ROS调控正常肝细胞增殖—静息转变的信号作用及机制研究

发布时间：2018-01-03 20:45

本文关键词：ROS调控正常肝细胞增殖—静息转变的信号作用及机制研究　出处：《第四军医大学》2011年博士论文　论文类型：学位论文

【摘要】：研究背景肝细胞通过调节自身增殖、静息状态的相互转变参与肝脏发育、再生、损伤修复等多种重要的生理病理过程。然而肝细胞增殖启动、终止的具体信号机制尚不清楚。初步研究显示,活性氧(ROS)在此动态转换过程中,具有不同的反应和作用。实验目的通过不同发育时期的肝细胞模型和肝切除动物模型,研究生长发育及肝再生过程中ROS水平变化与肝细胞增殖-静息转变的关系,阐明ROS信号在诱导肝细胞增殖-静息转变过程中的关键地位及作用机制,为进一步认识肝细胞增殖-静息转变的自我调控过程,寻找新的调控靶点奠定基础。实验方法与研究方案 1.观察大鼠肝脏发育过程中ROS的调控及ROS与肝细胞增殖→静息过渡的关系 ①分离不同发育阶段的肝细胞,检测ROS水平及细胞周期变化。 ②检测以Nrf2为核心的抗氧化产物的表达及活性变化。 2.体外实验研究ROS调控肝细胞增殖周期开、关的量效关系 ①根据前期实验的观察结果,分别选择胎龄15-16天和成年SD大鼠的肝细胞作为研究对象进行原代分离培养。 ②通过直接干预增殖或静息肝细胞内的ROS水平,观察胞内ROS水平的改变对细胞增殖、细胞周期及CyclinD、Rb、PCNA表达的影响,明确ROS是否在肝细胞增殖-静息转变中起着信号调控作用,同时阐明其作用的剂量-效应规律。 3. ROS对MAPKs的选择性磷酸化作用及ROS-MAPKs-CyclinD-Rb调控通路的研究 ①根据以上结果选择合适的干预剂量,通过降低增殖肝细胞内ROS水平诱导其静息或升高静息肝细胞内ROS水平诱导其增殖后,观察MAPKs磷酸化改变,证实ROS对MAPKs的选择性磷酸化激活作用。 ②干预胞内ROS浓度的同时,有针对性的采用特异性MAPKs抑制剂,观察胞内ROS水平、细胞增殖活性、细胞周期及相关蛋白表达、MAPKs磷酸化改变,进一步证实ROS信号的上游地位,探讨ROS-MAPKs-CyclinD-Rb调控通路在肝细胞增殖-静息状态转变中的作用。 4.体内实验研究成年大鼠肝脏微环境H_2O_2水平变化对细胞静息状态的影响及作用机制。 ①GOX处理大鼠,在生理范围内升高肝脏微环境的H_2O_2水平,观察肝脏增殖能力及ERK活性改变,以证明ROS在肝细胞增殖自我调控机制中起“阀门样”作用。 ②在GOX处理的基础上给予CAT清除肝内H_2O_2,观察能否对抗GOX的效应,以证明GOX的促增殖作用与其肝内代谢产物H_2O_2直接相关。 ③在GOX处理的基础上给予MEK1/2选择性抑制剂PD184161,观察抑制ERK通路能否遏制GOX的增殖启动效应,以证明ERK通路激活是H_2O_2启动肝细胞增殖的重要机制。 5.体内实验研究ROS信号对肝再生过程的调控采用成熟的大鼠70%肝切除模型,取不同时间点观察肝再生过程中ROS水平变化与肝组织增殖能力变化。同时正、反向干预肝再生早期的ROS水平,观察对肝再生及增殖相关信号通路的影响,探讨ROS平衡在肝再生启动中的作用。实验结果 1. SD大鼠发育过程中,出生后15天,80%以上的肝细胞处于G0期,PCNA、Ki67、CyclinD、AFP表达下降,Rb磷酸化水平明显降低,说明出生后2周左右,SD大鼠肝细胞结束增殖、过渡至稳定的静息状态。我们的研究发现,线粒体是发育过程中肝细胞内ROS的主要来源,Nrf2及下游的抗氧化产物在不同的发育时期选择性表达,两者共同作用的结果为,伴随着肝细胞由增殖过渡到静息,ROS水平显著降低,提示ROS与细胞增殖-静息转变密切相关。 2.采用NAC处理ROS水平相对较高的胚胎肝细胞,能显著降低ROS水平,抑制细胞增殖,诱导细胞静息,而p38抑制剂能显著阻断NAC诱导的细胞静息作用。采用H_2O_2处理ROS水平相对较低的成年肝细胞,能显著升高ROS水平,促进细胞增殖,而ERK抑制剂能显著阻断H_2O_2诱导的细胞增殖作用。以上结果说明,调控ROS可以通过选择性激活ERK或p38诱导细胞增殖或静息。 3. GOX可使肝脏ROS水平增高,改变肝脏的静息状态,促进增殖。CAT可以降低ROS水平,阻断GOX的增殖启动作用,说明GOX的促增殖作用与其肝内代谢产物H_2O_2直接相关。ERK特异性抑制剂可以显著阻断GOX的促增殖效应,但效果弱于CAT,说明ERK通路只是ROS启动细胞增殖的调控通路之一。 4. ROS水平在肝再生过程中变化先高后低,与肝切除后再生启动与终止密切相关。肝再生早期,GOX处理可以升高肝脏ROS水平,加重肝切除后氧化应激,抑制肝再生,而CAT处理可以降低肝脏ROS水平,减轻肝切除后氧化应激,但同样抑制肝再生。说明肝再生过程中的ROS水平被精确调控在“所需”范围内,可能是肝再生启动与进展的关键信号因素。结论: 本课题通过观察正常肝脏发育及肝细胞再生过程中ROS的变化规律,探讨ROS信号改变与肝细胞增殖-静息相互转变的内在联系,从“ROS水平的相对高低是维持肝细胞增殖或静息状态所必需的”这一新视角出发研究肝细胞周期自我调控的实现途径,证明了“ROS信号诱导肝细胞增殖-静息转变”的理论假设,初步阐明了ROS剂量依赖性调控G0期启动、G1期关闭的具体分子机制,为肝再生、发育的调控研究提供新的方向和思路。
[Abstract]:Research background
Liver cells through regulating their proliferation, transformation of resting state in liver development and regeneration, physiological and pathological process of injury repair and many other important. However, liver cell proliferation started, specific signaling mechanism termination is not clear. The preliminary study shows that the reactive oxygen species (ROS) in the dynamic process, with the reaction and effect of different.
Experimental purpose
Animal models of liver cells and liver model by different developmental stages of resection, ROS changes the level of the relationship between the growth and the process of liver regeneration and liver cell proliferation and resting, clarify the ROS signal in liver cell proliferation induced by status and function of the key resting during the transition to self regulate mechanism, further understanding of liver cell proliferation rest change process, lay the foundation for the regulation of new targets.
Experimental method and research scheme
1. the regulation of ROS during the development of rat liver and the relationship between ROS and hepatocyte proliferation and resting transition
(1) the liver cells of different developmental stages were isolated and the level of ROS and the changes of cell cycle were detected.
(2) detection of the expression and activity of antioxidant products at the core of Nrf2.
2. in vitro study of ROS regulating the proliferation cycle of hepatocytes and the relationship between the volume and effect of the liver cells
(1) according to the observation results of the previous experiments, the liver cells of 15-16 days of gestational age and adult SD rats were selected as the research object for primary isolation and culture.
Through the direct intervention of proliferation or resting liver cells ROS level, ROS level of observation of intracellular changes on cell proliferation, cell cycle and CyclinD, Rb, PCNA expression, to determine whether ROS changes in liver cell proliferation plays a regulatory role in resting signal, and to clarify the dose effect.
The selective phosphorylation of 3. ROS to MAPKs and the study of ROS-MAPKs-CyclinD-Rb regulation pathway
According to the above results, the appropriate intervention dose was selected according to the above results. By decreasing the level of ROS in the proliferating liver cells, inducing the resting or elevated ROS level in resting hepatocytes to induce the proliferation, we observed the phosphorylation of MAPKs and confirmed the selective phosphorylation activation of ROS on MAPKs.
At the same time, the intervention of the intracellular ROS concentration, the use of MAPKs specific inhibitor of ROS, to observe the level of intracellular, cell proliferation, cell cycle and expression of related protein, change the phosphorylation of MAPKs, further confirmed that the ROS signal of the upstream position, to explore the role of ROS-MAPKs-CyclinD-Rb pathway in liver cell proliferation changes in resting state.
4. the effect and mechanism of the changes of H_2O_2 level on the resting state of the liver microenvironment in adult rats were studied in vivo.
(1) GOX treatment in rats increased the level of H_2O_2 in the liver microenvironment in physiological range, and observed the proliferation ability and ERK activity of liver. It showed that ROS played a "valve like" role in the self-regulation mechanism of hepatocyte proliferation.
(2) on the basis of GOX treatment, CAT was cleared to remove H_2O_2 in the liver to observe whether it could antagonize GOX. It was proved that the proliferation promoting effect of GOX was directly related to its metabolite H_2O_2.
(3) on the basis of GOX treatment, we gave MEK1/2 selective inhibitor PD184161 to observe whether inhibiting ERK pathway could inhibit GOX's proliferation and priming effect, so as to prove that ERK pathway activation is an important mechanism for H_2O_2 to initiate hepatocyte proliferation.
The regeneration process control using the mature rat model of 70% hepatectomy for liver and 5. in vivo ROS signals at different time points to observe the changes of proliferation changes of ROS level in the process of liver regeneration and liver tissue. At the same time, the reverse intervention of liver regeneration early ROS level, observe the effects on liver regeneration and proliferation related signal pathway the study of ROS balance role in the initiation of liver regeneration.
experimental result
1. the development of SD rats, 15 days after birth, more than 80% of the liver cells are G0, PCNA, Ki67, CyclinD, AFP decreased the phosphorylation level of Rb decreased significantly, indicating about 2 weeks after birth, the liver cells of rat SD proliferation of resting state transition to the end, we study stability. Found that mitochondria are the main source of liver cells in ROS development process, oxidation products Nrf2 and downstream of the selective expression in different development period, the interaction between the two results, accompanied by liver cell proliferation by transition to resting, ROS level decreased significantly, which suggested ROS cell proliferation and transformation of resting closely related.
2. using NAC ROS with relatively high levels of fetal liver cells, can significantly reduce the ROS level, inhibit cell proliferation, induce cell resting, while the p38 inhibitor can significantly inhibit cell resting effect induced by NAC. ROS H_2O_2 is used to process the relatively low level of adult liver cells, can significantly increase the level of ROS and promote cell proliferation. ERK inhibitors can significantly inhibit cell proliferation induced by H_2O_2. These results suggest that the regulation of ROS can activate ERK or p38 by selective induction of cell proliferation or resting.
3. GOX can increase liver ROS level, the change of resting state in liver, promote the proliferation of.CAT can decrease the level of ROS, blocking the priming effect on proliferation of GOX, GOX proliferation and liver metabolite H_2O_2 is directly related to the specific inhibitor of.ERK could significantly reduce the proliferation promoting effect of GOX, but the effect is weaker than that of CAT. One of the only ROS ERK pathway regulation pathway cell proliferation.
4. the level of ROS in the process of liver regeneration changes from high to low, and regeneration after liver resection is closely related with the start end. Early liver regeneration, GOX treatment can increase the ROS level of liver after hepatectomy, increased oxidative stress, inhibiting liver regeneration, while CAT treatment can reduce the ROS level of liver after hepatectomy, reduce oxidative stress. But also inhibit liver regeneration. Liver regeneration process of the ROS level is in the precise control of the "desired" range, may be the key factor of signal initiation and progression of liver regeneration.
Conclusion:
Changes of this subject by observing the normal liver development and regeneration of liver cells during ROS, to study the relationship between ROS signal changes and the proliferation of liver cells and resting between changes in the way, from the relative level of ROS level is to maintain liver cell proliferation or resting state required for starting this new perspective on liver cell cycle self regulation, prove the theoretical assumptions of the ROS signal induced hepatocyte proliferation and resting transformation ", illustrates the ROS dose dependent regulation of G0 promoter, the molecular mechanism of G1 closed, for liver regeneration, provide new direction and ideas on the regulation of development.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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