GC-C信号通路在溃疡性结肠炎肠道屏障及炎症发生中的作用

发布时间：2018-05-09 14:48

本文选题：GC-C + Gn　；参考：《昆明医科大学》2017年博士论文

【摘要】：溃疡性结肠炎(ulcerativecolitis,UC)是一种主要累及结肠的慢性特发性肠道炎症性疾病,是炎症性肠病(inflammatory bowel disease,IBD)的主要类型,以反复发作的腹痛、腹泻和粘液脓血便为常见临床症状。由于其病程迁延反复和结肠癌高风险率而严重影响患者生活质量,耗费大量医疗资源。近年来中国的IBD发病率逐渐升高,尤其是UC。IBD发病机制尚未完全阐明。尽管目前随着生物制剂如英夫利西单抗的应用,IBD患者的治疗效果得到改善,但仍有部分患者对药物治疗不敏感,病情严重而需行结肠切除手术。因此,研究UC的病因学,寻找一种新的特异性强的治疗靶点是本学科亟待解决的问题。肠上皮细胞间紧密连接蛋白(tight junction proteins,TJPs)作用于肠上皮形成紧密连接的肠粘膜机械屏障。当肠粘膜屏障遭到破坏时,肠道通透性增加,易导致肠道炎症的发生。近年来研究发现鸟苷酸环化酶C(guanylate cyclase C,GC-C)信号转导通路调节肠上皮屏障功能和肠道炎症的发生。GC-C是一种主要表达于肠上皮细胞的跨膜受体,鸟苷蛋白(guanylin,Gn)和尿鸟苷素(uroguanylin,Ugn)是其内源性配体,亦表达于胃肠道上皮细胞。热稳定性肠毒素(heat-stable enterotoxin,STa)是GC-C的外源性配体,由产肠毒素大肠杆菌产生。当这些配体(Gn/Ugn/STa)与 GC-C 结合后细胞内环磷酸鸟苷(cyclic guanosine monophosphate cGMP)水平增高,激活GC-C信号通路,参与调节细胞内外正常的离子浓度和肠道水电解质平衡。然而,目前关于GC-C信号通路在肠道炎症损伤中的作用尚未明确。本实验研究首先发现UC患者结肠粘膜组织中GC-C及其内源性配体Gn和Ugn的表达量与健康对照人群相比均明显降低,且随着UC患者疾病活动度增加,GC-C、Gn和Ugn的表达下降更为显著。提示UC患者GC-C信号通路活性降低,其活性与UC疾病活动度呈负相关。GC-C信号通路可能参与了 UC的发生与发展。在上述发现的基础上,为了进一步说明GC-C信号通路在肠道屏障及炎症损伤中的作用,我们运用人肠上皮细胞Caco-2单层屏障模型进行实验研究,通过检测 GC-C 信号通路关键因子(GC-C、Gn、Ugn 和 cGMP)、TJPs(occludin、claudin-1和ZO-1)和促炎症因子(IL-8和TNF-α)的表达,发现细胞在白介素1β(Interleukin-1β,IL-1β)刺激下 GC-C、Gn、Ugn、cGMP、claudin-1 和 ZO-1 的表达明显降低,细胞活力和超氧化物歧化酶(superoxidedismutase,SOD)酶活性下降,IL-8和TNF-α的表达明显升高。经转染Gn过表达载体后,细胞活力、SOD酶活性、Gn、GC-C、cGMP、claudin-1和ZO-1的表达均明显升高,单层细胞的通透性、IL-8和TNF-α的表达明显降低。而经转染GC-CshRNA干扰载体的细胞在IL-1β的刺激下,细胞活力、SOD酶活性、claudin-1和ZO-1的水平下降更明显,单层细胞的通透性、IL-8和TNF-α水平升高更为显著。提示GC-C配体可激活处于静止状态的GC-C信号通路,活化的GC-C信号通路可减轻Caco-2细胞在IL-1β诱导下的屏障功能受损及炎症性损伤。最后,我们运用右旋糖酐硫酸酯钠(dextran sodium sulfate,DSS)诱导的UC小鼠模型进行GC-C信号通路的体内功能实验,发现经注射Gn过表达慢病毒的结肠炎小鼠精神、进食、活动好转,体重上升,稀便及粘液血便情况减轻,肠道通透性和结肠组织炎症性评分降低,结肠组织GC-C、Gn、Ugn、cGMP、claudin-1和ZO-1的表达均升高,结肠组织和外周血清IL-8和TNF-α水平均明显降低。提示Gn过表达慢病毒运用于UC小鼠可减轻小鼠肠道炎症损伤,修复受损的肠粘膜屏障。本研究结果表明UC患者肠粘膜GC-C、Gn和Ugn的表达与UC疾病活动度呈负相关,GC-C配体可激活处于静止状态的GC-C信号通路,活化的GC-C信号通路可减轻Caco-2细胞在IL-1β诱导下的屏障功能受损及炎症性损伤,并可减轻DSS诱导的小鼠肠道炎症。综合以上研究结果,我们认为GC-C信号通路在UC的发生与发展中发挥了重要作用。GC-C受体激动剂具有用于UC患者临床治疗的潜能,为寻求IBD新的治疗策略提供重要的实验依据。第一部分GC-C及其配体Gn、Ugn在UC患者中差异性表达分析[目的]研究不同疾病活动度的UC患者结肠粘膜组织GC-C及其内源性配体Gn和Ugn的表达差异,探讨GC-C信号通路与UC及其严重度的关系。[方法]收集60例UC患者病变的结肠粘膜组织和20例正常对照者正常的结肠粘膜组织,运用改良的Mayo评分系统对UC患者的疾病活动度进行评分,分为轻度活动组(18例)、中度活动组(23例)和重度活动组(19例)。采用实时荧光定量PCR(qRT-PCR)和蛋白质印迹法(Western blot)方法分别检测肠粘膜组织GC-C、Gn和UgnmRNA和蛋白质的表达。比较分析GC-C、Gn和Ugn的转录和表达水平与UC疾病严重程度的相关性。[结果]qRT-PCR检测结果显示:正常对照组和轻、中、重度UC组的结肠粘膜组织中 GC-C mRNA 相对表达量分别为(1.652±0.258)、(0.525±0.240)、(0.153±0.107)和(0.034±0.020);Gn mRNA 相对表达量分别为(1.484±0.628)、(0.243±0.063)、(0.097±0.051)和(0.056±0.014);Ugn mRNA 相对表达量分别为(1.196±0.609)、(0.334±0.058)、(0.126±0.066)和(0.082±0.023)。与正常对照组相比,UC 组 GC-C、Gn、UgnmRNA的相对表达量均明显降低,轻、中、重度UC组间GC-C、Gn、UgnmRNA的相对表达量亦有统计学差异,随着疾病活动度加重,GC-C、Gn和UgnmRNA的相对表达量均明显降低。运用Western blot方法检测的结果显示:各组间肠粘膜组织GC-C、Gn、Ugn蛋白相对表达量的变化与mRNA的变化一致。[结论]UC患者肠粘膜组织中GC-C、Gn和Ugn的转录和表达水平下调,与UC疾病活动度呈负相关。第二部分GC-C信号通路在Caco-2细胞屏障及炎症损伤中的作用研究[目的]运用Caco-2细胞单层屏障模型研究GC-C信号通路在肠上皮屏障和炎症性损伤中的作用。[方法]将Caco-2肠上皮细胞培养在Transwell小室构建单层细胞屏障模型,检测单层细胞的跨膜电阻确定屏障的完整性。运用IL-1β刺激Caco-2细胞模拟肠上皮炎症性细胞,并造成细胞间紧密连接结构的破坏。采用脂质体法分别转染GC-C shRNA干扰载体和Gn过表达载体至细胞。经过不同处理后,通过测定大分子物质FD-4通过细胞的速率检测Caco-2单层细胞的通透性;采用CCK-8法检测细胞活力;NBT法检测细胞SOD酶活性;ELISA方法检测细胞上清液IL-8、TNF-α水平;采用qRT-PCR和Western blot方法分别检测细胞GC-C信号通路关键因子(GC-C、Gn、Ugn 和 cGMP)、紧密连接蛋白(occludin、claudin-1 和 ZO-1)和促炎症因子(IL-8、TNF-α)的表达。[结果]Caco-2单层细胞在培养的第19天,跨膜电阻值稳定在250 Ω· cm2以上,表明肠屏障模型构建成功。转染Gn过表达载体48小时后,除了 Gn的表达明显升高外,细胞GC-C和cGMP的表达亦明显升高,Ugn的表达无明显变化。转染GC-CshRNA48小时后的细胞除了 GC-C的表达明显降低外,Gn、Ugn和cGMP的表达亦明显降低。IL-1 β刺激细胞后,细胞活力和SOD酶活性明显降低,GC-C、Gn、Ugn、cGMP、claudin-1、ZO-1的表达均明显降低,单层细胞的通透性、IL-8和TNF-α的水平均明显升高。在IL-1β刺激的细胞转染Gn过表达载体后,与过表达阴性对照组(vector control 1)相比,细胞活力、SOD酶活性、claudin-1和ZO-1的表达都明显升高,IL-8、TNF-α的表达和细胞通透性均明显降低。相反地,经转染GC-C shRNA干扰载体的细胞与干扰阴性对照组(vector control 2)相比,在IL-1β的诱导下IL-8、TNF-α的表达和细胞通透性升高更为显著,细胞活力、SOD酶活性、claudin-1和ZO-1下降更为显著。各组间occludin的表达无明显差异。[结论]GC-C及其配体Gn和Ugn的表达相互调节,其配体可激活处于静止状态的GC-C信号通路,活化的GC-C信号通路可减轻Caco-2细胞在IL-1 β诱导下的屏障功能受损及炎症性损伤。第三部分GC-C信号通路在UC小鼠肠道炎症发生中的作用研究[目的]运用葡聚糖硫酸钠(DSS)诱导的UC小鼠模型研究GC-C信号通路在肠道炎症发生中的作用。[方法]随机将60只Bal b/c小鼠分成5个组,每组有12只小鼠:(1)正常对照组(Control);(2)结肠炎组(DSS+ NS);(3)结肠炎+美沙拉嗪治疗组(DSS+Mesalamine);(4)结肠炎+Gn载体治疗组(DSS+ Gn);(5)结肠炎+美沙拉嗪联合Gn载体治疗组(DSS+ Mesalamine+ Gn)。给予小鼠自由饮用3%的DSS溶液1周进行UC模型的构建。UC模型构建成功后,美沙拉嗪治疗组的小鼠予30mg/kg的美沙拉嗪每日灌胃1次并持续1周,Gn载体治疗组通过尾静脉注射Gn过表达慢病毒每日1次治疗1周。实验第1日起观察记录小鼠的进食、活动、体重、大便性状及血便等情况。治疗结束后测量各组小鼠肠道对大分子物质FD-4的通透性。实验2周后处死小鼠,收集小鼠外周血清和结肠组织。通过HE染色在光学显微镜下观察结肠组织炎症损伤情况进行组织病理学评分。采用免疫组织化学方法检测结肠组织GC-C信号通路关键因子(GC-C、Gn、Ugn和cGMP)、紧密连接蛋白(occludin、claudin-1和ZO-1)和促炎症因子(IL-8、TNF-α)的表达。采用ELISA方法进行小鼠外周血清IL-8和TNF-α水平的检测。[结果]各治疗组小鼠精神、进食、活动较结肠炎组小鼠好转,体重均有升高,排稀便及粘液血便情况均减轻,其中美沙拉嗪联合Gn载体治疗组小鼠的改善效果最明显。组织病理学评分显示,结肠炎组小鼠的结肠组织学评分明显高于正常对照组,各治疗组的评分均低于结肠炎组,其中美沙拉嗪联合Gn载体治疗组的评分最低。与正常对照组相比,结肠炎组小鼠结肠组织GC-C、Gn、Ugn和cGMP的表达均明显降低,各治疗组的表达较结肠炎组小鼠均升高,以Gn载体治疗组升高最为显著。结肠炎组小鼠肠道通透性较正常对照组均明显升高,各治疗组小鼠的肠道通透性较结肠炎组均降低,以Gn载体治疗组降低最为显著。occludin、claudin-1和ZO-1在结肠炎组小鼠结肠组织中的表达较正常对照组均明显降低,各治疗组小鼠claudin-1和ZO-1的表达都明显升高,其中美沙拉嗪联合Gn载体治疗组升高最明显。结肠炎组小鼠结肠组织和外周血清中IL-8和TNF-α的表达水平较正常对照组均明显升高,各治疗组的表达较结肠炎组均明显降低,以Gn载体治疗组降低最为显著。[结论]Gn过表达慢病毒单独运用或联合美沙拉嗪用于UC小鼠中可减轻小鼠肠道炎症损伤,修复小鼠受损的肠粘膜屏障功能,进一步证明GC-C信号通路在肠道中的保护性作用。
[Abstract]:Ulcerativecolitis (UC) is a chronic and idiopathic intestinal inflammatory disease involved in the colon. It is the main type of inflammatory bowel disease (IBD), with recurrent abdominal pain, diarrhea and mucus purulent stool as a common clinical symptom. In recent years, the incidence of IBD in China has been increasing, especially the pathogenesis of UC.IBD has not been fully elucidated. Although with the application of biological agents such as inflixi monoclonal antibody, the treatment effect of IBD patients has been improved, but some patients are still insensitive to drug treatment, and the condition is strict. It is important to undergo colectomy. Therefore, the study of the etiology of UC and the search for a new and specific therapeutic target are an urgent problem in this subject. The tight junction proteins (TJPs) is a closely linked intestinal mucosal mechanical barrier in the intestinal epithelium. When the intestinal mucosal barrier is destroyed, The intestinal permeability is increased and the intestinal inflammation is easy to occur. In recent years, the study found that the guanylate cyclase C (GC-C) signal transduction pathway regulates the intestinal epithelial barrier function and the occurrence of intestinal inflammation,.GC-C is a transmembrane receptor mainly expressed in intestinal epithelial cells, guanylin, Gn, and urinary guanosine (uroguanyli) (uroguanyli). N, Ugn) is an endogenous ligand and is also expressed in gastrointestinal epithelial cells. Heat-stable enterotoxin (STa) is a exogenous ligand of GC-C, which is produced by enterotoxigenic Escherichia coli. When these ligand (Gn/Ugn/STa) and GC-C are combined with GC-C, the level of cyclic guanosine (cyclic guanosine monophosphate cGMP) increases and activates it. -C signaling pathway is involved in regulating the normal ionic concentration and the balance of water and electrolyte in the intestinal tract. However, the role of the GC-C signaling pathway in intestinal inflammation is not clear. First, we found that the expression of GC-C and its endogenous ligands, Gn and Ugn, in the colon mucosa of UC patients is compared with those of the healthy control population. The expression of GC-C, Gn and Ugn decreased more significantly with the increase of the degree of disease activity in UC patients. It was suggested that the activity of GC-C signaling pathway in UC patients decreased and the activity of the UC disease activity was negatively related to the.GC-C signaling pathway, which might be involved in the occurrence and development of UC. On the basis of the above occurrence, the GC-C signaling pathway was further explained. In the intestinal barrier and the role of inflammatory damage, we used the human intestinal epithelial cell Caco-2 monolayer barrier model for experimental study. By detecting the key factors of the GC-C signaling pathway (GC-C, Gn, Ugn and cGMP), TJPs (occludin, claudin-1 and ZO-1) and the expression of inflammatory factors (IL-8 and TNF- alpha), the cells were found in the interleukin 1 beta. IL-1 beta stimulated the expression of GC-C, Gn, Ugn, cGMP, claudin-1 and ZO-1, and the activity of cells and the activity of superoxide dismutase (superoxidedismutase, SOD) decreased, and the expression of IL-8 and TNF- alpha was significantly increased. The permeability of monolayer cells, the expression of IL-8 and TNF- alpha, decreased significantly. Cell viability, SOD enzyme activity, claudin-1 and ZO-1 levels decreased more significantly under the stimulation of IL-1 beta by transfected GC-CshRNA interfering carriers. The permeability of monolayer cells, IL-8 and TNF- alpha levels increased more significantly. The GC-C ligands were activated in a state of rest. GC-C signaling pathway, activated GC-C signaling pathway can reduce the barrier function and inflammatory damage induced by IL-1 beta induced by Caco-2 cells. Finally, we use the UC mouse model induced by sodium dextran (dextran sodium sulfate, DSS) to carry out the functional experiment of the GC-C signaling pathway in vivo, and find that the injection Gn overexpression of the lentivirus has been found. The expression of GC-C, Gn, Ugn, Ugn, cGMP, claudin-1 and ZO-1 in colonic tissues increased and the levels of IL-8 and TNF- alpha in colonic tissue and peripheral blood decreased significantly. The results of this study showed that the expression of GC-C, Gn and Ugn in the intestinal mucosa of UC patients was negatively correlated with the degree of UC disease activity. The GC-C ligand could activate the GC-C signaling pathway in the static state, and the activated GC-C signal pathway could reduce the screen of Caco-2 cells under IL-1 beta induced by UC. The impairment of the barrier function and inflammatory injury can reduce the intestinal inflammation induced by DSS in mice. Combined with the above results, we believe that the GC-C signaling pathway plays an important role in the development and development of UC, and the.GC-C receptor agonist has the potential for the clinical treatment of UC patients and provides an important experimental basis for the search for a new therapeutic strategy for IBD. The first part GC-C and its ligand Gn, Ugn in UC patients' differential expression analysis [Objective] to study the difference in the expression of GC-C and its endogenous ligand Gn and Ugn in the colon mucosa of UC patients with different degree of activity of disease, and to explore the relationship between the GC-C signal pathway and the UC and its severity. [Methods] collect 60 cases of the colon mucosa of the UC patients and 20 cases. The normal colon mucosa tissue of normal controls was divided into mild activity group (18 cases), moderate activity group (23 cases) and severe activity group (19 cases) by modified Mayo scoring system (23 cases) and severe activity group (19 cases). The GC-C in intestinal mucosa was detected by real-time fluorescence quantitative PCR (qRT-PCR) and egg white mass imprinting (Western blot). The expression of Gn and UgnmRNA and protein. The correlation between the transcriptional and expression levels of GC-C, Gn and Ugn and the severity of UC disease was compared. [results]qRT-PCR detection results showed that the relative expression of GC-C mRNA in the colon mucosa of the normal control group and the moderate, severe UC group was (1.652 + 0.258), (0.525 + 0.240), (0.153 + 0.107) and 0. (0.), respectively). The relative expression of Gn mRNA was (1.484 + 0.628), (0.243 + 0.063), (0.097 + 0.051) and (0.056 + 0.014), and the relative expression of Ugn mRNA was (1.196 + 0.609) and (0.334 + 0.058) and (0.243). Compared with the normal control group, the relative expression of GC-C, Gn and UgnmRNA in the UC group decreased obviously, light, medium, and severe UC The relative expressions of GC-C, Gn and UgnmRNA between groups were also statistically different. The relative expression of GC-C, Gn and UgnmRNA decreased with the increase of the degree of disease activity. The results of Western blot methods showed that the changes in the relative expression of GC-C, Gn, Ugn protein in the intestinal mucosa of each group were in accordance with the changes of mRNA. The transcriptional and expression levels of GC-C, Gn and Ugn in the tissues are down regulated and negatively correlated with the degree of UC disease activity. Second the role of the GC-C signaling pathway in the Caco-2 cell barrier and inflammatory damage [Objective] to study the role of GC-C signaling pathway in the intestinal barrier and inflammatory injury by the Caco-2 cell monolayer barrier model. [Methods] Caco- 2 intestinal epithelial cells were cultured in the Transwell compartment to construct a monolayer cell barrier model to detect the integrity of the barrier of transmembrane resistance in monolayer cells. IL-1 beta was used to stimulate Caco-2 cells to simulate enteric dermatitis and to cause the destruction of intercellular close connection structure. Liposome method was used to transfect GC-C shRNA interference vector and Gn over table respectively. The carrier to the cell. After different treatment, the permeability of Caco-2 monolayer cells was detected by the determination of the macromolecular substance FD-4 through the cell rate; the cell viability was detected by the CCK-8 method; the NBT method was used to detect the activity of the cell SOD enzyme; the ELISA method was used to detect the IL-8, TNF- a level of the cell supernatant, and the qRT-PCR and Western blot were used to detect the cell GC-. The key factors of C signaling pathway (GC-C, Gn, Ugn and cGMP), the expression of tight connexin (occludin, claudin-1 and ZO-1) and the proinflammatory factor (IL-8, TNF- alpha). [results]Caco-2 monolayer cells in nineteenth days of culture, the transmembrane resistance value is stable at 250 Omega cm2, indicating that the intestinal barrier model is constructed successfully. After transfection of over expression vector 48 hours, except for 48 hours. The expression of GC-C and cGMP increased obviously, and the expression of GC-C and cGMP increased obviously, and the expression of Ugn had no obvious change. The expression of Gn, Ugn and cGMP decreased obviously after GC-CshRNA48 hours after transfection, and the activity of cell and SOD enzyme were obviously reduced after the expression of Gn, Ugn and cGMP. The expression of ZO-1 was significantly reduced, the permeability of monolayer cells and the level of IL-8 and TNF- alpha were significantly increased. After transfection of IL-1 beta stimulated cells to Gn overexpressed vector, the expression of cell activity, SOD enzyme activity, claudin-1 and ZO-1 increased significantly, IL-8, TNF- alpha expression and cell permeability compared with the overexpressed negative control group (vector control 1). On the contrary, the cells of the transfected GC-C shRNA interference carrier and the negative control group (vector control 2) were compared with the IL-1 beta induced IL-8, the expression of TNF- A and the cell permeability increased more significantly. The cell viability, the activity of SOD enzyme, the claudin-1 and ZO-1 decreased more significantly. There was no significant difference in the expression of occludin between the groups. [conclusion the expression of]GC-C and its ligand Gn and Ugn regulates each other and its ligand activates the GC-C signaling pathway in the static state. The activated GC-C signaling pathway reduces the barrier function and inflammatory damage induced by IL-1 beta induced Caco-2 cells. The role of the third partial GC-C signaling pathway in the intestinal inflammation in the UC rat [Objective] A UC mouse model induced by dextran sodium sulfate (DSS) was used to study the role of GC-C signaling pathway in the occurrence of intestinal inflammation. [Methods] 60 Bal b/c mice were randomly divided into 5 groups, 12 mice in each group: (1) normal control group (Control); (2) colitis group (DSS+ NS); (3) colitis + mesalazine treatment group (DSS+Mesalamine); (4) +Gn colitis +Gn The carrier treatment group (DSS+ Gn); (5) colitis + methalazine combined with Gn carrier therapy group (DSS+ Mesalamine+ Gn). After the construction of the UC model of free drinking of 3% DSS in mice for 1 weeks construction of the.UC model of UC, the mice of methalazine treatment group were given 1 times daily for 1 weeks of methalazine to 30mg/kg, and the Gn carrier treatment group passed tail. The Gn overexpression of lentivirus was treated 1 times a day for 1 weeks. The mice's feeding, activity, weight, stool character and blood stool were observed on the first day of the experiment. After the treatment, the permeability of the large molecular substance FD-4 in the mice was measured. After 2 weeks, the mice were killed and the mice were collected and the peripheral blood and colon tissues were collected. HE was stained in the light. Histopathological scores were observed under the microscope. The key factors of GC-C signaling pathway in colon tissue (GC-C, Gn, Ugn and cGMP), the expression of tight connexin (occludin, claudin-1 and ZO-1) and the expression of pro-inflammatory factors (IL-8, TNF- alpha) were detected by immunohistochemical method. [results] the level of serum IL-8 and TNF- alpha was detected. [results] the mice in the treatment group were better than the mice in the colitis group, and the body weight was better than those in the colitis group. The dilute stool and the mucus and blood stool were all lightened. The improvement effect of the methalazine combined with the Gn carrier group was the most obvious. The score of the fabric was significantly higher than that of the normal control group. The scores of all the treatment groups were lower than those of the colitis group. The score of the methalazine combined with the Gn carrier group was the lowest. Compared with the normal control group, the expression of GC-C, Gn, Ugn and cGMP in colonic tissue in the colitis group was significantly lower than that in the colitis group. The expression of each treatment group was higher than that of the colitis group and Gn loaded mice. The intestinal permeability in the colitis group was significantly higher than that in the normal control group. The intestinal permeability of the mice in the treatment group was lower than that of the colitis group, and the most significant.Occludin in the Gn carrier group was reduced. The expression of claudin-1 and ZO-1 in the intestinal tissue of the colitis group was significantly lower than that of the normal control group. The expression levels of claudin-1 and ZO-1 in the treatment group were significantly higher than those in the treatment group, and mesalazine combined with Gn carrier group was the most significant.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574.62

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