【作者】 谭灿；

【导师】 张建湘；

【作者基本信息】 中南大学 ， 人体解剖与组织胚胎学， 2012， 博士

【摘要】 缺氧/缺血性损伤广泛存在于包括癌症、中风以及神经退行性疾病等多种疾病中,再供氧/再灌注会导致DNA损伤的积累从而加重缺氧/缺血造成的损伤。缺氧/缺血-再供氧/再灌注会导致非正常折叠或受损蛋白的积聚,这些蛋白大多被泛素-蛋白酶体系统清除。泛素介导的蛋白降解作用由底物蛋白的泛素化以及26S蛋白酶体参与的蛋白降解构成。泛素化包括了分别由E1激活酶、E2结合酶及E3连接酶催化的泛素激活、耦联以及转移三个级联步骤。其中,E3连接酶决定了泛素化修饰的底物专一性。以cullin环为基础的E3是E3酶中最大的家族之一,Cullin4A (CUL4A)是该家族的核心成员之一。CUL4A通过参与p53、p27和Cdt1等相关蛋白的降解过程,在肿瘤抑制、细胞成活、DNA损伤响应、细胞增殖以及基因组稳定性的维持等方面发挥了重要的作用。尽管有证据暗示表达上调的Cul4A有可能参与到细胞对缺氧/缺血-再供氧/再灌注导致的损伤的响应中,但是CUL4A在这一过程中的具体作用仍不得而知。在本研究中,我们构建了人源CUL4A(human CUL4A, hCUL4A)的腺病毒表达载体,通过病毒转染将hCUL4A成功导入了PC12细胞以及大鼠脑中。在随后的研究中,我们对CUL4A在缺氧/缺血-再供氧/再灌注导致的损伤中的功能及其作用机制作了初步探索。一、缺氧-再供氧诱导PC12细胞CM4A基因表达目的：检测PC12细胞中内源Cul4A基因对缺氧-再供氧(hypoxia-reoxygenation, H/R)处理的响应。方法：我们首先根据前人研究中的描述,建立PC12细胞H/R体系。通过检测缺氧后培养液氧分压(partial pressure of oxygen, pO2)的变化及细胞缺氧后缺氧诱导因子1α(hypoxia-inducible factor-1alpha,HIF-1α)蛋白的表达确认缺氧体系的有效性。然后,在PC12细胞缺氧1.5h,分别再供氧0.5、1、2、3、4和8h提取细胞总RNA进行半定量RT-PCR检测内源Cul4A表达。结果：缺氧后无细胞培养液中pO2升高,PC12细胞缺氧3h后细胞内HIF-1α蛋白表达升高。半定量RT-PCR的结果显示,PC12细胞中内源Cul4A在H/R处理1-4h之内被诱导至正常水平的三倍左右,随后在8h时下降到接近正常水平。结论：细胞缺氧系统是有效、稳定可靠的。PC12细胞内源Cul4A基因受H/R诱导上调表达,Cul4A可能会参与到PC12细胞对H/R损伤的细胞响应中。二、人CUL4A重组腺病毒载体的构建及其在PC12细胞中的表达特点目的：为研究CUL4A的表达上调在PC12细胞H/R损伤中的作用,我们需要首先构建携带有绿色荧光蛋白(green fluorescence protein,GFP)报告基因及hCUL4A基因的腺病毒表达载体,并研究其在PC12细胞中的表达特点。方法：扩增hCUL4A基因,并通过In-Fusion PCR克隆技术构建穿梭质粒pDC315-hCUL4A-EGFP,利用AdMaxTM腺病毒包装系统将该穿梭质粒与腺病毒表达载体骨架质粒pBHGlox ΔE1,E3Cre共转染HEK293细胞,经GFP荧光检测和Western印迹检测确认hCUL4A的表达后,进一步通过病毒扩增及纯化得到hCUL4A重组腺病毒载体Ad-hCUL4A-GFP。将该载体转染PC-12细胞,观察hCUL4A-GFP融合蛋白在PC-12细胞中的表达情况。结果：成功得到了较高滴度的腺病毒载体Ad-hCUL4A-GFP (1.6×1012pfu/ml)。荧光结果表明,Ad-hCUL4A-GFP在转染滴度大于1.6x109pfu/mL情况下,在转染8h后即可在PC12细胞中检测到表达。Hoechst33342染色的结果还表明转染的hCUL4A主要在PC12细胞的细胞质中表达。GFP荧光信号的检测以及western blot的结果还表明,hCUL4A在PC12细胞中的转染效率及蛋白表达水平依赖于腺病毒的转染时长和转染滴度。结论：腺病毒载体Ad-hCUL4A-GFP构建成功,掌握了其在PC12细胞中的最佳转染条件及时空表达模式。三、过表达人CUL4A对PC12细胞缺氧-再供氧损伤的保护作用及其机制探讨目的：初步探讨过表达hCUL4A在PC12细胞H/R损伤中的作用及机制。方法：将Ad-hCUL4A-GFP转染PC12细胞,再对细胞进行H/R处理。通过WST-1检测细胞活力、Hoechst33342染色细胞核研究细胞所处的凋亡阶段、加入蛋白酶体抑制剂MG132处理细胞、单细胞凝胶电泳检测DNA损伤及western blot检测凋亡相关蛋白caspase3、Bc1-2、p53、p27等的表达,对CUL4A在H/R中的作用及机制进行研究。结果：WST-1检测结果显示,PC12细胞H/R后,与空载体对照vector组相比,hCUL4A转染组的细胞活力明显增加(87.25%±1.75%vs.70.94%±3.39%,P<0.01)。Hoechst33342染色结果表明,H/R后,相对于vector组,hCUL4A转染组的PC12细胞处于凋亡Ⅰ期和Ⅱa期的细胞明显减少,总凋亡率得到显著抑制(24.77%±1.55%vs.40.73%±2.34%,P<0.01)。加入蛋白酶体抑制剂MG132(50μmol/L)后,hCUL4A过表达对总凋亡细胞数的影响作用受到抑制。单细胞凝胶电泳检测结果表明,PC12细胞经H/R处理后,hCUL4A过表达组细胞中带彗星尾的细胞(即DNA损伤细胞)在总体细胞中所占比例比vector组少60.39%(中位数=11.11%vs.28.05%,P<0.01)；并且过表达hCUL4A组尾距(tail moment, TM)值比vector组明显降低(中位数=4.88vs.10.43,P<0.01)。western blot结果显示,在PC12细胞中,由H/R诱导的caspase3活性上调明显被hCUL4A的过表达所抑制；与此相对应, hCUL4A的过表达组中抗凋亡因子Bcl-2的蛋白水平也明显比vector组高。另外,过表达hCUL4A还能有效抑制由H/R引起的p53和p27表达上调。结论：在H/R引起的PC12细胞损伤中,hCUL4A过表达可能通过调节细胞凋亡相关蛋白,如上调抗凋亡因子Bcl-2,抑制caspase3的活性,下调p53和p27的蛋白表达,并减少DNA的损伤,从而使细胞凋亡率下降,细胞活力升高。四、过表达hCUL4A对大鼠脑缺血再灌注损伤的保护作用目的：初步探索hCUL4A在大鼠脑缺血-再灌注(ischemia-reperfusion, I/R)损伤中的作用。方法：将Ad-hCUL4A-GFP通过大鼠右侧侧脑室立体定位注射转染大鼠脑,经基因表达及蛋白表达检测确认病毒注射后3d时hCUL4A能在大鼠脑中表达后,我们选取病毒注射后3d进行大鼠大脑中动脉梗塞(middle cerebral artery occlusion, MCAO)模型建立。大鼠MCAO再灌注后24h取脑进行TTC染色检测大脑梗塞体积及水肿程度；3d取脑对缺血中心区及缺血半影区的大脑皮层进行神经组织病理学检测(HE染色)；对MCAO再灌注后2-72h的大鼠连续进行神经功能缺损评分(neurological severity score, NSS, Longa评分法)。结果：TTC染色结果显示,与空载体对照vector组相比,hCUL4A组的脑梗塞体积减少约54.35%(中位数=187.42vs.410.60mm3,P<0.01),脑水肿程度也明显减轻(中位数=1.069vs.1.110,P<0.05)。HE染色结果显示,与vector组相比,hCUL4A组的大鼠脑缺血中心区以及缺血半影区的水肿程度较轻；红色神经元或鬼影细胞较少,神经元主要发生缺氧性损伤的改变而非vector组中表现的神经元坏死；CUL4A组中出现了较多增生肥大的星形胶质细胞,同时伴有毛细血管密度的增加(比vector组增加1.7倍)。NSS评分结果显示,与vector组相比,hCUL4A过表达组的大鼠在MCAO再灌注24、48和72h后的NSS评分明显下降。结论：hCUL4A过表达能有效减少由I/R造成的大鼠脑梗塞体积及减轻脑水肿程度,有可能会延缓神经元坏死的进程,并且促进脑I/R损伤后神经组织的修复,从而促进脑I/R后大鼠神经行为的有效恢复。综上所述,我们的结果揭示了过表达hCUL4A在PC12细胞以及大鼠脑中对缺氧/缺血-再供氧/再灌注造成的损伤具有保护作用。中风等疾病是典型的以缺氧/缺血-再供氧/再灌注为特征的疾病,我们的发现提供了一种可能,即CUL4A上调表达有可能在中风等疾病中也具有有益的作用。我们的研究为将来在中风等疾病的体内模型或者临床研究中进一步揭示CUL4A的作用机制提供了有价值的参考信息。更多还原

【Abstract】 Hypoxic/ischemic injury has been implicated in many diseases, including various types of cancer, stroke and neurodegenerative diseases. Reoxygenation/reperfusion leads to the accumulation of DNA damage and therefore exacerbates hypoxic/ischemic injury. Hypoxia/ischemia-reoxygenation/reperfusion leads to the accumulation of misfolded or damaged proteins that are generally degraded by the ubiquitin proteasome system (UPS). The process is initiated by ubiquitination through a cascade of three enzymes, the E1activating enzyme, the E2conjugating enzyme and the E3ubiquitin ligase. Ubiquitinated proteins are then subjected to degradation by the26S proteasome. The ubiquitin E3ligase determines the specificity for ubiquitination substrates. Cullin RING-based E3is one of the largest ubiquitin E3families. Cullin4A (CUL4A) is a key member of the cullin family. By participating in the degradation of relevant proteins such as p53, p27and Cdtl, CUL4A is thought to be crucial in diverse cellular processes, such as tumor suppression, cell survival, DNA damage response, proliferation and genome stability. It has been reported that the expression of CUL4A can be induced by hypoxic injury. However, the effect of the elevated CUL4A expression on hypoxia-reoxygenation (H/R) injury is currently unclear. In this study, an adenoviral expression vector baring human CUL4A (hCUL4A) gene was constructed. hCUL4A was then successfully transfected to PC12cells and the rat brain. In the following studies, we explored the functions and mechanism of CUL4A in the H/R-or ischemia/reperfusion (I/R)-induced injury.Chapter1Cul4A gene expression in PC12cells was induced by hypoxia-reoxygenation injuryObjective:To investigate the response of endogenouse Cul4A gene to hypoxia-reoxygenation (H/R).Methods:We first established H/R experimental system in PC12cells according to the previouse report. After detecting the partial pressure of oxygen (pO2) in the hypoxic media and detecting the protein expression of hypoxia-inducible factor-1alpha (HIF-1α)in hypoxic cells to confirm the efficiency of the hypoxic procedure,PC12cells were subjected to1.5h of hypoxia followed by0.5,1,2,3,4or8h of reoxygenation. Then total RNA was isolated and semi-quantitative RT-PCR was performed to detect endogenous Cul4A expression.Results:The pO2was elevated in the media without cells after H/R. HIF-la was expressed in PC12cells subjected to3h of hypoxia. The semi-quantitative RT-PCR results shown that the expression of endogenous Cul4A increased up to3-fold during the first1-4h after H/R and then declined to normal levels8h after H/R.Conclusion:The hypoxic system is effective and stable. Cul4A gene was elevated after H/R. These results suggest the involvement of the Cul4A gene in the response to H/R in PC12cells.Chapter2Construction of the human CUL4A recombinant adenovirus vector and its expression patterns in PC12cellsObjective:To examine the function of the elevated CUL4A in the response to H/R in PC12cells, adenovirus expression vector carrying green fluorescence protein (GFP) and hCUL4A was first needed to be constructed. Then the expression patterns of the vector in PC12cells were studied.Methods:Human CUL4A gene was amplified and cloned into shuttle vector (pDC315-hCUL4A-EGFP) by In-Fusion PCR cloning technology. The skeleton plasmid of adenovirus expressioin vector, pBHG lox△E1, E3Cre, was then co-transfected with pDC315-hCUL4A-EGFP into HEK293cells using AdMaxTM vector system. After confirming the expression of hCUL4A through GFP fluorescence detection and western blot, hCUL4A adenovirus expression vector Ad-CUL4A-GFP was obtained through further viral amplification and purification. The expression patterns of hCUL4A in PC-12cells were studied after transfecting Ad-hCUL4A-GFP into PC-12cells.Results:High titer hCUL4A adenovirus expression vector Ad-hCUL4A-GFP (1.6×1012pfu/mL) was obtained. Fluorescent results shown that since the transfecting titer was more than1.6×109pfu/mL, the expression of hCUL4A-GFP could be detected after8h post-transfection. Hoechst33342staining results shown that the transfected hCUL4A mainly expressed in the cytoplasm of PC12cells. GFP fluorescence detection and western blot shown that the transfection efficiency and the expression level of hCUL4A was time and tranfectional titer dependent.Conclusion:The adenovirus vector Ad-hCUL4A-GFP was successfully constructed. That the optimal transfection titer and the spatio-temporal expression pattern of hCUL4A revealed in this study paved the way for the functional study of hCUL4A in PC12cells in the future.Chapter3The protective effects and mechanism of hCUL4A overexpression on hypoxia-reoxygenation injury in PC12cellsObjective:To investigate the functions and mechanism of hCUL4A in H/R injury in PC12cells.Methods:PC12cells were transfected by Ad-hCUL4A-GFP, then were subjected to H/R. The functions and mechanism of CUL4A in H/R were studied by detecting cell viability with WST-1, analyzing differnent stages of apoptosis with Hoechst33342staining, dealing the cells with proteasome inhibitor MG132, detecting DNA injury with single cell gel electrophoresis (SCGE), and detecting the apoptosis related protein expression (caspase3, Bcl-2, p53, p27) through western blot.Results:Cell viability was measured using the WST-1assay. Compared with the vector control group (70.94%±3.39%), cell viability increased after H/R in the hCUL4A group (87.25%±1.75%, P<0.01). Hoechst33342staining results shown that, compared with the adenoviral vector control, transfection with hCUL4A significantly suppressed H/R-induced apoptosis (24.77%±1.55%versus40.73%±2.34%, P<0.01) by decreasing the presence of stage I and stage IIa apoptotic cells. However, with the addition of the proteasome inhibitor MG132(50μmol/L), the percentage of total apoptotic cells was no longer significantly different after H/R between the two groups. Results of the SCGE shown that the percentage of DNA-damaged cells, which is determined by the percentage of nuclei with tails, was60.39%lower in the hCUL4A group than in the control group (median=11.11%versus28.05%, P<0.01) after H/R; Moreover, the tail moment decreased significantly in the hCUL4A group compared to the control group. Results of western blot shown that, H/R-induced up-regulation of caspase3activity was significantly suppressed in hCUL4A transgenic PC12cells. Consistent with the decreased caspase3activity, elevated Bcl-2levels were also observed in hCUL4A transgenic cells after H/R. Moreover, the elevated expression of p53and p27induced by H/R could be inhibited by hCUL4A overexpression.Conclusion:Overexpression of hCUL4A may protect PC12cells from H/R-induced injury by modulating apoptosis-related proteins, such as up-regulating Bcl-2, suppressing the activity of caspase3, down-regulating p53and p27. CUL4A can also suppress H/R-induced DNA damage, therefore decreasing apoptosis cells and increasing cell viability.Chapter4The protective effects of hCUL4A overexpression on ischemia-reperfusion injury in the rat brainObjective:To explore the functions of hCUL4A in ischemia-reperfusion (I/R)-induced brain injury in rat.Methods:Adenovirus Ad-hCUL4A-GFP was infused into rat brains by intracerebroventricular injection. After confirming the hCUL4A gene expression and protein expression in the rat brain3days post adenoviral injection, the middle cerebral artery occlusion (MCAO) model in rat was performed on this day. TTC staining for the assessment of the brain infarct volume and brain edema was performed at24h after MCAO/reperfusion. The histopathology of cortical neurons in the ischemic core and in the ischemic penumbra was examined3days after reperfusion using H&E staining. A widely used5-point NSS system was used to evaluate neurological behaviors from2to72h after MCAO/reperfusion.Results:TTC results shown that compared with the adenoviral vector control group (median=410.60mm3), the brain infarct volume caused by transient MCAO in the hCUL4A group (median=187.42mm3, P<0.01) was significantly decreased by54.35%. Brain edema was also reduced in the hCUL4A group compared to the vector group (median=1.069vs.1.110, P<0.05). In H&E staining brain slides, compared with the vector group, a reduction of brain edema was observed in rats in hCUL4A group, and a few red neurons and ghost cells were found. Most of the neurons were undergoing swelling, instead of the necrosis observed in the vector groups. Moreover, the proliferation of astrocytes and an increase in capillary density (up to1.7-fold vs. vector group), were also found in the hCUL4A group. NSS score results shown that compared with the vector group, significant decreases of the NSS in the hCUL4A group were observed at24,48and72h after reperfusion.Conclusion:After I/R in rat, hCUL4A overexpression can dramatically decrease brain edema and brain infarct volume, might suppress neuron necrosis and promote the repair of neural tissue, therefore promoting neurological functional recovery.In summary, our results reveal the protective effects of hCUL4A in PC12cells and rat brain upon hypoxia/ischemia-reoxygenation/reperfusion injury. Diseases such as stroke are typically characterized by hypoxia/ischemia-reoxygenation/reperfusion induced injury. Our finding raises the possibility that elevated CUL4A may exert a beneficial role in stroke disease. It will be of interest to further explore more detailed mechanisms for the protective function of the elevated hCUL4A in ischemia-reperfusion injury in in vivo stroke models or stroke disease.更多还原