自噬与肿瘤发生及其治疗的相关研究进展

正自噬是将细胞内的长寿命蛋白或者受损细胞器降解为基本生物分子的溶酶体降解途径。最新研究结果表明,细胞失去自噬功能可能导致对代谢压力、基因损伤敏感,从而诱导肿瘤的发生~([1])。然而,科学家们在多种肿瘤如乳腺癌、卵巢癌以及前列腺癌中发现自噬相关基因Beclin1缺失,说明自噬也可能发挥抑制肿瘤的作用~([2-3])。在肿瘤治疗领域,自噬一方面是肿瘤细胞凋亡缺陷情况下的另一种程序性死亡形     

细胞自噬与脓毒症

自噬可分为3种类型：微自噬(microautophagy)、分子伴侣介导的自噬(chaperone mediated autophagy,CMA)和巨自噬(macroautophagy)。巨自噬(简称自噬autophagy)是一种基因调控的、高度保守的细胞降解过程,也是一种重要的自我平衡的过程,发生于所有真核细胞中［1-3］。在针对应激的过程中,自噬常常扮演着细胞应答的角色［4］。而且,自噬无论在先天免疫还是适应性免疫机制中均有着不可忽视的重要作用［2］。自噬与各种疾病的病理及生理过程广泛相关（例如感染、肿瘤、神经退行性疾病、心血管及肺部疾病等）,近期的研究［5-6］也表明,自噬在脓毒症中发挥一定的保护作用。本文从细胞病理学和分子生物学角度就细胞自噬在脓毒症发生发展中的作用作一综述,为寻找脓毒症治疗的新手段提供依据与线索。     

细胞自噬与人乳头瘤病毒感染研究进展

自噬是细胞在适应营养缺乏时,通过溶酶体降解自身成分来维持自稳态的一个重要过程,在炎症、神经退行性疾病、肿瘤和抗病毒感染中都有重要作用.HPV是噬上皮组织的双链DNA病毒,能够引起良性增生或增加恶性病变风险.目前已知自噬在肿瘤发生和病毒感染中有重要作用,自噬相关的Beclin1,p62,LC3等基因在HPV感染的组织样本和细胞系中,表达均有明显改变;而且HPV的早期和晚期基因也能够引起自噬过程和自噬流的改变.本文就细胞自噬与HPV感染相关进展进行综述.     

自噬与糖尿病性视网膜病变的研究进展

随着生活水平的不断提高,糖尿病患者的数量逐年增多。作为糖尿病患者严重的并发症之一,糖尿病视网膜病变的患者也日益增多。目前对于糖尿病视网膜病变的机制,仍然不明确。近年来自噬在糖尿病视网膜病变的发生发展中所起的作用成为了临床研究的热点。在糖尿病视网膜病变的发生和发展中,自噬的调控与生长因子、胰岛素、营养物质及能量水平等多种因素引起的多条细胞内反应途径密切相关。自噬的基本过程主要是通过自噬体的作用来调节细胞内的动态平衡,其中通过几种不同的途径来保证调控的精确性。在糖尿病视网膜病变中,自噬发挥着双重作用,其中线粒体在其中发挥了重要作用。此外在缺血和缺氧条件下,缺氧可激活低氧诱导因子(HIF-l),通过转录因子Bnip3及Bnip3L的过表达,使Bcl-2蛋白释放出Beclin 1,从而激活Beclin 1相关的自噬通路。本文综述了自噬在糖尿病视网膜病变中作用的研究进展,为探明糖尿病视网膜病变的发病机制提供帮助。     

自噬基因Beclin1过表达诱导人卵巢癌细胞SKOV3自噬和凋亡的研究

目的:探讨自噬基因Beclin 1在人卵巢癌SKOV3细胞中过表达对其体外生长活性的影响。方法:构建自噬基因Beclin 1的真核表达载体pcDNA3.1/Beclin1,将其稳定转染SKOV3细胞,实现Beclin 1在SKOV3中的过表达;用MTT法分析Beclin1过表达对SKOV3增殖的影响,用流式细胞仪检测凋亡和自噬情况,电镜和荧光显微镜下观察自噬现象。结果:pcD-NA3.1/Beclin1转染SKOV3细胞后Beclin 1在mRNA和蛋白水平均高于空质粒pcDNA3.1转染组和未转染组。Beclin 1在SKOV3中的稳定过表达后G1期细胞明显增多,S期细胞比例明显减少,细胞增殖受到抑制,凋亡率为(21.26±3.89)%,高于空质粒pcDNA3.1转染组和未转染组,差异有统计学意义(P<0.05);流式细胞仪检测pcDNA3.1/Beclin1转染SKOV3细胞后MDC标记的自噬囊泡平均荧光强度高于pcDNA3.1转染组和未转染组。pcDNA3.1/Beclin1转染SKOV3细胞后在电镜下可见大量自噬囊泡形成。在荧光显微镜下见pcDNA3.1/Beclin1转染后SKOV3细胞中MDC标记的自噬囊泡明显增加。结论:自噬基因Beclin1过表达可诱导人卵巢癌细胞SKOV3自噬和凋亡,针对自噬基因Beclin1的卵巢癌基因治疗可能具有可行性。     

射线诱发的细胞自噬效应的研究进展

电离辐射是职业有害因素中重要的物理因素,包括α射线、β射线、χ射线、γ射线、质子束及其他粒子束等.射线可以杀伤细胞.最近研究发现,细胞自噬在射线诱发细胞死亡中起了非常重要的作用.射线常用于肿瘤治疗,最近几年,射线诱发细胞自噬在肿瘤治疗中研究较多.为此,我们就放射治疗中细胞自噬效应的研究进展作简要的介绍,为职业卫生中开展细胞自噬效应研究提供思路.     

桔梗皂苷D在体外对人肝癌细胞株HepG2的杀伤作用机制研究

目的研究桔梗皂苷D在体外对人肝癌细胞株Hep G2的杀伤作用及其机制。方法 MTT法检测桔梗皂苷D对Hep G2细胞增殖的抑制作用,台盼蓝染色法检测桔梗皂苷D对Hep G2细胞死亡的诱导作用。Western blot检测自噬标记蛋白LC3-Ⅱ的表达,并用荧光定量PCR法检测自噬相关基因Beclin1的表达。荧光定量PCR法检测Hep G2细胞线粒体膜电位相关蛋白BNIP3的表达水平。JC-1染料处理Hep G2细胞,并用流式细胞术检测桔梗皂苷D对线粒体膜电位的影响。结果桔梗皂苷D可显著抑制Hep G2细胞的增殖并杀伤肿瘤细胞。桔梗皂苷D处理后Hep G2细胞的Beclin1水平和LC3-Ⅱ表达显著增高,BNIP3表达水平显著增高并诱导其线粒体肿胀,膜电位降低。结论桔梗皂苷D上调BNIP3表达,诱导Hep G2细胞发生自噬性死亡。     

顺铂作用下卵巢癌敏感细胞A2780与耐药细胞A2780/DDP凋亡与自噬水平的变化及意义

目的:通过分析自噬对卵巢癌细胞顺铂敏感性的影响,探讨自噬与卵巢癌顺铂耐药性的关系,为卵巢癌治疗提供依据。方法:培养卵巢癌细胞株A2780和A2780/DDP,进行卵巢癌细胞系A2780及A2780/DDP顺铂敏感性的测定,流式细胞仪测定顺铂作用下A2780和A2780/DDP细胞的凋亡率;将10μM顺铂作用48h的A2780细胞和40μM顺铂作用48h的A2780/DDP细胞经处理,采用Western blot法检测Beclin1蛋白的表达;电镜下观察10μM顺铂作用48h的A2780细胞和40μM顺铂作用48h的A2780/DDP细胞经固定染色处理的自噬细胞活性。结果:顺铂主要通过诱导凋亡对卵巢癌耐药细胞A2780/DDP及敏感细胞A2780发挥细胞毒效应;顺铂作用下,耐药细胞的自噬活性明显增强,而敏感细胞自噬活性无明显增强。结论:顺铂诱导耐药卵巢癌细胞发生凋亡的能力较亲代敏感细胞降低,在其发生过程中,反应性自噬活性增强可能与其顺铂耐药性的形成有关,提示对肿瘤细胞的自噬水平进行监测及调控可能为逆转卵巢癌铂耐药提供新的思路。     

mTOR信号通路在细胞自噬和凋亡调节中的作用

自噬及凋亡广泛存在于细胞中,是细胞内生物大分子的降解再循环过程,在细胞生长代谢中发挥着重要作用。两者相互作用,共同推动和影响细胞的程序性死亡,维持机体自身稳态及外界环境刺激下的应激反应。mTOR信号通路是一条经典的调控自噬凋亡信号通路,在细胞代谢中发挥重要作用。结合mTOR信号通路探讨自噬及凋亡在细胞代谢及生物体生长发育过程中的作用及相关研究进展,对其在细胞增殖、老化及肿瘤发生等进行综述,对肿瘤等疾病的诊疗提供借鉴。     

PI3K在MG132诱导卵巢癌细胞自噬中作用的研究

目的:研究PI3K通路在蛋白酶体抑制剂MG132诱导卵巢癌细胞自噬中的作用。方法:MG132处理多种卵巢癌细胞(SKOV3、OVCAR3、A2870)后,光镜观察胞质内囊泡的形成,吖啶橙(AO)染色观察酸性囊泡的形成,Western blot检测自噬特异性蛋白LC3的变化。PI3K抑制剂(渥曼青霉素与3-MA)联合MG132处理多种卵巢癌细胞后,AO染色及West-ern blot检测自噬的变化。采用shRNA技术下调OVCAR3细胞的Beclin 1表达,MG132处理细胞,AO染色观察酸性囊泡的形成。结果:在多种卵巢癌细胞系中,MG132可诱导酸性囊泡形成及LC3-Ⅰ向LC3-Ⅱ的转化增强,PI3K抑制剂与MG132联合应用与单独应用MG132相比自噬水平无改变。下调卵巢癌细胞中Beclin 1表达后,MG132诱导的囊泡形成无改变。结论:MG132通过PI3K非依赖性途径诱导多种卵巢癌细胞发生自噬。     

Autophagy in aging and in neurodegenerative disorders

Autophagy (ATG) is the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles. Cellular homeostasis requires continuous removal of worn-out components and replacement with newly synthesized ones. Studies in yeast and other mammalian systems have increased our knowledge of the molecular mechanism of autophagy and the role of autophagy in various pathological conditions. Discovery of the genes involved in the process of autophagy has provided insight into the involvement of various molecular pathways. Growing evidence has indicated that diminished autophagic activity may play a pivotal role in the aging process. Cellular aging is characterized by a progressive accumulation of nonfunctional cellular components owing to oxidative damage and a decline in turnover rate and housekeeping mechanisms. Lysosomes are key organelles in the aging process due to their involvement in both macroautophagy and other housekeeping mechanisms. Autophagosomes themselves have limited degrading capacity and rely on fusion with lysosomes. Accumulation of defective mitochondria also appears to be critical in the progression of aging. Inefficient removal of nonfunctional mitochondria by lysosomes constitutes a major issue in the aging process. Autophagy has been associated with a growing number of pathological conditions, including cancer, myopathies, and neurodegenerative disorders. In this review, we discuss the cellular and molecular mechanisms involved in autophagy, the mechanisms of aging, and the possible role of autophagy in this process. Understanding the mechanisms by which autophagy impacts aging may provide useful molecular targets for pharmaceuticals designed to delay aging or correct conditions of premature aging.     

Combination of radiotherapy and adenovirus-mediated p53 gene therapy for MDM2-overexpressing hepatocellular carcinoma

The p53 gene plays a determinant role in radiation-induced cell death and its protein product is negatively regulated by MDM2. We investigated whether adenovirus-mediated modified p53 gene transfer, which blocks p53-MDM2 binding, is effective for radiation-induced cell death in hepatocellular carcinoma (HCC) at different MDM2 cellular levels. Human hepatocellular carcinoma cell lines expressing MDM2 at low levels (Huh7) and high levels (SK-Hep1) were used. Ad-p53 and Ad-p53vp are replication-deficient adenoviral vectors containing human wild-type or modified p53, respectively. The anti-tumor effect was highest for Ad-p53 + radiotherapy (RT) in the low-level MDM2 cells, whereas this effect was highest for Ad-p53vp + RT in the MDM2-overexpressing cells. In Huh-7 cells, Ad-p53 + RT decreased cell viability (32%) in vitro and inhibited tumor growth (enhancement factor, 1.86) in vivo. Additionally, p21 expression and apoptosis were increased. In contrast, in SK-Hep1 cells, Ad-p53vp + RT showed decreased cell viability (51%) in vitro and inhibition of tumor growth (enhancement factor, 3.07) in vivo. Caspase-3 expression and apoptosis were also increased. Adenovirus-expressing modified p53, which blocks p53-MDM2 binding, was effective in killing tumor cells overexpressing MDM2. Furthermore, the combination strategy for disruption of the p53-MDM2 interaction with RT demonstrated enhanced anti-tumor effects both in vitro and in vivo.     

抑制自噬基因Beclin 1的表达对耐药卵巢癌细胞A2780/DDP顺铂敏感性的影响及相关机制研究

[目的]观察抑制自噬基因Beclin1的表达对卵巢癌耐药细胞A2780/DDP顺铂敏感性的影响并探讨其中机制。[方法]利用脂质体将自噬基因Beclin1RNA干扰质粒pSUPER-Beclin1和对照质粒pSUPER-non分别转染耐药卵巢癌细胞A2780/DDP,筛选稳定表达株,检测顺铂作用下各组细胞(pSUPER-Beclin1组、pSUPER-non组和未转染组)生长抑制率、半数抑制浓度(IC50)、自噬和凋亡率的变化。[结果]A2780/DDP细胞转染干扰质粒pSUPER-Be-clin1后,细胞内Beclin1蛋白的表达明显下降;比较3组细胞顺铂48hIC50,pSUPER-Beclin1转染组最低(P﹤0.01);对3组细胞自噬与凋亡水平的检测显示,抑制Beclin1蛋白表达,在顺铂作用下,细胞自噬水平明显下降,而凋亡细胞比例明显升高,凋亡蛋白Caspase-3的表达亦明显上调,实验组与对照组相比差异有统计学意义(P﹤0.01)。[结论]抑制耐药细胞A2780/DDP自噬基因Beclin1的表达,可通过抑制自噬,增强凋亡提高耐药细胞对顺铂的敏感性。     

Beclin1-induced Autophagy Abrogates Radioresistance of Lung Cancer Cells by Suppressing Osteopontin

Osteopontin (OPN) serves as an indicator of resistance to radiotherapy. However, the role of OPN in the development of acquired radioresistance in human lung cancer cells has not yet been fully elucidated. Therefore, the potential importance of OPN as a marker of lung cancer with a potential significant role in the development of radioresistance against repeated radiotherapy has prompted us to define the pathways by which OPN regulates lung cancer cell growth. In addition, autophagy has been reported to play a key role in the radiosensitization of cancer cells. Here, we report that increased OPN expression through induction of nuclear p53 following irradiation was inhibited by exogenous beclin-1 (BECN1). Our results clearly show that BECN1 gene expression led to induction of autophagy and inhibition of cancer cell growth and angiogenesis. Our results suggest that the induction of autophagy abrogated the radioresistance of the cancer cells. Interestingly, we showed that knockdown of OPN by lentivirus-mediated shRNA induced the autophagy of human lung cancer cell. Taken together, these results suggest that OPN and BECN1 can be molecular targets for overcoming radioresistance by controlling autophagy.     

Resveratrol protects intestinal epithelial cells against radiation-induced damage by promoting autophagy and inhibiting apoptosis through SIRT1 activation

Intrinsic autophagy is important for the maintenance of intestinal homeostasis and intestinal regeneration. Ionizing radiation suppresses intrinsic autophagy and reduces damage-induced regeneration in the intestine, resulting in intestinal injury. Resveratrol, a sirtuin 1 (SIRT1) agonist, promotes autophagy and exerts radioprotective effect. In this study, the protective effect of resveratrol against radiation-induced intestinal injury and its potential mechanism were investigated. Intestinal epithelial cells (IEC-6) were exposed to 10 Gy ionizing radiation and resveratrol (0.1–40.0 μM). Cell viability was investigated using Cell Counting Kit 8 (CCK8), apoptosis was observed by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry, and the expression of apoptotic and autophagic proteins was determined by western blotting. Resveratrol exerted a high toxicity against IEC-6 cells, but at low concentrations, it inhibited ionizing radiation-induced apoptosis. Resveratrol increased SIRT1 expression after irradiation and inhibited ionizing radiation-induced p53 acetylation and pro-apoptotic protein, Bax, expression. Furthermore, resveratrol promoted autophagy via the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, thereby protecting IEC-6 cells against radiation-induced damage. These results suggest that resveratrol reduces radiation-induced IEC-6 cell damage by inhibiting apoptosis and promoting autophagy via the activation of SIRT1, and that the PI3K/AKT/mTOR signaling pathway is involved in the induction of autophagy.