【作者】 刘玉和；

【导师】 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的:探讨溶酶体-线粒体途径在氧化应激诱导的人宫颈癌细胞死亡过程中的作用及其机制。方法:(1)MTT法检测VK3诱导Hela细胞损伤过程中的细胞生存率;电镜观察细胞超微结构;DCFH-DA免疫荧光方法检测细胞内活性氧的水平;Hoechst33342免疫荧光方法检测细胞凋亡,RT-PCR、Western blot检测凋亡相关蛋白Bcl-2、Bax mRNA和Cyt C及caspase3蛋白的表达及自吞噬相关蛋白Beclin 1、LC3-II蛋白的表达。(2)AO染色检测溶酶体的稳定性、Rhodamine 123染色检测线粒体膜电势的变化; RT-PCR检测mTOR信号转导途径p70S6K mRNA、线粒体分裂融合基因mRNA表达的变化和Western blot检测Akt、mTOR蛋白的表达;MDC荧光染色检测自噬泡的聚集。(3)BCECF-AM荧光染色流式细胞仪分析Hela细胞损伤时细胞浆的pH变化。结果:(1)MTT检测VK3 (15μM、30μM、60μM)作用人子宫颈癌Hela细胞6h,12h,24h细胞生存率逐渐降低,并呈剂量时间相关性;细胞内活性氧增加;Hoechst33342染色检测VK3组细胞核呈现凋亡细胞核形态;RT-PCR和Western blot结果显示,VK3能引起Hela细胞Caspase-3、CytC蛋白表达增强以及Bax/Bcl-2 mRNA比值明显增高;同时发现VK3能引起Hela细胞自吞噬相关蛋白Beclin 1、LC3-II表达增加;电镜结果显示VK3能引起Hela细胞内自噬泡聚集。(2)与对照组比较,溶酶体稳定性在VK3作用Hela细胞3h开始降低、线粒体膜电势在作用6h开始降低,Akt、mTOR及mTOR底物p70S6K mRNA表达和磷酸化Akt和mTOR蛋白表达减低,线粒体分裂融合基因Mfn1、Opa1、MTP18 mRNA表达下降,而Mfn2、Drp1及Fist1 mRNA变化不明显;(3)与VK3组比较,联合应用VK3和NAC后,抑制了细胞的溶酶体稳定性、线粒体膜电势降低,Akt、mTOR及mTOR底物p70S6K mRNA表达和磷酸化Akt和mTOR蛋白表达均无明显降低。各组线粒体分裂融合基因表达无明显变化;(4)联合应用VK3和NH4Cl组细胞生存率降低,凋亡细胞数量增多,溶酶体膜稳定性及线粒体膜电势明显降低,磷酸化的Akt和mTOR蛋白表达明显下降,Drp1和MTP18 mRNA表达明显降低,Mfn1、Opa1基因表达增高,Mfn2和Fist1无明显变化,自噬泡数量增加,caspase3蛋白表达增高。(5)BCECF-AM荧光染色结果显示,与对照组相比,VK3组Hela细胞损伤时细胞浆的pH值降低。与VK3组比较,联合应用VK3和NAC组细胞浆pH值降低不明显;联合应用VK3和NH4Cl组细胞浆pH值明显降低。结论:(1)通过观察VK3诱导的Hela细胞氧化应激损伤时,caspase3裂解片段增多;cytosolic cyt C蛋白释放增加;Bax mRNA表达逐渐增强;Bcl-2 mRNA表达逐渐减弱;细胞核呈现典型凋亡形态;Beclin 1蛋白表达逐渐增强;LC3-II蛋白表达逐渐增强;细胞内自噬泡形成,联合应用VK3和NAC后活性氧的释放率减少;凋亡细胞核表现和自噬泡减少;提示VK3诱导Hela细胞死亡中,除了与I型程序性细胞死亡(凋亡)有关外,可能也与自噬有关。(2)通过观察不同时间点VK3诱导Hela细胞死亡过程,发现细胞浆红色荧光减弱、绿色荧光增强,说明溶酶体稳定性出现下降,而且溶酶体膜稳定性下降开始于VK3作用后第3h,线粒体膜电势下降开始于第6h。联合应用VK3和NAC后,溶酶体膜稳定性的及线粒体膜电势的下降均不明显;说明在VK3诱导的Hela细胞氧化应激损伤时,溶酶体稳定性降低先于线粒体膜电势下降,表明VK3可能通过影响溶酶体后导致线粒体损伤。(3)通过观察VK3诱导的Hela细胞氧化应激损伤时,Akt,mTOR和p70S6K基因表达明显下降;磷酸化Akt(ser-473)和mTOR(ser-2481)蛋白的表达明显下降;联合应用VK3和NH4Cl后,磷酸化的Akt和mTOR蛋白表达下降更明显;联合应用VK3和NAC后, Akt,mTOR和p70S6K基因和磷酸化的Akt和mTOR蛋白表达的下降不明显,提示在VK3诱导的Hela细胞死亡过程中, Akt/ mTOR信号途径的变化可能参与了I型程序性细胞死亡(凋亡)和自噬的发生过程。(4) VK3诱导的细胞氧化应激损伤时,细胞线粒体融合基因Mfn1和Opa1及线粒体分裂基因MTP18 mRNA表达明显降低;联合应用NAC可以抑制VK3诱导的Mfn1、Opa1及MTP18基因表达降低,表明线粒体融合基因和分裂基因表达异常可能与氧化应激诱导的细胞损伤有关。(5)联合应用VK3和NH4Cl后,与单独应用VK3相比,MTT结果显示细胞生存率明显降低,溶酶体稳定性明显下降,线粒体膜电势也明显降低。Mfn1、Opa1基因表达增加,Drp1和MTP18 mRNA表达明显降低,提示NH4Cl可能具有协同VK3诱导氧化应激细胞的溶酶体及线粒体损伤作用,导致溶酶体稳定性明显下降及线粒体融合基因和分裂基因mRNA表达异常,进一步表明溶酶体影响线粒体细胞死亡途径是氧化应激诱导Hela细胞死亡的主要机制。溶酶体与线粒体之间的平衡对细胞生存具有十分重要的影响。(6)通过观察联合应用NH4Cl和VK3的细胞内自噬泡聚集明显增多,凋亡相关蛋白caspase3表达增高,提示NH4Cl可能抑制了自噬泡和溶酶体的融合过程,进而凋亡作用增强。(7) VK3诱导的细胞氧化应激时, FL1/FL2比值降低,细胞浆pH值降低,酸性pH的细胞数增多(13.6%)。联合应用VK3和NAC,FL1/FL2比值增高,细胞浆pH值升高,酸性pH的细胞数减少(5.4%)。联合应用VK3和NH4Cl,FL1/FL2比值明显降低,细胞浆pH值明显降低,酸性pH的细胞数明显增多(43.8%),提示氧化应激诱导Hela细胞死亡可能与细胞浆酸化有关。细胞酸化即可能是细胞凋亡的伴随现象,同时又可能导致溶酶体、线粒体损伤,加重溶酶体-线粒体途径引起的细胞死亡,其机制有待于进一步研究。更多还原

【Abstract】 Programmed cell death was divided by Apoptosis,Autophgy and Necroptosis. Genes controlling programmed cell death were two sorts, in which one is inhibiting cell death, the other is promoting cell death.The two sorts of genes interact with each other in order to control cell normal death.Vitamin K is a kind of necessary vitamin. Study on Vitamin K suggested that it may have anti-tumor activity. At present, VK3 anti-tumor may be related to oxidative stress.ROS is increased in oxidative stress,which results in injury widely.Lately,study on apoptosis suggested that there was intracelluar abnormal signaling transduction. Intracellular acidification in these abnormal signaling, is a character of apoptosis. Intracellular pH adjust maybe tightly related to apoptosis.In order to investigate the roles of oxidative stress in inducing cell death, we make use of VK3 to induce Hela oxidative stress and cytosolic acidification, to observe mechanisms of oxidative stress in cell death.Methods:(1)To detect the apoptosis and autophagy mechanisms of VK3-induced Hela cells injury, by MTT、Hoechst33342、DCFH-DA immunofluorescent、RT-PCR、Western blot and transmission Electron Microscope.(2)To investigate the effect of oxydative stress througy lysosome on mitochondria in Hela cells death by AO, Rh123 immunofluorescent,MTT,RT-PCR and Western blot analysis and transmission Electron Microscope.(3)To observe role of intracellular acidification in Hela cells injuride by oxydative stress by BCECF-AM Flowcytometry analysis.Results:(1)During the VK3-induced Hela cells injuries, the survival rate was decreased and apoptosis appeared,with the increased level of ROS.The Caspase3 and cytC proteins expression were increased significantly. The ratio of Bax/Bcl-2 mRNA and protein was increased obviously. The expression of autophagy-associated Beclin 1 protein and LC3-II was up-regulated. Autophagic vacuoles was observed.(2)During the VK3-induced Hela cells injuries,lysosome was instabilized at 3h and mitochondrial△Ψm was decreased at 6h. Akt,mTOR and p70S6K mRNA expression and phospho-Akt/mTOR were all decreased.Mfn1、Opa1、MTP18 mRNA were all decreased and Mfn2、Drp1及Fist1 mRNA appeared no obvious change. In the VK3 combined with NAC group,lysosome stabilization and mitochondrial△Ψm were all not decreased. Akt,mTOR and p70S6K mRNA expression and phospho-Akt/mTOR were all not decreased.Mfn1、Opa1、MTP18 mRNA were all not decreased.In the VK3 combined with NH4Cl group,lysosome stabilization and mitochondrial△Ψm were all decreased obviously.Mfn1 mRNA expression was not decreased and Drp1,MTP18 mRNA expression was decreased.(3)BCECF-AM staining appeared cytoplasmic acidification in VK3 group. Compared to VK3 group,cytoplasmic acidification in VK3 combined with NAC group was decreased,while that in VK3 combined with NH4Cl group was increased severely.Conclusions:(1)By observing that cleavaged caspase3 and cytosolic CytC protein level were inceresed, Bax mRNA level was increased, Bcl-2 mRNA level was decreased,apoptosis cellular nuclear morphology of VK3-induced Hela oxydative stress,as well as Beclin1 and LC3-II protein expressions were increased with autophgosomes formation , while ROS level decresed,apoptosis nuclear appearance and autophagosome formation decreased induced by combinated with VK3 and NAC,suggests that ROS was related to programme I cell death(apoptosis) and also autophagy in Hela cells injuries.(2)By observing the death process of VK3-induced Hela cells in different time point,it is appeared that decreasing in red fluroscence and increasing in green fluroscence in the cytoplasm,it suggestes that lysosomal appeares instability.Moreover, lysosomal instability began at 3h induced by VK3, and mitochondrial clapse began at 6h,while lysosomal instability and mitochondrial△Ψm clapse not obvious induced by combinated with VK3 and NAC.It suggests that lysosomal stability was ahead of mitochondrial clapse. VK3 maybe injury mitochondria through lysosome.(3) By observing that the Akt,mTOR and p70S6K mRNA expressions obviously decreased,phospho-Akt at Ser-473 with phospho-mTOR at Ser-2481 decreased induced by VK3,then phospho-Akt,phospho-mTOR protein expressions decreased in combined with VK3 and NH4Cl,while Akt,mTOR, p70S6K mRNA expressions and phospho-Akt,phospho-mTOR were not decresed in combined with VK3 and NAC,it indicates that Akt/mTOR signal pathway may be involved in programme I cell death(apoptosis) and also autophagy in Hela cells injuries induced by VK3.(4)Mitochondrial mission genes Mfn1 and Opa1 as well as fussion genes MTP18 mRNA expressions were obviously decreased. Mfn1,Opa1 and MTP18 mRNA expressions were increase in combined with VK3 and NAC. It suggests that mitochondrial mission genes and fussion genes abnormal expressions may be involved in mitochondrial pathway death mechanism induced by oxydative stress.(5)Compared with VK3, cell viability by MTT,lysosomal stability and mitochondrial△Ψm were all decreased,as well as mitochondrial mission Mfn1,Opa1 genes espressions increasing and fussion gene Drp1 and MTP18 mRNA expressions decreasing in VK3 combined with NH4Cl group Hela cells. It suggests that lysosome through affecting mitochondrial death pathway maybe the main mechanism in ROS-induced Hela cells death.The balance between lysosome and mitochondria is important to cell survival.(6)By observing increase in autuphagosome and caspase3 protein expression in VK3 combined with NH4Cl group Hela cells,suggests that VK3 combined with NH4Cl may inhibit fussion process between autophagosome and lysosome,in turn strongly induces apoptosis.(7)FL1/FL2 ratio and intracellular pH were all decreased,and cell number with acidic intracellular pH increase(13.6%)induced by VK3. FL1/FL2 ratio and intracellular pH were all increased,and cell number with acidic intracellular pH decreased(5.4%)induced by combined with VK3 and NAC. FL1/FL2 ratio and intracellular pH were all obvious decreased and cell number with acidic intracellular pH increase(43.8%)induced by combined with VK3 and NH4Cl.It suggests that oxydative stress induces Hela cells death may be related to cytosolic acidification. Cytosolic acidification maybe along with apoptosis, moreover also result in lysosomal and mitochondrial injury aggravating lysosome-mitochondrial cell death pathway,which mechanism remain be further investigated.更多还原