本文選題：水溶性輔酶Q10　切入點(diǎn)：魚藤酮　出處：《寧夏醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的探討水溶性輔酶Q10(CoQ10)對(duì)魚藤酮(Rotenone,Rot)誘導(dǎo)帕金森病(PD)細(xì)胞模型細(xì)胞凋亡的保護(hù)作用,以及改善線粒體功能障礙及線粒體融合裂解動(dòng)態(tài)失衡的機(jī)制研究,為水溶性CoQ10用于PD的治療提供理論依據(jù)。研究方法1.采用已經(jīng)由神經(jīng)生長因子誘導(dǎo)分化的PC12細(xì)胞傳代培養(yǎng),選取合適的Rot濃度處理對(duì)數(shù)生長期的PC12細(xì)胞建立PD細(xì)胞模型。2.確定水溶性CoQ10濃度作為治療濃度,將實(shí)驗(yàn)分為溶劑對(duì)照(vehicle組)組,Rot組,CoQ10組和CoQ10治療組。3.cck-8法檢測(cè)細(xì)胞活性,并在倒置顯微鏡下觀察細(xì)胞形態(tài)。4.ROS檢測(cè)試劑盒檢測(cè)各組細(xì)胞內(nèi)ROS變化。5.JC1-Mitochondrial膜Potential Assay試劑盒染色后使用流式細(xì)胞儀分析各組線粒體膜電位(MMP)變化。6.MitoTracker線粒體熒光探針標(biāo)記線粒體,激光共聚焦顯微鏡觀察線粒體形態(tài)變化。7.Western blotting檢測(cè)各組細(xì)胞凋亡以及線粒體融合分裂相關(guān)蛋白表達(dá)變化。結(jié)果1.Rot處理24h后的PC12細(xì)胞存活率顯著降低并與劑量呈負(fù)相關(guān),CoQ10可改善Rot處理誘導(dǎo)的PC12細(xì)胞存活率降低。2.Rot引起ROS水平升高,CoQ10可降低Rot處理后細(xì)胞內(nèi)的ROS水平。3.Rot誘導(dǎo)PC12細(xì)胞線粒體膜電位(MMP)降低,導(dǎo)致線粒體片段化,水溶性CoQ10可改善Rot處理誘導(dǎo)的PC12細(xì)胞MMP降低和線粒體片段化。4.Western blotting實(shí)驗(yàn)表明CoQ10可降低Rot引起的Caspase-9、active Caspase-3以及Bax的表達(dá)增多,上調(diào)Bcl-2的表達(dá),阻止AIF向核內(nèi)轉(zhuǎn)移;水溶性CoQ10可降低Rot引起的線粒體分裂蛋白Drp1表達(dá)增多,上調(diào)線粒體融合蛋白Mfn2、OPA1的表達(dá),對(duì)Mfn1、Fis1的表達(dá)無明顯影響。結(jié)論1.水溶性CoQ10可改善Rot致PC12細(xì)胞MMP降低及線粒體片段化。2.水溶性CoQ10對(duì)Rot致PC12細(xì)胞凋亡具有保護(hù)作用。其機(jī)制可能是通過降低細(xì)胞氧化應(yīng)激以及線粒體通路細(xì)胞凋亡實(shí)現(xiàn)的。3.水溶性CoQ10對(duì)Rot致PC12細(xì)胞線粒體融合、分裂紊亂具有保護(hù)作用。其機(jī)制可能是通過上調(diào)Mfn2、OPA1的表達(dá),下調(diào)Drp1的表達(dá)實(shí)現(xiàn)。
[Abstract]:Objective to investigate the protective effect of water-soluble coenzyme Q10 CoQ10 on apoptosis induced by rotenonerotenone rotator (rotenonerotenone Rot) and to investigate the mechanism of improving mitochondrial dysfunction and dynamic imbalance of mitochondrial fusion cleavage. To provide theoretical basis for the treatment of PD with water-soluble CoQ10. Methods 1. PC12 cells, which have been induced by nerve growth factor (NGF), were subcultured. PD cell model was established by selecting appropriate Rot concentration to treat PC12 cells in logarithmic growth phase. 2. The concentration of water-soluble CoQ10 was determined as the therapeutic concentration. The experiment was divided into two groups: Rot group, Rot group, CoQ10 group and CoQ10 treatment group, and the cell activity was detected by the method of 3.cck-8. Cell morphology was observed under inverted microscope. 4. Ros assay kit was used to detect the changes of intracellular ROS. 5. JC1-Mitochondrial membrane Potential Assay kit was stained. The changes of mitochondrial membrane potential were analyzed by flow cytometry. 6. MitoTracker mitochondrial fluorescence probe was used to label mitochondria. The morphologic changes of mitochondria were observed by laser confocal microscope. 7. Western blotting was used to detect apoptosis and the expression of mitochondrial fusion mitogen-associated protein. 1. The survival rate of PC12 cells after 24 hours of Rot treatment decreased significantly and showed negative phase with dose. 2. CoQ10 can improve the survival rate of PC12 cells induced by Rot. 2.Rot induced increase in ROS level. CoQ10 can reduce the ROS level of PC12 cells after Rot treatment. 3. Rot induced mitochondrial membrane potential of PC12 cells. Water soluble CoQ10 could improve the MMP reduction and mitochondrial fragmentation of PC12 cells induced by Rot. 4. Western blotting experiment showed that CoQ10 could decrease the expression of Caspase-9 active Caspase-3 and Bax induced by Rot, upregulate the expression of Bcl-2 and prevent AIF from transferring to nucleus. Water soluble CoQ10 could decrease the expression of mitochondrial mitogen Drp1 induced by Rot, and up-regulate the expression of Mfn2OPA1, a mtDNA fusion protein. Conclusion 1. Water soluble CoQ10 can improve the decrease of MMP and mitochondrial fragmentation in PC12 cells induced by Rot. 2. Water-soluble CoQ10 can protect PC12 cells from apoptosis induced by Rot. The mechanism may be by reducing cell oxidation. 2. Stress and apoptosis of mitochondrial pathway. 3. Rot induced mitochondrial fusion of PC12 cells by water-soluble CoQ10. The mechanism may be by up-regulating the expression of MfN2 OPA1 and down-regulating the expression of Drp1.
【學(xué)位授予單位】：寧夏醫(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)】：R742.5

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本文編號(hào)：1585210