本文關(guān)鍵詞：ROS調(diào)控正常肝細(xì)胞增殖—靜息轉(zhuǎn)變的信號作用及機(jī)制研究　出處：《第四軍醫(yī)大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： ROS 細(xì)胞增殖 細(xì)胞靜息 肝再生 MAPKs

【摘要】：研究背景 肝細(xì)胞通過調(diào)節(jié)自身增殖、靜息狀態(tài)的相互轉(zhuǎn)變參與肝臟發(fā)育、再生、損傷修復(fù)等多種重要的生理病理過程。然而肝細(xì)胞增殖啟動(dòng)、終止的具體信號機(jī)制尚不清楚。初步研究顯示,活性氧(ROS)在此動(dòng)態(tài)轉(zhuǎn)換過程中,具有不同的反應(yīng)和作用。 實(shí)驗(yàn)?zāi)康?通過不同發(fā)育時(shí)期的肝細(xì)胞模型和肝切除動(dòng)物模型,研究生長發(fā)育及肝再生過程中ROS水平變化與肝細(xì)胞增殖-靜息轉(zhuǎn)變的關(guān)系,闡明ROS信號在誘導(dǎo)肝細(xì)胞增殖-靜息轉(zhuǎn)變過程中的關(guān)鍵地位及作用機(jī)制,為進(jìn)一步認(rèn)識肝細(xì)胞增殖-靜息轉(zhuǎn)變的自我調(diào)控過程,尋找新的調(diào)控靶點(diǎn)奠定基礎(chǔ)。 實(shí)驗(yàn)方法與研究方案 1.觀察大鼠肝臟發(fā)育過程中ROS的調(diào)控及ROS與肝細(xì)胞增殖→靜息過渡的關(guān)系 ①分離不同發(fā)育階段的肝細(xì)胞,檢測ROS水平及細(xì)胞周期變化。 ②檢測以Nrf2為核心的抗氧化產(chǎn)物的表達(dá)及活性變化。 2.體外實(shí)驗(yàn)研究ROS調(diào)控肝細(xì)胞增殖周期開、關(guān)的量效關(guān)系 ①根據(jù)前期實(shí)驗(yàn)的觀察結(jié)果,分別選擇胎齡15-16天和成年SD大鼠的肝細(xì)胞作為研究對象進(jìn)行原代分離培養(yǎng)。 ②通過直接干預(yù)增殖或靜息肝細(xì)胞內(nèi)的ROS水平,觀察胞內(nèi)ROS水平的改變對細(xì)胞增殖、細(xì)胞周期及CyclinD、Rb、PCNA表達(dá)的影響,明確ROS是否在肝細(xì)胞增殖-靜息轉(zhuǎn)變中起著信號調(diào)控作用,同時(shí)闡明其作用的劑量-效應(yīng)規(guī)律。 3. ROS對MAPKs的選擇性磷酸化作用及ROS-MAPKs-CyclinD-Rb調(diào)控通路的研究 ①根據(jù)以上結(jié)果選擇合適的干預(yù)劑量,通過降低增殖肝細(xì)胞內(nèi)ROS水平誘導(dǎo)其靜息或升高靜息肝細(xì)胞內(nèi)ROS水平誘導(dǎo)其增殖后,觀察MAPKs磷酸化改變,證實(shí)ROS對MAPKs的選擇性磷酸化激活作用。 ②干預(yù)胞內(nèi)ROS濃度的同時(shí),有針對性的采用特異性MAPKs抑制劑,觀察胞內(nèi)ROS水平、細(xì)胞增殖活性、細(xì)胞周期及相關(guān)蛋白表達(dá)、MAPKs磷酸化改變,進(jìn)一步證實(shí)ROS信號的上游地位,探討ROS-MAPKs-CyclinD-Rb調(diào)控通路在肝細(xì)胞增殖-靜息狀態(tài)轉(zhuǎn)變中的作用。 4.體內(nèi)實(shí)驗(yàn)研究成年大鼠肝臟微環(huán)境H_2O_2水平變化對細(xì)胞靜息狀態(tài)的影響及作用機(jī)制。 ①GOX處理大鼠,在生理范圍內(nèi)升高肝臟微環(huán)境的H_2O_2水平,觀察肝臟增殖能力及ERK活性改變,以證明ROS在肝細(xì)胞增殖自我調(diào)控機(jī)制中起“閥門樣”作用。 ②在GOX處理的基礎(chǔ)上給予CAT清除肝內(nèi)H_2O_2,觀察能否對抗GOX的效應(yīng),以證明GOX的促增殖作用與其肝內(nèi)代謝產(chǎn)物H_2O_2直接相關(guān)。 ③在GOX處理的基礎(chǔ)上給予MEK1/2選擇性抑制劑PD184161,觀察抑制ERK通路能否遏制GOX的增殖啟動(dòng)效應(yīng),以證明ERK通路激活是H_2O_2啟動(dòng)肝細(xì)胞增殖的重要機(jī)制。 5.體內(nèi)實(shí)驗(yàn)研究ROS信號對肝再生過程的調(diào)控采用成熟的大鼠70%肝切除模型,取不同時(shí)間點(diǎn)觀察肝再生過程中ROS水平變化與肝組織增殖能力變化。同時(shí)正、反向干預(yù)肝再生早期的ROS水平,觀察對肝再生及增殖相關(guān)信號通路的影響,探討ROS平衡在肝再生啟動(dòng)中的作用。 實(shí)驗(yàn)結(jié)果 1. SD大鼠發(fā)育過程中,出生后15天,80%以上的肝細(xì)胞處于G0期,PCNA、Ki67、CyclinD、AFP表達(dá)下降,Rb磷酸化水平明顯降低,說明出生后2周左右,SD大鼠肝細(xì)胞結(jié)束增殖、過渡至穩(wěn)定的靜息狀態(tài)。我們的研究發(fā)現(xiàn),線粒體是發(fā)育過程中肝細(xì)胞內(nèi)ROS的主要來源,Nrf2及下游的抗氧化產(chǎn)物在不同的發(fā)育時(shí)期選擇性表達(dá),兩者共同作用的結(jié)果為,伴隨著肝細(xì)胞由增殖過渡到靜息,ROS水平顯著降低,提示ROS與細(xì)胞增殖-靜息轉(zhuǎn)變密切相關(guān)。 2.采用NAC處理ROS水平相對較高的胚胎肝細(xì)胞,能顯著降低ROS水平,抑制細(xì)胞增殖,誘導(dǎo)細(xì)胞靜息,而p38抑制劑能顯著阻斷NAC誘導(dǎo)的細(xì)胞靜息作用。采用H_2O_2處理ROS水平相對較低的成年肝細(xì)胞,能顯著升高ROS水平,促進(jìn)細(xì)胞增殖,而ERK抑制劑能顯著阻斷H_2O_2誘導(dǎo)的細(xì)胞增殖作用。以上結(jié)果說明,調(diào)控ROS可以通過選擇性激活ERK或p38誘導(dǎo)細(xì)胞增殖或靜息。 3. GOX可使肝臟ROS水平增高,改變肝臟的靜息狀態(tài),促進(jìn)增殖。CAT可以降低ROS水平,阻斷GOX的增殖啟動(dòng)作用,說明GOX的促增殖作用與其肝內(nèi)代謝產(chǎn)物H_2O_2直接相關(guān)。ERK特異性抑制劑可以顯著阻斷GOX的促增殖效應(yīng),但效果弱于CAT,說明ERK通路只是ROS啟動(dòng)細(xì)胞增殖的調(diào)控通路之一。 4. ROS水平在肝再生過程中變化先高后低,與肝切除后再生啟動(dòng)與終止密切相關(guān)。肝再生早期,GOX處理可以升高肝臟ROS水平,加重肝切除后氧化應(yīng)激,抑制肝再生,而CAT處理可以降低肝臟ROS水平,減輕肝切除后氧化應(yīng)激,但同樣抑制肝再生。說明肝再生過程中的ROS水平被精確調(diào)控在“所需”范圍內(nèi),可能是肝再生啟動(dòng)與進(jìn)展的關(guān)鍵信號因素。 結(jié)論: 本課題通過觀察正常肝臟發(fā)育及肝細(xì)胞再生過程中ROS的變化規(guī)律,探討ROS信號改變與肝細(xì)胞增殖-靜息相互轉(zhuǎn)變的內(nèi)在聯(lián)系,從“ROS水平的相對高低是維持肝細(xì)胞增殖或靜息狀態(tài)所必需的”這一新視角出發(fā)研究肝細(xì)胞周期自我調(diào)控的實(shí)現(xiàn)途徑,證明了“ROS信號誘導(dǎo)肝細(xì)胞增殖-靜息轉(zhuǎn)變”的理論假設(shè),初步闡明了ROS劑量依賴性調(diào)控G0期啟動(dòng)、G1期關(guān)閉的具體分子機(jī)制,為肝再生、發(fā)育的調(diào)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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363

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