease of PC12 cells induced by MPP +, and Dkk1 may play a role by inhibiting the classical Wnt signaling pathway protein.
OBJECTIVE: The first part of the experiment showed that Dkk1 induced expression preceded cell death in MPP + induced PC12 cell injury model. The aim of this experiment was to explore the mechanism of Dkk1 induced PC12 cell injury in MPP + induced PC12 cell injury model.
Methods: After 48 hours of exogenous administration of rhDkkl combined with MPP + for PC12 cells, the cell survival rate and the expression of Wnt signaling pathway related proteins were detected.
RESULTS: The results showed that rhDkkl aggravated the damage of MPP + on PC12 cells, but LiCl pretreatment for 1 h could not significantly improve the damage of PC12 cells induced by the combination of hDkk1 (100ng/ml) and MPP +. hDkk1 could promote the decrease of TH in PC12 cells induced by MPP +, and MPP + alone treated PC12 cells. Dkk1-siRNA can significantly reduce the apoptosis of PC12 cells induced by MPP +. 4. Dkk1-siRNA can effectively increase the expression of beta-catenin and p-Ser9-GSK-3 beta, but also significantly increase the level of TH.
CONCLUSION: Dkkl participates in the apoptosis of PC12 cells induced by MPP +, which may be related to the inhibition of classical Wnt signaling pathway.
Objective: To observe the effect of Dkkl on Parkinson's disease model in 6-OHDA injured rats.
METHODS: Parkinson's disease model was established in rats injured with 6-OHDA by brain stereotactic localizer and 6-OHDA by LiCl pretreatment for 7 days. In rats injured with 6-OHDA by rhDkk1 and MFB in substantia nigra, the damage of dopaminergic neurons in substantia nigra was detected by perfusion immunohistochemistry 35 days after operation. The changes of TH, Dkk1, beta catenin and p-Ser9-GSK-3 beta in ventral midbrain of rats were detected by Western Blot.
Results: The number of TH-positive neurons decreased significantly in 6-OHDA injury group, while the number of TH-positive neurons increased significantly in LiC1 preconditioning group. The number of TH-positive neurons decreased more significantly in rhDkk1 and 6-OHDA combined injury group than that in 6-OHDA alone injury group. (3) Inhibition of the classical Wnt signaling pathway was also detected in the ventral midbrain of rats with 6-OHDA injury, including the decrease of beta-catenin and p-Ser9-GSK-3 beta, both of which could be elevated by LiCl; rhDkkl combined with 6-OHDA could further aggravate the decrease of beta-catenin and p-Ser9-GSK-3 beta.
CONCLUSION: Dkkl is induced to express in the ventral mesencephalon of 6-OHDA-injured rats, which may promote the damage of dopaminergic neurons in substantia nigra of 6-OHDA-injured rats by inhibiting the classical Wnt signaling pathway.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R742.5

【共引文獻(xiàn)】

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