【作者】 冀强；

【导师】 岑瑛；

【作者基本信息】 四川大学 ， 整形烧伤学（专业学位）， 2022， 博士

【摘要】 目的:婴幼儿血管瘤(infant hemangioma,IH),是婴幼儿时期最常见的良性血管源性肿瘤,以内皮细胞的异常增殖和血管的异常结构为特点。IH通常在婴幼儿出生时不存在,出生后的4周内出现,数月间迅速增大,其中在5-8周时生长最快,消退阶段长达6年,分为增生期、稳定期和消退期三个阶段。虽然可口服普萘洛尔、外用皮质类固醇、咪喹莫特、辅助脉冲染料激光等方法治疗,但部分瘤体仍有残留的畸形,如皮肤萎缩疤痕、解剖结构功能畸形、纤维脂肪团,因此需手术切除治疗。关于细胞代谢已经研究了近百年,但内皮细胞(EC)代谢也是近十多年才受到越来越多的关注。氧化磷酸化提供ATP的糖代谢是多数正常体细胞的主要代谢方式,然而研究发现肿瘤细胞和EC能量代谢的特点表现为积极摄取葡萄糖和谷胺酰胺,进行有氧糖酵解,这种代谢重编程也被称为Warburg效应(Warburg效应被认为是肿瘤细胞或血管内皮细胞的重要特征)。研究发现,婴幼儿血管瘤内皮细胞(HemEC)与脐静脉内皮细胞(HUVEC)比较,前者的糖酵解相关分子在m RNA和蛋白质水平上均高度表达。Sirtuins蛋白家族是NAD~+依赖性脱乙酰酶,被分割于不同的细胞器中。它们从酵母到人类的进化上是保守的,在序列和他们的细胞功能方面具有高度的同源性,尤其在感知和调节细胞对外压力方面发挥了重要的生理和病理作用。烟酰胺核糖(NR)是外源性NAD~+前体,在多种细胞类型中具有广泛的活性,包括调节细胞粘附、极性、迁移、增殖和分化,最重要的是对细胞具有去分化功能。NR的作用机制主要是通过提高各细胞器内NAD~+水平,激活SIRT1/SIRT3等通路完成其细胞生物活性。虽然关于婴幼儿血管瘤的基础与临床研究较为丰富,然而关于Sirtuins蛋白家族在HemEC中的糖代谢机制研究鲜见报道,因此我们拟通过NR诱导和sh RNA转染HemEC,影响SIRT1/SIRT3蛋白表达,研究HemEC细胞表型及糖代谢变化,为IH的治疗提供新途径。材料和方法:本研究通过临床获取病例标本,体外原代培养、分选婴幼儿血管瘤内皮细胞(HemEC),观察细胞内SIRT1/SIRT3和糖酵解限速酶PKM2等酶蛋白表达;应用烟酰胺核糖(NR)诱导SIRT1/SIRT3表达,观察HemEC细胞增殖、迁移、周期、凋亡、线粒体膜电位和自噬等细胞表型变化,并应用Seahorse能量代谢检测仪探讨SIRT3对HemEC糖酵解、线粒体氧化磷酸化的作用机制,通过体外转染sh RNA使SIRT3低表达,进一步研究SIRT3对HemEC细胞内糖代谢重编程的影响,并通过在裸鼠体表建立婴幼儿血管瘤模型,证实SIRT1/SIRT3在IH中体内、体外作用机制。结果:1、通过对增殖期婴幼儿血管瘤新鲜标本组织块的酶解,细胞的收集、培养、传代、通过CD31免疫磁珠分选,能够获得足量满足要求的HemEC。婴幼儿血管瘤内皮细胞(HemEC)与脐静脉内皮细胞(HUVEC)联合注入裸鼠皮下,可形成较为可靠的血管瘤动物模型。2、增殖期HemEC与HUVEC比较,酶蛋白SIRT1、SIRT3、PKM2、HIF-1a和SOD2高表达,说明HemEC增殖活跃,糖酵解水平高,具有较强的细胞内抗氧化能力。3、通过不同剂量NR诱导HemEC实验表明,在生理低剂量NR环境中,主要激活SIRT1/SIRT3通路完成其细胞内生物活性,促进糖酵解和增强细胞氧化磷酸化,促进血管新生和生长。在高剂量NR环境中,SIRT3过表达,抑制糖酵解、细胞氧化磷酸化和细胞增殖,增加细胞凋亡,阻滞细胞周期(处于G0/G1期),降低线粒体膜电位。研究还发现NR在一定剂量范围内可能抑制HemEC细胞自噬。4、诱导实验还证实,NR可促进HemEC糖酵解限速酶PKM2表达,但PKM2对HemEC增殖影响较小,更可能影响IH血管发生与形成。5、shRNA转染HemEC后,通过SIRT3低表达,降低糖酵解限速酶PKM2表达,抑制细胞糖酵解水平和HemEC迁移,增强了HemEC自噬通量。6、血管瘤动物模型提示,低剂量NR喂食组,裸鼠血管瘤瘤体血管组织较丰富;而高剂量喂食组提示,血管瘤体组织内缺少条索状或团块状细胞增生,新生血管密度弱于低剂量NR组和对照组,但与后两组缺乏统计学差异。结论:我们的数据确定线粒体中SIRT1/SIRT3在调控HemEC糖酵解和线粒体氧化磷酸化水平方面发挥重要作用。表明SIRT3被外源性NAD~+前体烟酰胺核糖(NR)诱导后,可能抑制HemEC增殖,降低线粒体膜电位,阻滞细胞周期循环。同时我们的研究证明SIRT3是HemEC的一种自噬调节因子。可以预期,SIRT3可能会在IH发展的三个阶段发挥不同的作用,成为治疗IH的潜在靶点。更多还原

【Abstract】 Objective:Infant hemangioma(IH)is the most common benign vascular tumor in infants and young children,characterized by abnormal proliferation of endothelial cells and abnormal structure of blood vessels.IH usually does not exist in infants and young children at birth,appears in the first 4 weeks after birth,and increases rapidly within a few months,with the fastest growth at 5-8 weeks,and a regression stage of up to 6 years.three stages:period and regression period.Although oral propranolol,topical corticosteroids,imiquimod,and auxiliary pulsed dye laser can be used for treatment,some tumors still have residual deformities,such as skin atrophy scars,anatomical structural and functional deformities,and fibrous cellulite.Surgical excision is required.Since infantile hemangioma is dominated by endothelial cell proliferation,its energy metabolism must be that it provides ATP energy in the main form of glycolysis for vascular growth and migration.At present,many studies have proved this metabolic mode,such as cantelmo,A.R.and others believe that tumor endothelial cells(tumor cells)have high glycolysis metabolism and mediate the intermediate product of nucleotide synthesis.Cell metabolism has been studied for nearly a hundred years,but endothelial cell(EC)metabolism has only received more and more attention in the past decade.Glucose metabolism that provides ATP by oxidative phosphorylation is the main way of metabolism in most normal somatic cells.However,studies have found that tumor cells and ECs are characterized by active uptake of glucose and glutamine,and aerobic glycolysis,which is characterized by energy metabolism.Programming is also known as the Warburg effect.The study found that compared with umbilical vein endothelial cells(HUVEC),the glycolysis-related molecules of infant hemangioma endothelial cells(HemEC)were highly expressed at both mRNA and protein levels.The sirtuins family of proteins are NAD+-dependent deacetylases that are segregated into different organelles.They are evolutionarily conserved from yeast to humans,have high homology in sequence and their cellular functions,and play important physiological and pathological roles in sensing and regulating post-cellular external pressures.Nicotinamide ribose(NR)is an exogenous NAD+precursor with a wide range of activities in multiple cell types,including regulation of cell adhesion,polarity,migration,proliferation and differentiation,and most importantly,dedifferentiation of cells.The mechanism of action of NR is mainly by increasing the level of NAD+in each organelle and activating the SIRT1/SIRT3 pathway to complete its cellular biological activity.Although the basic and clinical research on infantile hemangioma is abundant,there are few reports on the glucose metabolism mechanism of the sirtuins protein family in HemEC.Therefore,we intend to induce NR and transfect HemEC with shRNA to affect the expression of SIRT1/SIRT3 protein.To study the changes of HemEC cell phenotype and glucose metabolism,and provide a new way for the treatment of IH.Materials and Methods:In this study,we obtained clinical specimens,primary cultured and sorted HemEC in vitro,and observed intracellular SIRT1/SIRT3 and glycolysis rate-limiting enzyme PKM2 and other enzyme protein expressions.The expression of SIRT1/SIRT3 was induced by nicotinamide ribose(NR),and the phenotypic changes of HemEC cells such as proliferation,migration,cycle,apoptosis,mitochondrial membrane potential and autophagy were observed.The mechanism of action of SIRT3 was reduced by transfection of shRNA in vitro,and the effect of SIRT1/SIRT3 on the reprogramming of glucose metabolism and metabolism in HemEC cells was further studied.Internal and external mechanisms of action.Results:1.Sufficient amount of cells can be obtained by separating,culturing,and collecting,expanding,passaged,and sorting by CD31 immunomagnetic beads of fresh clinical specimens of infantile hemangioma in the proliferative stage.Infant hemangioma endothelial cells(HemEC)and umbilical vein endothelial cells(HUVEC)were injected subcutaneously into nude mice to form a reliable hemangioma animal model.2.Compared with HUVEC in the proliferation stage,HemEC showed high expression of enzyme proteins SIRT1,SIRT3,PKM2,HIE-1a and SOD2,indicating that HemEC proliferates actively,has a high level of glycolysis,and has strong intracellular antioxidant capacity.3.The experiment of intervening HemEC with different doses of NR showed that in the low-dose NR environment,the SIRT1/SIRT3 pathway was mainly activated to complete its intracellular biological activity,promoting glycolysis and enhancing cellular oxidative phosphorylation.In a high-dose NR environment,overexpression of SIRT3 inhibits glycolysis,cellular oxidative phosphorylation and cell proliferation,increases apoptosis,arrests cell cycle(in G0/G1 phase),reduces mitochondrial membrane potential,and inhibits autophagy.4.NR induction experiments also confirmed that NR can promote the expression of HemEC glycolysis rate-limiting enzyme PKM2,but PKM2 has little effect on HemEC proliferation,and is more likely to affect IH angiogenesis and formation.5.shRNA knockdown of SIRT3 reduced the expression of glycolytic enzyme PKM2,inhibited the level of glycolysis and HemEC migration,and enhanced the autophagic flux of HemEC.6.The animal model of hemangioma showed that in the low-dose NR feeding group,the tumor vascular tissue of the hemangioma in nude mice was more abundant;while the high-dose feeding group showed that the hemangioma tissue lacked cord-like or clump-like cell proliferation,and new growth was observed in the high-dose feeding group.Vessel density was weaker than that of the low-dose NR group and the control group,but was not statistically different from the latter two groups.Conclusion:Our data identify important roles for SIRT1/SIRT3 in mitochondria in regulating HemEC glycolysis and mitochondrial oxidative phosphorylation levels.It is suggested that SIRT3 may inhibit HemEC proliferation,reduce mitochondrial membrane potential and block cell cycle after being induced by exogenous NAD+precursor nicotinamide ribose(NR).At the same time,our study proved that SIRT3 is an autophagy regulator of HemEC.It can be expected that SIRT3 may play different roles in the three stages of IH development and become a potential target for the treatment of IH.更多还原