Analysis of CRC Methods and Potential Data Integrity Exploits
与结直肠癌相关的粪菌移植研究现状
Journal of Colorectal&AnalSurgery结直肠肛门外科2020年12月第26卷第6期与结直肠癌相关的粪菌移植研究现状*崔曼曼1,马晓飞1,胡家丽1,胡丽霞1,张磊昌2△1江西中医药大学临床医学院江西南昌3300042江西中医药大学附属医院肛肠科江西南昌330006DOI:10.19668/ki.issn1674-0491.2020.06.037结直肠癌(colorectal cancer,CRC)为常见恶性肿瘤之一,在恶性肿瘤中发病率及死亡率均较高[1]。
CRC的发生、发展与多种因素相关,包括遗传因素、精神压力、饮食结构等[2]。
也有研究表明,结直肠癌患者肠道菌群的数量及种类均发生了变化[3-6]。
正常情况下,肠道菌群对维持肠道内环境稳态有重要作用,对机体进行正常生命活动具有重要的影响,肠道菌群失调可能引发一系列的不适或疾病,而粪菌移植(fecal microbiota transplantation,FMT)可以通过将健康供体的粪便作用于受体消化道来达到治疗目的,是调节肠道菌群失衡的方法之一。
本文围绕与CRC相关的FMT研究现状综述如下。
1FMT的应用与发展在东晋医学家葛洪编写的《肘后备急方》中已有“野葛芋毒、山中毒菌欲死者:并饮粪汁一升,即活”的描述[7],这是我国医学史上对粪汁应用的已知的最早记载。
1958年Eiseman等报道,对4例伪膜性肠炎患者进行治疗时将患者各自健康家属的粪便制成粪水用来灌肠[8],此为现代早期报道FMT参与治疗获得成功的案例。
2013年,Van Nood等[9]的研究证实,FMT对复发性艰难梭菌肠炎的治疗效果优于万古霉素,这一研究结果使得FMT得到了医学界更为广泛的关注[10]。
经过多年的发展与临床应用,目前复发性艰难梭菌感染(Clostridium difficile infec⁃tion,CDI)成为其公认适应证,FMT已经被列入CDI的治疗指南[11]。
【计算机专业文献翻译】题库管理系统
附录一英文材料While you’re going through the development process, the most important issue is this: Don’t get lost. It’s easy to do. Most of the analysis and design methods are intended to solve the largest of problems. Remember that most projects don’t fit in to that category, so you can usually have successful analysis and design with a relatively small subset of what a method recommends. But some sort of process, no matter how limited, will generally get you on your way in a much better fashion than simply beginning to code.It’s also easy to get stuck, to fall into “analysis paralysis,” where you feel like you can’t move forward because you haven’t nailed down every little detail at the current stage. Remember, no matter how much analysis you do, there are so me things about a system that won’t reveal themselves until design time, and more things that won’t reveal themselves until you’re coding, or not even until a program is up and running. Because of this, it’s crucial to move fairly quickly through analysis and design, and to implement a test of the proposed system.This point is worth emphasizing. Because of the history we’ve had with procedural languages, it is commendable that a team will want to proceed carefully and understand every minute detail before moving design and implementation. Certainly, when creating a DBMS, it pays to understand a customer’s needs thoroughly. But a DBMS is in a class of problems that is very well-posed and well-understood; in many such programs, the database structure is the problem to be tackled. The class of programming problem discussed in this chapter is of the “wild-card” (my term) variety, in which the solution isn’t simply re-forming a well-known solution, but instead involves one or more “wild-card factors”-elements for which there is no well-understood previous solution, and for which research is necessary. Attempting to thoroughly analyze a wildcard problem before moving into design andimplementation results in analysis paralysis because you don’t have enough inform ation to solve this kind of problem during the analysis phase. Solving such a problem requires iteration through the whole cycle, and that requires risk-taking behavior (which makes sense, because you’re trying to do something new and the potential rewards are higher). It may seem like the risk is compounded by “rushing” into a preliminary implementation, but it can instead reduce the risk in a wild-card project because you’re finding out early whether a particular approach to the problem is viable. Product development is risk management.It’s often proposed that you “build one to throw away.” With OOP, you may still throw part of it away, but because code is encapsulated into classes, during the first iteration you will inevitably produce some useful class designs and develop some worthwhile ideas about the system design that do not need to be thrown away. Thus, the first rapid pass at a problem not only produces critical information for the next analysis, design, and implementation iteration, it also creates a code foundation for that iteration.That said, if you’re looking at a methodology that contains tremendous detail and suggests many steps and documents, it’s still difficult to know when to stop. Keep in mind what you’re trying to discover: What are the objects? (How do you partition your project into its component parts?)What are their interfaces? (What messages do you need to be able to send to each object?)If you come up with nothing more than the objects and their interfaces, then you can write a program. For various reasons you might need more descriptions and documents than this, but you can’t get away with any less.The process can be undertaken in five phases, and a phase 0 that is just the initial commitment to using some kind of structure.Phase 0: Make a planYou must first decide what steps you’re going to have in your process. It sounds simple (in fact, all of this sounds simple) and yet people often don’t make this decision before they start coding. If your plan is “let’s jump in and start coding,” fine. (Sometimes that’s appropriat e when you have well-understoodproblem.) At least agree that this is the plan.You might also decide at this phase that some additional process structure is necessary, but not the whole nine yards. Understandab ly enough, some programmers like to work in “vacation mode” in which no structure is imposed on the process of developing their word; “It will be done when it’s done.” This can be appealing for awhile, but I’ve found that having a few milestones along the way helps to focus and galvanize your efforts around those milestones instead of being stuck with the single goal of “finish the project.” In addition, it divides the project into more bite-sized pieces and makes it seem less threatening (plus the milestones offer more opportunities for celebration).When I began to study story structure (so that I will someday write a novel) I was initially resistant to the idea of structure, feeling that when I wrote I simply let it flow onto the page. But I later realized that when I write about computers the structure is clear enough so that I don’t think much about it. But I still structure my work, albeit only semi-consciously in my head. So even if you think that your plan is to just start coding, you still somehow go through the subsequent phases while asking and answering certain questions.The mission statementAny system you build, no matter how complicated, has a fundamental purpose, the business that it’s in, and the basic need that it satisfies. If you can look past the user interface, the hardware- or system-specific details, the coding algorithms and the efficiency problems, you will eventually find the core of its being, simple and straightforward. Like the so-called high concept from a Hollywood movie, you can describe it in one ore two sentences. This pure description is the starting point.The high concept is quite important because it sets the tone for your project; it’s a mission statement. You won’t necessarily get it right the first time (you may be in a later phase of the project before it becomes completely clear), but keep trying until it feels right. For example, in an air-traffic control system to a very small airfield; perhaps there’s only a human controller or none at all. A more useful model won’t concern the solution you’re creating as much as it describes the problem: “Aircraft arrive, unload, service and reload, and depart.”Phase 1: What are we making?It’s necessary to stay focused on the heart of what you’re trying to accomplish in this phase: determine what the system is supposed to do. The most valuable tool for this is a collection of what are called “use cases.” Use cases identify key features in the system that will reveal some of the fundamental classes you’ll be using. These are essentially descriptive answers to questions like:“Who will use this system?”“What can those actors do with the system?”“How does this actor do that with this system?”“How else might this work if someone else were doing this, or if the same actor had a different objective?”(to reveal variations)“What problems might happen while doing this with the system?”(to reveal exceptions)If you are designing an auto-teller, for example, the use case for a particular aspect of the functionality of the system is able to describe what the auto-teller does in every possible situation. Each of these “situations” is referred to as a scenario, and a use case can be considered a collection of scenarios. You can think of a scenario as a question that starts with: “What does the system do if…?” For example, “What does the auto-teller do if a customer has just deposited a check within 24 hours and there’s not enough in the account without the check to provide the desired withdrawal?”Use case diagrams are intentionally simple to prevent you from getting bogged down in system implementation details prematurely:The use cases produce the requirements specifications by determining all the interactions that the user may have with the system. You try to discover a full set of use cases f or your systems, and once you’ve done that you have the core of what the system is supposed to do. The nice thing about focusing on use cases is that they always bring you back to the essentials and keep you from drifting off into issues that aren’t critic al for getting the job done. That is, if you have a full set of use cases you can describe your system and move onto the next phase. You probably won’t get it all figured out perfectly on the first try, but that’s OK. Everything will reveal itself in time, and if you demand a perfect system specificationat this point you’ll get stuck.Phase 2: How will we build it?In this phase you must come up with a design that describes what the classes look like and how they will interact. An excellent technique in determining classes and interactions is the Class-Responsibility-Collaboration (CRC) card. Part of the value of this tool is that it’s so low-tech: you start out with a set of blank 3” by 5” cards, and you write on them. Each card represents a single class, and on the card you write:The name of the class. It’s important that this name capture the essence of what the class does, so that it makes sense at a glance.The “responsibilities” of the class: what it should do. This can typically be summarized by ju st stating the names of the member functions (since those names should be descriptive in a good design), but it does not preclude other notes. If you need to seed the process, look at the problem from a lazy programmer’s standpoint: What objects would you like to magically appear to solve your problem?The “collaborations” of the class: what other classes does it interact with? “Interact” is an intentionally broad term; it could mean aggregation or simply that some other object exists that will perform services for an object of the class. Collaborations should also consider the audience for this class. For example, if you create a class Firecracker, who is going to observe it, a Chemist or a Spectator? The former will want to know what chemicals go into the construction, and the latter will respond to the colors and shapes released when it explodes.You may feel like the cards should be bigger because of all the information you’d like to get on them, but they are intentionally small, not only to keep your classes small but also to keep you from getting into too much detail too early. If you can’t fit all you need to know about a class on a small card, the class is too complex (either you’re getting too detailed, or you should create more than one class). The i deal class should be understood at a glance. The idea of CRC cards is to assist you in coming up with a first cut of the design so that you can get the big picture and then refine your design.One of the great benefits of CRC cards is in communication. It’s best done real-time, in a group, without computers. Each person takes responsibility for several classes (which at first have no names or other information). You run a live simulation by solving one scenario at a time, deciding which messages are sent to the various objects to satisfy each scenario. As you go through this process, you discover the classes that you need along with their responsibilities and collaborations, and you fill out the cards as you do this. When you’ve moved through all the use cases, you should have a fairly complete first cut of your design.Before I began using CRC cards, the most successful consulting experiences I had when coming up with an initial design involved standing in front of a team, who hadn’t built an OOP project bef ore, and drawing objects on a whiteboard. We talked about how the objects should communicate with each other, and erased some of them and replaced them with other objects. Effectively, I was managing all the “CRC cards” on the whiteboard. The team (who kne w what the project was supposed to do) actually created the design; they “owned” the design rather than having it giving it given to them.All I was doing was guiding the process by asking the right questions, trying out the assumptions, and taking the feedback from the team to modify those assumptions. The turn beauty of the process was that the team learned how to do object-oriented design not by reviewing abstract examples, but by working on the one design that was most interesting to them at that moment: theirs.Once you’ve come up with a set of CRC cards, you may want to create a more formal description of your design using UML. You don’t need to use UML .but it can be helpful, especially if you want to put up a diagram on the wall for everyone to ponder, which is a good idea. An alternative to UML is a textual description of the objects and their interfaces, or, depending on your programming languages, the code itself.UML also provides an additional diagramming notation for describing the dynamic model of your system. This is helpful in situations in which the state transitions of a system or subsystem are dominant enough that they need their own diagrams (such as in a control system). You may also need to describe the data structures, for systems or subsystems in which data is a dominant factor (such as a database),You’ll know you’re done with phase 2 when you have described the objects and their interfaces.Well, most of them –there are usually a few that slip through the cracks and don’t make them selves known until phase 3. But that’s OK. All you are concerned with is that you eventually discover all of your objects. It’s nice to discover them early in the process but OOP provides enough structure so that it’s not so bad if you discover them later. In fact, the design of an object tends to happen in five stages, throughout the process of program development.Phase 3: Build the coreThis is the initial conversion from the rough design into a compiling and executing body of code that can be tested, and especially that will prove or disprove your architecture. This is not a one-pass process, but rather the beginning of a series of steps that will iteratively build the system, as you’ll see in phase4.Your goal is to find the core of your system architecture that needs to be implemented in order generate a running system, no matter how incomplete that system is in this initial pass. You’re creating a framework that you can build upon with further iterations. You’re also performing the first of many system integrations and tests, and giving the stakeholders feedback about what their system will look like and how it is progressing. Ideally, you are also exposing some of the critical risks. You’ll probably also discover changes and improvements that can be made to your original architecture – things you would not have learned without implementing the system.Part of building the system is the reality check that you get from testing against your requirements analysis and system specification (in whatever form they exist). Make sure that your tests verify the requirements and use cases. When the core of the system is stable, you’re ready to move on and add more functionality.Phase 4: Iterate the use casesOnce the core framework is running, each feature set you add is a small project in itself. You add a feature set during an iteration, a reasonably short period of development.How big is an iteration? Ideally, each iteration lasts one to three weeks (this can vary based on the implementation language). At the end of that period, you have an integrated, tested system with more functionality than it had before. But what’s particularly interesting is the basis for the iteration: a single use case. Each use case is a package of related functionality that you build into the system all at once, during one iteration. Not only does this give you a better idea of what the scope of a use case should be, but it also gives more validation to the idea of a use case, since the concept isn’t discarded after analysis and design, but instead it is a fundamental until of development throughout the software-building process.You stop iterating when you achieve target functionality or an external deadline arrives and the customer can be satisfied with the current version. (Remember, software is a subscription business.) Because the process is iterative, you have many opportunities to ship a product instead of a single endpoint; open-source projects work exclusively in an iterative, high-feedback environment, which is precisely what makes them successful.An iterative development process is valuable for many reasons. You can reveal and resolve critical risks early, the customers have ample opportunity to change their minds, programmer satisfaction is higher, and the project can be steered with more precision. But an additional important benefit is the feedback to the stakeholders, who can see by the current state of the product exactly where everything lies. This may reduce or eliminate the need for mind-numbing status meetings and increase the confidence and support from the stakeholders.Phase 5: EvolutionThis is the point in the development cycle that has traditionally been called “maintenance,” a catch-all term that can mean everything from “getting it to work the way it was really supposed to in the first place” to “adding features that the customer forgot to mention” to the more traditional “fixing the bugs that show up” and “adding new features as the need arises.” So many misconceptions have been applied to the term “maintenance” t hat it has taken on a slightly deceiving quality, partly because it suggests that you’ve actually build a pristine program and all you need to do is change parts, oil it, and keep it from rusting. Perhaps there’s a better term to describe what’s going on.I’ll use the term evolution. That is, “You won’t get it right the first time, so give yourself the latitude to learn and to go back and make changes.” You might need to make a lot of changes as you learn and understand the problem more deeply. The elegance you’ll produce if you evolve until you get it right willpay off, both in the short and the long term. Evolution is where your program goes from good to great, and where those issues that you didn’t really understand in the first pass become clear. It’s also where your classes can evolve from single-project usage to reusable resources.What is it means to “get it right” isn’t just that the program works according to the requirements and the use cases. It also means that the internal structure of the code makes sense to you, and feels like it fits together well, with no awkward syntax, oversized objects, or ungainly exposed bits of code. In addition, you must have some sense that the program structure will survive the changes that it will inevitably go through during its lifetime, and that those changes can be made easily and cleanly. This is no small feat. You must not only understand what you’re building, but also how the program will evolve (what I call the vector of change). Fortunately, object-oriented programming languages are particularly adapt at supporting this kind of continuing modification –the boundaries created by the objects are what tend to keep the structure from breaking down. They also allow you to make changes –ones that would seem drastic in a procedural program –without causing earthquakesthroughout your code. In fact, support for evolution might be the most important benefit of OOP.英文文献译文整个开发过程时,最重要的问题是:不要迷路。
具核梭杆菌促进小鼠结肠肿瘤发生及其机制研究
具核梭杆菌促进小鼠结肠肿瘤发生及其机制研究李菁;于亚男;姜晓娜;路艳艳;杨林;荆雪;田字彬【摘要】背景:越来越多的证据表明结直肠癌(CRC)与肠道菌群密切相关.近年研究显示具核梭杆菌在CRC发生中可能发挥重要作用,但具体作用机制尚不明确.目的:探讨具核梭杆菌与CRC发生的相关性及其可能作用机制.方法:选用野生型C57BL/6小鼠和肠道多发性腺瘤APC(Min/+)小鼠,分别予具核梭杆菌菌液灌胃、皮下注射致癌剂1,2-二甲基肼二盐酸盐(DMH)或两者联合等不同处理,观察结肠异常隐窝灶(ACF)(8周)或肿瘤(20周)生成情况.以Roche 454 GS FLX焦磷酸测序分析各组野生型C57BL/6小鼠肠道菌群结构,Bio-Plex ProTM技术检测肠黏膜免疫因子表达.结果:与无具核梭杆菌定植的DMH处理组野生型C57BL/6小鼠或APC(Min/+)小鼠相比,具核梭杆菌定植小鼠结肠内ACF或肿瘤数量显著增多(P<0.05).具核梭杆菌定植小鼠肠道菌群结构改变明显,表现为蓝藻菌门含量减少,软皮菌门、疣微菌门含量增加(P均<0.05),肠黏膜肿瘤相关免疫因子IL-21、IL-22、IL-31、CD40L表达亦显著增高(P<0.05).结论:具核梭杆菌在肠道定植可促进小鼠结肠肿瘤发生,其机制可能主要为引起肠道菌群失衡和调控肠黏膜肿瘤相关免疫因子表达.%Background:Accumulating evidence links colorectal cancer (CRC) with the gut microbiota.Fusobacterium nucleatum (F.nucleatum) has been revealed to be involved in the development of CRC, however, the mechanism of F.nucleatum in mediating colorectal tumorigenesis is still poorly understood.Aims:To investigate the effect and potential mechanism of F.nucleatum on CRC.Methods:Wild type C57BL/6 mice and APC(Min/+) mice characterized by multiple intestinal neoplasia were used in this animal study.After administered with F.nucleatum intragastrically and/or1,2-dimethylhydrazine (DMH, a carcinogen) subcutaneously, the aberrant crypt foci (ACF) and colonic tumor were counted at 8th and 20th week, respectively.Structural alteration of intestinal microbiota and mucosal immune factors were detected in wild type C57BL/6 mice receiving different interventions by using Roche 454 GS FLX pyrosequencing and Bio-Plex ProTM cytokine assay, respectively.Results:In DMH-treated wild type C57BL/6 mice or APC(Min/+) mice, number of ACF and colonic tumor in those administered with F.nucleatum were significantly higher than those without (P<0.05).F.nucleatum colonization significantly altered the lumen microbial structure, with decreased Cyanobacterium and increased Tenericutes and Verrucomicrobia (P all <0.05).Furthermore, F.nucleatum up-regulated expressions of tumor-related immune factors in colonic mucosa, such as IL-21, IL-22, IL-31 and CD40L(P<0.05).Conclusions:F.nucleatum colonization in intestine may prompt colonic tumorigenesis in mice via inducing intestinal dysbiosis and modulating tumor-related immune factors expression.【期刊名称】《胃肠病学》【年(卷),期】2017(022)007【总页数】6页(P396-401)【关键词】结直肠肿瘤;肠道菌群;具核梭杆菌;免疫,黏膜【作者】李菁;于亚男;姜晓娜;路艳艳;杨林;荆雪;田字彬【作者单位】青岛大学附属医院消化内科 266003;青岛大学附属医院消化内科266003;海军青岛第一疗养院;青岛大学附属医院消化内科 266003;青岛大学附属医院消化内科 266003;青岛大学附属医院消化内科 266003;青岛大学附属医院消化内科 266003【正文语种】中文背景:越来越多的证据表明结直肠癌(CRC)与肠道菌群密切相关。
用于术前预测结直肠癌淋巴结转移的放射性诺模图的发展和验证(英文正文)
© 2016 by American Society of Clinical Oncology
0732-183X/16/3418w-2157w/$20.00
DOI: 10.1200/JCO.2015.65.9128
ABSTRACT
Purpose To develop and validate a radiomics nomogram or preoperative prediction of lymph node (LN) metastasis in patients with colorectal cancer (CRC).
Patients and Methods The prediction model was developed in a primary cohort that consisted of 326 patients with clinicopathologically confirmed CRC, and data was gathered from January 2007 to April 2010. Radiomic features were extracted from portal venous–phase computed tomography (CT) of CRC. Lasso regression model was used for data dimension reduction, feature selection, and radiomics signature building. Multivariable logistic regression analysis was used to develop the predicting model, we incorporated the radiomics signature, CT-reported LN status, and independent clinicopathologic risk factors, and this was presented with a radiomics nomogram. The performance of the nomogram was assessed with respect to its calibration, discrimination, and clinical usefulness. Internal validation was assessed. An independent validation cohort contained 200 consecutive patients from May 2010 to December 2011.
载5-氟尿嘧啶细胞囊泡对结直肠癌细胞株HCT116_的杀伤作用
胞内功能,包括自噬㊁线粒体功能障碍和细胞凋亡[11]㊂Xu 等[5]发现上调Sirt1/FoxO1信号轴可以缓解DM 大鼠肾损伤㊂本研究结果显示,与NR 组相比,HG 组p -Sirt1/Sirt1㊁p -FoxO1/FoxO1蛋白水平显著降低,而CT 使p -Sirt1/Sirt1㊁p -FoxO1/FoxO1蛋白水平显著上调,暗示CT 可以通过激活Sirt1/FoxO1信号轴激活细胞自噬以及细胞凋亡,进而缓解高糖诱导的GEC 炎性损伤以及氧化应激损伤㊂本研究用Sirt1/FoxO1信号轴抑制剂和CT 共同处理GEC ,结果发现SIRT1-IN -1逆转了CT 对GEC 的有利作用㊂综上所述,CT 可以通过上调Sirt1/FoxO1信号轴激活自噬,减轻高糖诱导的炎性损伤㊁氧化应激损伤㊂本文不足是研究机制较浅,我们将在下步实验深入研究㊂ʌ参考文献ɔ[1]㊀Orr P ,Shank BC ,Hickson S ,et al.Clinical management ofglomerular diseases [J ].Nurs Clin North Am ,2018,53(4):551-567.[2]㊀Daehn IS ,Duffield JS.The glomerular filtration barrier :astructural target for novel kidney therapies [J ].Nat RevDrug Discov ,2021,20(10):770-788.[3]㊀Sun L ,Sun C ,Zhou S ,Zhang L ,et al.Tamsulosin attenuateshigh glucose -induced injury in glomerular endothelial cells[J ].Bioengineered ,2021,12(1):5184-5194.[4]㊀Song M ,Chen L ,Zhang L ,et al.Cryptotanshinone enhanceswound healing in type 2diabetes with modulatory effects on inflammation ,angiogenesis and extracellular matrix remodel-ling [J ].Pharm Biol ,2020,58(1):845-853.[5]㊀Xu J ,Liu LQ ,Xu LL ,et al.Metformin alleviates renal injuryin diabetic rats by inducing Sirt1/FoxO1autophagic signalaxis [J ].Clin Exp Pharmacol Physiol ,2020,47(4):599-608.[6]㊀Shan MY ,Dai Y ,Ren XD ,et al.Berberine mitigates nonalco-holic hepatic steatosis by downregulating SIRT1-FoxO1-SREBP2pathway for cholesterol synthesis [J ].Integr Med ,2021,19(6):545-554.[7]㊀Lo SH ,Hsu CT ,Niu HS ,et al.Cryptotanshinone inhibitsSTAT3signaling to alleviate cardiac fibrosis in type 1-like diabetic rats [J ].Phytother Res ,2017,31(4):638-646.[8]㊀Mukohara S ,Mifune Y ,Inui A ,et al.In vitro and in vivotenocyte -protective effectiveness of dehydroepiandrosteroneagainst high glucose -induced oxidative stress [J ].BMCMusculoskelet Disord ,2021,22(1):519-529.[9]㊀Tang PM ,Nikolic -Paterson DJ ,Lan HY.Macrophages :ver-satile players in renal inflammation and fibrosis [J ].Nat Rev Nephrol ,2019,15(3):144-158.[10]㊀Koch EAT ,Nakhoul R ,Nakhoul F ,et al.Autophagy in dia-betic nephropathy :a review [J ].Int Urol Nephrol ,2020,52(9):1705-1712.[11]㊀He W ,Zhang A ,Qi ,et al.FoxO1,a potential therapeutictarget ,regulates autophagic flux oxidative stress mitochon-drial dysfunction and apoptosis in human cholangiocarcino-ma QBC939cells [J ].Cell Physiol Biochem ,2018,45(4):1506-1514.ʌ文章编号ɔ1006-6233(2023)06-0893-07载5-氟尿嘧啶细胞囊泡对结直肠癌细胞株HCT116的杀伤作用孙建海,㊀晏㊀菲,㊀魏武杰,㊀邓㊀洁,㊀李㊀黎,㊀刘㊀莉,㊀马燕凌(江汉大学附属湖北省第三人民医院肿瘤科,㊀湖北㊀武汉㊀430000)ʌ摘㊀要ɔ目的:探讨载5-氟尿嘧啶细胞囊泡对结直肠癌细胞株HCT116的杀伤作用㊂方法:设HCT116组㊁细胞囊泡组(EVs 混悬液5mL ㊁106个/mL )㊁5-氟尿嘧啶组(5-氟尿嘧啶5mL ㊁30μg /mL )㊁5-氟尿嘧啶载药囊泡组(EVs 混悬液5mL ㊁106个/mL +5-氟尿嘧啶5mL ㊁30μg /mL ),以上各组细胞每孔设6个平行样,培养72h ㊂培养结束后,测定细胞增殖侵袭㊁凋亡水平,RT -PCR 法及蛋白印迹法测定各组细胞miR -128㊁PIK3水平㊂结果:细胞囊泡组OD 值㊁存活率㊁单克隆形成数目㊁穿膜数㊁迁移距离㊁凋亡率㊁miR -128水平㊁PI3K mRNA 和蛋白水平与HCT116组比较无统计学差异(P >0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组OD 值㊁存活率㊁单克隆形成数目㊁穿膜数㊁迁移距离㊁PI3KmRNA 和蛋白水平明显低于HCT116组㊁细胞囊泡组(P <0.05),凋亡率㊁miR -128水平明显高于HCT116组㊁细胞囊泡组(P <0.05);5-氟尿嘧啶载药囊泡组OD 值㊁存活率㊁单克隆形成数目㊁穿膜数㊁迁移距离㊁PI3K mRNA 和蛋白水平明显低于5-氟尿嘧啶组,凋亡率㊁miR -128水平明显高于5-氟尿嘧啶组(P <0.05)㊂结论:细胞外㊃398㊃ʌ基金项目ɔ湖北省卫健委2019年科研项目,(编号:WJ2019F184)ʌ通讯作者ɔ马燕凌囊泡装载5-氟尿嘧啶能明显抑制结直肠癌细胞株HCT116增殖㊁侵袭水平,增强其凋亡水平,其机制可能与细胞外囊泡装载5-氟尿嘧啶能促进结直肠癌细胞株HCT116高表达miR-128,低表达PI3K有关㊂ʌ关键词ɔ㊀载5-氟尿嘧啶细胞囊泡;㊀结直肠癌;㊀细胞增殖ʌ文献标识码ɔ㊀A㊀㊀㊀㊀㊀ʌdoiɔ10.3969/j.issn.1006-6233.2023.06.03Killing Effect of5-Fluorouracil Cell Vesicles onColorectal Cancer Cell Line HCT116SUN Jianhai,YAN Fei,WEI Wujie,et al(Hubei Third People's Hospital Affiliated to Jianghan University,Hubei Wuhan430000,China)ʌAbstractɔObjective:To explore the killing effect of5-fluorouracil cell vesicles on colorectal cancer cell line HCT116.Methods:The HCT116group,cell vesicles group(EVs suspension5mL,106cells/mL), 5-fluorouracil group(5-fluorouracil5mL,30μg/mL),and5-fluorouracil drug-loaded vesicles group(EVs suspension5mL,106cells/mL+5-fluorouracil5mL,30μg/mL)were set up,and6parallel samples of each well of the above groups were incubated for72h.After incubation,the levels of cell proliferation,invasion and apoptosis were determined,and the levels of miR-128and PIK3were determined by RT-PCR and western blotting.Results:The OD value,survival rate,number of monoclonal formation,number of membrane pene-tration,migration distance,apoptosis rate,miR-128level,PI3K mRNA and protein levels in cell vesicles group were not significantly different from those in HCT116group(P>0.05).OD value,survival rate,num-ber of monoclonal formation,number of membrane penetration,migration distance,PI3KmRNA and protein levels in5-fluorouracil group and5-fluorouracil drug-loaded vesicles group were significantly lower than those in HCT116group and cell vesicles group(P<0.05),and the apoptosis rate and the level of miR-128were significantly higher than those in HCT116group and cell vesicle group(P<0.05).OD value,survival rate, number of monoclonal formation,number of membrane penetration,migration distance,PI3K mRNA and pro-tein levels in5-fluorouracil drug-loaded vesicles group were significantly lower than those in5-fluorouracil group,and the apoptosis rate and miR-128level were significantly higher than those in5-fluorouracil group (P<0.05).Conclusion:Extracellular vesicles loaded with5-fluorouracil can significantly inhibit the prolif-eration and invasion level of colorectal cancer cell line HCT116,and enhance its apoptosis level,which may be related to the mechanism that extracellular vesicles loaded with5-fluorouracil can promote the high expres-sion of miR-128and low expression of PI3K in colorectal cancer cell line HCT116.ʌKey wordsɔ㊀5-fluorouracil;㊀Colorectal cancer;㊀Proliferation of cells㊀㊀结直肠癌(CRC)可导致正常结肠黏膜恶化为浸润性肠黏膜[1],遗传和环境因素等许多风险因素都会导致CRC的发生[2]㊂手术是早期CRC病例的金标准治疗方法,但绝大多数CRC患者术后生存率较低㊂细胞外囊泡(EV)是一种球形脂质双层,EV可以将蛋白质㊁糖蛋白㊁脂质㊁核酸和细胞因子从母体细胞转移到受体细胞,以促进受体细胞的表型变化,并在细胞间通讯中发挥重要作用㊂EV可以携带母体细胞成分以影响各种生物过程,包括:DNA转移㊁细胞代谢物输出㊁细胞间通讯等㊂细胞外囊泡由于其可修饰性㊁有效的装载能力和天然的肿瘤靶向特性而被广泛用作抗肿瘤药物的递送工具[3,4]㊂研究发现,5-氟尿嘧啶与结直肠癌患者来源的EV有机结合就形成了载5-氟尿嘧啶细胞囊泡,其具有高效靶向性㊁特异性高以及副作用小等特点㊂微小RNA(miRNA)是19-24个核苷酸的小型非编码RNA,已被确定为多种癌症类型的潜在标志物,包括CRC[5]㊂大量的miRNA与CRC的发展或总体存活率低有关㊂例如,在转移性CRC患者中,miR-30d-5p血浆水平有所提高㊂基于miRNA的微阵列显示miR-128是CRC细胞中最显著上调的miRNA之一,miR-128过表达有助于促进细胞迁移和侵袭[6]㊂本研究拟探讨载5-氟尿嘧啶细胞囊泡对结直肠癌细胞株HCT116的杀伤作用,为结直肠癌的治疗提供依据㊂1㊀材料与方法1.1㊀试剂及仪器:DMEM-F12(Sigma-Aldrich Chemi-㊃498㊃cal Company),FBS(System Biosciences),1ˑ抗生素和抗真菌剂(Life technologies),10%胎牛血清(FBS;Hy-clone;GE Healthcare Life Sciences),Dulbecco改良Ea-gle培养基(DMEM)完全培养基(Sigma),链霉素㊁青霉素(Gibco;Thermo Fisher Scientific),CCK-8溶液(Ye-asen),FITC-膜联蛋白㊁碘化丙锭(PI)(BioLegend), Transwell室(BD Biosciences)㊁Matrigel(Corning),TR-Izol试剂盒(Invitrogen),miScript逆转录试剂盒(Qia-gen),SYBR Premix EX Taq(TaKaRa Otsu Shiga),逆转录试剂盒(Promega),IQTM SYBR Green Supermix试剂盒(Bio-Rad),Radio Immunoprecipitation Assay裂解缓冲液㊁二辛可宁酸蛋白质测定试剂盒(Thermo Fisher),一抗PIK3兔多克隆抗体㊁GAPDH兔铵多克隆抗体(Abcam,Cambridge),辣根过氧化物酶标记的山羊抗兔IgG(Abcam Cambridge)㊂SpectraMax M5酶标仪(Molecular Devices),流式细胞仪(BD Biosciences), Cell QuestPro软件(BD Biosciences),Applied Biosys-tems7500系统(Applied Biosystems)㊂1.2㊀细胞外囊泡分离:结直肠癌细胞株HCT116在DMEM-F12中生长,含有10%EV消耗的FBS和1ˑ抗生素和抗真菌剂培养48h后,通过不同的离心方法将EV从培养基中分离出来㊂将收集的培养基以300g离心10min,然后以2000g离心25min㊂通过0.22μm过滤器收集上清液㊂使用获得的培养基在100000g和4ħ下离心60min来制造EV㊂然后弃去上清液㊂在相同条件下,将培养基超速离心并用磷酸盐缓冲盐水(PBS)重悬用于后续分析㊂提取的EV由日立JEM-2100透射电子显微镜(日本电子有限公司)鉴定㊂1.3㊀细胞培养及分组:结直肠癌细胞株HCT116购自Be Na Culture Collection㊂细胞系均在含有10%胎牛血清的Dulbecco改良Eagle培养基(DMEM)完全培养基中培养,添加100U/mL链霉素和100U/mL青霉素㊂所有细胞系均置于37ħ㊁5%CO2恒温培养箱中培养备用㊂HCT116组㊁细胞囊泡组㊁5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组培养方法HCT116组的培养方法如前所述;细胞囊泡组加入EV混悬液5mL(106个/mL);5 -氟尿嘧啶组加入5-氟尿嘧啶5mL(30μg/mL);5-氟尿嘧啶载药囊泡组加入载药囊泡混悬液5mL,载药囊泡混悬液的制备方法如下:取5-氟尿嘧啶30μg,溶于1mL EVs混悬液中,在37ħ恒温培养箱中孵育1h, 9168r/min离心10min,经0.22μm过滤器过滤; 30900r/min离心70min,将沉淀溶于PBS,即得载药囊泡混悬液㊂各组设6个平行样,培养72h㊂1.4㊀HCT116细胞活力及单克隆形成数目检测:将细胞接种到96孔板中,每孔1ˑ103个细胞,在5%CO2, 37ħ环境下孵育72h,将10μL CCK-8溶液加入每个孔中㊂在37ħ下孵育1.5h后,使用SpectraMax M5检测450nm处的光密度值㊂细胞存活率=(实验组OD-空白组OD)/(HCT116组OD-空白组OD)㊂将各组结直肠癌细胞株HCT116用0.25%胰蛋白酶消化成单细胞,以4ˑ105细胞/孔的密度接种到6孔培养板中, 24h后,将50个细胞接种于6孔组织培养板中,继续培养2周,然后将细胞用结晶紫-福尔马林溶液染色10min并计数㊂1.5㊀HCT116细胞凋亡测定:使用FITC-膜联蛋白V 和碘化丙锭(PI)通过流式细胞术分析细胞凋亡㊂将2mL细胞(5ˑ104个细胞/mL)接种到6孔板中,48h 后,用PBS洗涤细胞两次,消化并重悬于100μL结合缓冲液中㊂细胞密度调整为0.5ˑ106个/mL细胞,使用5μL Annexin V/FITC在室温下黑暗染色细胞10min,加入100μL结合缓冲液,将细胞在室温下在黑暗中用5μL PI染色5min㊂用流式细胞术分析细胞凋亡率,用Cell Quest Pro软件计算凋亡细胞百分比㊂1.6㊀HCT116细胞侵袭㊁迁移测定:通过Transwell小室评估细胞的侵袭能力㊂Transwell室底部膜的孔径为8μm,底室充满600μL含有10%FBS的DMEM营养液,顶室的大小为200μL,接种5ˑ105个细胞,在37ħ㊁5%CO2的培养箱中培养24h,将Transwell小室取出并固定在由甲醇和冰醋酸(3:1)组成的液体中30min,用PBS洗涤腔室,用0.5%结晶紫染色并最后固定,在显微镜下随机取5个视野观察并计数染色细胞数㊂迁移水平测定,将约5ˑ104个细胞加入涂有500ng/mL Ma-trigel的24孔Transwell上室㊂下室充满10%FBS补充的DMEM培养基,将细胞在37ħ㊁5%CO2的培养箱中培养㊂24h后,取出Transwell插入物,使用无菌镊子移除插入物以产生均匀的500μm无细胞间隙,用4%多聚甲醛固定30min,在显微镜下计算迁移距离㊂1.7㊀HCT116细胞miR-128㊁PIK3mRNA水平测定: TRIzol试剂盒提取总RNA㊂为了量化miR-128的表达,采用miScript逆转录试剂盒(Qiagen)合成互补DNA,并使用SYBRPremixEXTaq以U6作为内标进行qRT-PCR参考miR-128㊂为了量化PIK3mRNA的表达,使用逆转录试剂盒(Promega)生成cDNA,并应用IQTM SYBR Green Supermix试剂盒以GAPDH作为内源对照进行扩增㊂结果用2-ΔΔCt值进行量化和归一化㊂qRT-PCR检测均在Applied Biosystems7500系统上进行㊂1.8㊀结直肠癌细胞株HCT116PIK3蛋白表达水平测㊃598㊃定:不同处理组的细胞转染72h后,用Radio Immuno-precipitation Assay裂解缓冲液在冰上裂解10min,二辛可宁酸蛋白质测定法定量蛋白质,将蛋白质在100V 电压下加载到十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中,转移到聚偏二氟乙烯膜上,用5%BSA/TBST封闭60min,将膜与一抗PIK3兔多克隆抗体和GAPDH兔铵多克隆抗体在4ħ下孵育过夜,在室温下用1ˑTBST 溶液洗涤每次5min,重复3次㊂室温下,将膜与辣根过氧化物酶标记的山羊抗兔IgG孵育1h,并用TBST 洗涤3次㊂每20min应用电化学发光检测发光反应,并拍摄蛋白质印记以供观察㊂1.9㊀统计学分析:SPSS24.0软件进行统计分析,定量数据以均值ʃ标准差表示,多组均数比较采用单因素方差分析,两组间比较采用LSD-t检验,P<0.05表示差异有统计学意义㊂2㊀结㊀果2.1㊀各组结直肠癌细胞株HCT116OD值㊁存活率比较:细胞囊泡组OD值㊁存活率与HCT116组比较无统计学差异(P>0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组OD值㊁存活率明显低于HCT116组㊁细胞囊泡组(P<0.05),5-氟尿嘧啶载药囊泡组OD值㊁存活率明显低于5-氟尿嘧啶组(P<0.05)㊂见表1㊂表1㊀各组结直肠癌细胞株HCT116OD值存活率比较组别复孔数OD值存活率(%) HCT116组60.80ʃ0.0773.52ʃ5.73细胞囊泡组60.81ʃ0.0872.85ʃ5.37 5-氟尿嘧啶组60.54ʃ0.05b52.10ʃ3.27b 5-氟尿嘧啶载药囊泡组60.24ʃ0.05bc30.12ʃ4.04bc F19.65821.365 P0.0000.000㊀㊀注:b与HCT116组相比P<0.05;c与5-氟尿嘧啶组相比P<0.052.2㊀各组结直肠癌细胞株HCT116单克隆形成数目比较:细胞囊泡组单克隆形成数目与HCT116组比较无统计学差异(P>0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组单克隆形成数目明显低于HCT116组㊁细胞囊泡组(P<0.05),5-氟尿嘧啶载药囊泡组单克隆形成数目明显低于5-氟尿嘧啶组(P<0.05)㊂见表2㊁图1㊂表2㊀各组结直肠癌细胞株HCT116克隆形成数目比较组别复孔数单克隆形成数目HCT116组6647.77ʃ99.47细胞囊泡组6652.59ʃ95.35 5-氟尿嘧啶组6453.54ʃ55.87b 5-氟尿嘧啶载药囊泡组6124.29ʃ22.47bc F25.297 P0.000㊀㊀注:b与HCT116组相比P<0.05;c与5-氟尿嘧啶组相比P<0.05图1㊀各组结直肠癌细胞株HCT116克隆形成数目比较(结晶紫染色,ˑ400)(A:HCT116组B:细胞囊泡组C:5-氟尿嘧啶组C:5-氟尿嘧啶载药囊泡组)2.3㊀各组结直肠癌细胞株HCT116凋亡率比较:细胞囊泡组凋亡率与HCT116组比较无统计学意义(P>0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组凋亡率明显高于HCT116组㊁细胞囊泡组(P<0.05),5-氟尿嘧啶载药囊泡组凋亡率明显高于5-氟尿嘧啶组(P<0.05)㊂见表3㊁图2㊂表3㊀各组结直肠癌细胞株HCT116凋亡率比较组别复孔数凋亡率(%) HCT116组6 2.06ʃ0.32细胞囊泡组6 2.02ʃ0.33 5-氟尿嘧啶组6 3.04ʃ0.52b 5-氟尿嘧啶载药囊泡组614.75ʃ2.14bc F15.632 P0.000㊀㊀注:b与HCT116组相比P<0.05;c与5-氟尿嘧啶组相比㊃698㊃P<0.05图2㊀各组结直肠癌细胞株HCT116凋亡率比较流式细胞图(A:HCT116组B:细胞囊泡组C:5-氟尿嘧啶组C:5-氟尿嘧啶载药囊泡组)2.4㊀各组结直肠癌细胞株HCT116侵袭能力比较:细胞囊泡组穿膜数与HCT116组比较无统计学差异(P> 0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组穿膜数明显低于HCT116组㊁细胞囊泡组(P<0.05),5-氟尿嘧啶载药囊泡组穿膜数明显低于5-氟尿嘧啶组(P< 0.05)㊂见表4㊁图3㊂表4㊀各组结直肠癌细胞株HCT116穿膜数比较组别复孔数穿膜数(个) HCT116组62654.69ʃ484.30细胞囊泡组62701.74ʃ411.88 5-氟尿嘧啶组61247.02ʃ205.59b 5-氟尿嘧啶载药囊泡组6423.63ʃ85.89bc F29.667 P0.000㊀㊀注:b与HCT116组相比P<0.05;c与5-氟尿嘧啶组相比P<0.05图3㊀各组结直肠癌细胞株HCT116穿膜数数目比较(结晶紫染色,ˑ400)(A:HCT116组B:细胞囊泡组C:5-氟尿嘧啶组C:5-氟尿嘧啶载药囊泡组)2.5㊀各组结直肠癌细胞株HCT116迁移能力比较:细胞囊泡组迁移距离与HCT116组比较无统计学差异(P >0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组迁移距离明显低于HCT116组㊁细胞囊泡组(P<0.05),5-氟尿嘧啶载药囊泡组迁移距离明显低于5-氟尿嘧啶组(P<0.05)㊂见表5㊁图4㊂表5㊀各组结直肠癌细胞株HCT116迁移距离比较组别复孔数迁移距离(μm) HCT116组628.92ʃ5.02细胞囊泡组629.02ʃ5.03 5-氟尿嘧啶组615.80ʃ2.00b 5-氟尿嘧啶载药囊泡组6 6.41ʃ1.32bc F36.257 P0.000㊀㊀注:b与HCT116组相比P<0.05;c与5-氟尿嘧啶组相比P<0.05图4㊀各组结直肠癌细胞株HCT116迁移距离比较(ˑ400) (A:HCT116组B:细胞囊泡组C:5-氟尿嘧啶组C:5-氟尿嘧啶载药囊泡组)2.6㊀各组结直肠癌细胞株HCT116miR-128㊁PI3K mRNA和蛋白水平比较:细胞囊泡组miR-128水平与HCT116组比较无统计学差异(P>0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组miR-128高于HCT116组㊁细胞囊泡组(P<0.05),5-氟尿嘧啶载药囊泡组miR-128高于5-氟尿嘧啶组(P<0.05)㊂细胞囊泡组PI3K mRNA和蛋白与HCT116组比较无统计学差异(P>0.05);5-氟尿嘧啶组㊁5-氟尿嘧啶载药囊泡组PI3K mRNA和蛋白低于HCT116组㊁细胞囊泡组(P< 0.05),5-氟尿嘧啶载药囊泡组PI3KmRNA和蛋白低㊃798㊃于5-氟尿嘧啶组(P<0.05)㊂见表6㊂表6㊀各组结直肠癌细胞株HCT116miR -128PI3K mRNA 和蛋白表达水平比较组别复孔数miR -128PI3KmRNA PI3K 蛋白(/GAPDH )HCT116组6 1.17ʃ0.23 3.85ʃ0.65 3.95ʃ0.79细胞囊泡组6 1.15ʃ0.26 3.90ʃ0.63 3.89ʃ0.785-氟尿嘧啶组6 3.68ʃ0.57b 2.54ʃ0.44b 2.34ʃ0.47b 5-氟尿嘧啶载药囊泡组65.42ʃ0.98bc1.63ʃ0.28bc1.44ʃ0.21bcF 29.85732.25818.578P0.0000.0000.000㊀㊀注:b 与HCT116组相比P<0.05;c 与5-氟尿嘧啶组相比P<0.053㊀讨㊀论EV 作为一种纳米级的膜结构,主要负责携带各种内容物,通过质膜融合㊁内吞㊁与细胞表面受体结合等机制广泛参与肿瘤的生物学过程㊂EV 作为肿瘤侵袭转移的分子基础之一,对肺癌的早期诊断和靶向治疗具有重要意义㊂研究发现,来源于高转移肺癌细胞和晚期肺癌患者血清的EV 诱导波形蛋白表达,诱导HBECs 上皮-间质转化,诱导非转移癌细胞的迁移㊁侵袭和增殖[7];与来自早期非小细胞肺癌(NSCLC )细胞的EV 相比,来自转移性SCLC 细胞的EV 对肿瘤细胞迁移和侵袭的影响更大㊂特别是在缺氧条件下,转移性小细胞肺癌细胞EV 中与肿瘤细胞迁移和侵袭密切相关的转化生长因子-β和白细胞介素-10含量增加[8,9]㊂EV 作为细胞间信息交换的重要物质,通过自分泌或远距离传播的方式将相关信号分子传递给靶细胞,从而产生一系列生物学效应㊂因此,EV 靶向肿瘤治疗方法可能具有广阔的前景㊂已经确定了EV 中常见的三个蛋白质家族,分别是热休克蛋白70(HSP70)㊁S -腺苷同型半胱氨酸酶和甘油醛3-磷酸脱氢酶㊂在这项研究中,我们对EV 进行了蛋白质组学分析,发现了所有三种蛋白质,进一步证实了EV 的纯化㊂同时,我们在EV 中发现了MAP30㊁MAP30可以增加细胞内Ca 2+离子浓度,从而通过细胞凋亡触发ROS 介导的癌细胞凋亡㊂本研究发现5-氟尿嘧啶载药囊泡处理后细胞凋亡的产生,这可能是由EV 中的MAP30蛋白介导的㊂氟尿嘧啶可诱导线粒体ROS ,导致癌细胞增殖㊁侵袭能力下降,ROS 的产生增强了5-氟尿嘧啶对癌细胞的抗肿瘤作用㊂而抗氧化剂降低了5-氟尿嘧啶在结肠癌中的凋亡作用㊂本研究发现载5-氟尿嘧啶细胞囊泡能明显抑制结直肠癌细胞株HCT116增殖㊁侵袭水平,增强其凋亡水平㊂PI3K 蛋白家族是由调节亚基p85和催化亚基p110组成的二聚体,广泛参与调控细胞增殖㊁分化㊁凋亡和迁移等表型㊂有报道[10,11]称PI3K 信号的异常激活参与5-氟尿嘧啶耐药的发生发展,而EV 通过激活细胞凋亡信号通路和负向调节PI3K /Akt 信号通路,抑制5-氟尿嘧啶耐药NSCLC 细胞增殖并诱导细胞凋亡,延缓5-氟尿嘧啶耐药NSCLC 细胞的增殖和凋亡㊂此外,炎症是肿瘤进展的重要组成部分㊂化疗增强炎症可能导致治疗失败和转移㊂PI3K 是先天免疫系统的重要组成部分之一,在癌症中起着重要作用㊂许多因素可以激活PI3K ,包括K +流出㊁细胞内钙㊁内质网(ER )应激和ROS [12]㊂之前的研究结果表明,5-氟尿嘧啶治疗增加了口腔癌(OSCC )中PI3K 的表达,从而介导了5-氟尿嘧啶耐药耐药性,而5-氟尿嘧啶耐药可以促进OSCC 中的肿瘤生长和转移㊂Fer 样家族成员4(Ferrostatin -4)是PI3K 的下游调控因子,其在肺癌细胞系A549和95D 中的过表达抑制集落形成㊁细胞增殖和迁移,导致细胞中PI3K /Akt 表达降低,同时使用小分子抑制剂激活PI3K /Akt 信号磷酸酶和张力蛋白同系物逆转了Ferrostatin -4对细胞增殖和转移的抑制作用㊂同样,miR -128表达在肺癌组织或细胞中下调,miR -128的敲低通过激活PI3K /Akt 通路促进A549细胞活力㊁集落形成和侵袭,加速肿瘤转移和复发㊂本研究结果与上述研究结果一致,发现细胞外囊泡装载5-氟尿嘧啶能促进结直肠癌细胞株HCT116高表达miR -128,低表达PI3K ㊂本研究未探讨细胞外囊泡装载5-氟尿嘧啶对直肠癌细胞株HCT116Ferrostatin -4表达的影响,这将在后续研究中进行㊂㊃898㊃综上所述,细胞外囊泡装载5-氟尿嘧啶能明显抑制结直肠癌细胞株HCT116增殖㊁侵袭水平,增强其凋亡水平,其机制可能与细胞外囊泡装载5-氟尿嘧啶能促进结直肠癌细胞株HCT116高表达miR -128,低表达PI3K 有关㊂ʌ参考文献ɔ[1]㊀Xi Y ,Xu P.Global colorectal cancer burden in 2020and pro-jections to 2040[J ].Transl Oncol ,2021,14(10):101174.[2]㊀Biller LH ,Schrag D.Diagnosis and treatment of metastaticcolorectal cancer :a review [J ].JAMA ,2021,325(7):669-685.[3]㊀Herrmann IK ,Wood MJA ,Fuhrmann G.Extracellular vesiclesas a next -generation drug delivery platform [J ].Nat Nano-technol ,2021,16(7):748-759.[4]㊀Verweij F J ,Balaj L ,Boulanger CM ,et al.The power of ima-ging to understand extracellular vesicle biology in vivo [J ].Nat Methods ,2021,18(9):1013-1026.[5]㊀Guo Y ,Guo Y ,Chen C ,et al.Circ3823contributes to growth ,metastasis and angiogenesis of colorectal cancer :involvementof miR -30c -5p /TCF7axis [J ].Mol Cancer ,2021,20(1):93.[6]㊀He F ,Song Z ,Chen H ,et al.Long noncoding RNA PVT1-214promotes proliferation and invasion of colorectal cancerby stabilizing Lin28and interacting with miR -128[J ].On-cogene ,2019,38(2):164-179.[7]㊀Wang H ,Wang X ,Zhang H ,et al.The HSF1/miR -135b -5paxis induces protective autophagy to promote oxaliplatin re-sistance through the MUL1/ULK1pathway in colorectal cancer [J ].Oncogene ,2021,40(28):4695-4708.[8]㊀Zhang W ,Wang Z ,Cai G ,et al.Circ _DOCK1regulatesUSP11through miR -132-3p to control colorectal cancerprogression [J ].World Surg Oncol ,2021,19(1):67.[9]㊀Zhang X ,Li T ,Han Y N ,et al.miR -125b promotes colorec-tal cancer migration and invasion by dual -targeting CFTR and CGN [J ].Cancers ,2021,13(22):5710.[10]㊀Zheng J ,Yang T ,Gao S ,et al.miR -148a -3p silences theCANX /MHC -I pathway and impairs CD8+T cell -mediated immune attack in colorectal cancer [J ].FASEB ,2021,35(8):e21776.[11]㊀Zhou L ,Li J ,Tang Y ,et al.Exosomal LncRNA LINC00659transferred from cancer -associated fibroblasts promotescolorectal cancer cell progression via miR -342-3p /ANXA2axis [J ].Transl Med ,2021,19(1):8.[12]㊀Bai L ,Gao Z ,Jiang A ,et al.Circular noncoding RNA circ_0007334sequestrates miR -577to derepress KLF12and ac-celerate colorectal cancer progression [J ].Anticancer Drugs ,2022,33(1):409-422.ʌ文章编号ɔ1006-6233(2023)06-0899-08CircRNA YAP1对股骨头微血管内皮细胞血管生成的作用研究日夏提㊃帕尔哈提,㊀翟㊀生,㊀吕㊀青(新疆医科大学第五附属医院骨科中心,㊀新疆㊀乌鲁木齐㊀830000)ʌ摘㊀要ɔ目的:探讨环状RNA (circRNA )YAP1对激素性股骨头坏死(SONFH )大鼠股骨头微血管内皮细胞(MVECs )血管生成的作用与机制㊂方法:成年雄性SD 大鼠接受甲基强的松龙(MPS ,20mg ㊃kg -1㊃d -1肌肉注射3周,以诱导SONFH 模型(n =30只)㊂Ctrl 组(n =30只)注射等量生理盐水㊂用mi-cro -CT 扫描和HE 染色评估是否造模成功㊂分离大鼠的股骨头MVECs ㊂并用CD31蛋白作为标志物用免疫荧光化学法(IF )鉴定股骨头MVECs ㊂将MVECs 分为过表达circRNA YAP1组(pcDNA -YAP1组)㊁阴性对照组(pcDNA -NC 组)㊁以及pcDNA -YAP1联合Wnt1/β-catenin 通路拮抗剂dickkopf -1处理组(pcDNA -YAP1+dickkopf -1组)㊂用qPCR 检测circRNA YAP1表达,Western blot 法分别检测Wnt1㊁β-catenin ㊁VEGF -A 和vimentin 的表达㊂用CCK -8实验检测各组细胞的增殖活性㊂Transwell 小室检测细胞的迁移率㊂管形成实验检测细胞的血管生成能力㊂结果:HE 染色结果显示Ctrl 组表现为健康状态,无骨坏死,SONFH 组大鼠均重度股骨头坏死,股骨头恶化㊂micro -CT 扫描的结果显示Ctrl 组表现骨小梁致密规则,骨小梁数量和形态均正常,SONFH 组伴空骨陷窝,骨小梁减少,骨小梁缩短㊂与Ctrl 组(1.00ʃ0.08;1.00ʃ0.12;1.00ʃ0.15)比较,SONFH 组股骨组织中circRNA YAP1(0.27ʃ0.04)㊁Wnt1(0.49ʃ0.03)㊁β-catenin (0.33ʃ0.03)表达量均显著减少(t =12.158,P =0.009;t =10.596,P =0.012;t =10.080,P =0.014)㊂另外,成功分离Ctrl 组和SONFH 组的股骨头MVECs ㊂与Ctrl 组(1.00ʃ0.㊃998㊃ʌ基金项目ɔ新疆维吾尔自治区自然科学基金项目,(编号:2021D01C426)ʌ通讯作者ɔ吕㊀青。
慢性饥饿应激通过增强ITGB1表达促进结直肠癌细胞迁移
doi:10.3971/j.issn.1000-8578.2024.23.1031慢性饥饿应激通过增强ITGB1表达促进 结直肠癌细胞迁移李思雨,曹静桦,王凤伟Chronic Starvation Stress Promotes Migration Ability of Colorectal Cancer Cells byInducing ITGB1 UpregulationLI Siyu, CAO Jinghua, WANG FengweiState Key Laboratory of Oncology in South China, Guangdong Provincial Clinical ResearchCenter for Cancer, Sun Yat-sen University Cancer Center, Guangzhou 510060, China CorrespondingAuthor:WANGFengwei,E-mail:********************.cnAbstract: Objective To investigate the effects of chronic starvation stress on the proliferation and migration of colorectal cancer cells, as well as the underlying mechanisms. Methods By using prolonged serum starvation to simulate chronic starvation stress in tumor cells, we established enduring serum-deprived models of SW480 and DLD-1 cells and observed cellular morphological change. Effects of prolonged serum starvation on SW480 and DLD-1 proliferative and migratory capabilities were assessed using CCK-8 and Transwell assays. Differential gene-expression analysis on SW480 cultured with 1% FBS or 10% FBS medium was followed by GO and KEGG pathway assessments. Migration-related protein interactions were explored using String database and Metascape software, leading to 16 genes being selected for RT-qPCR validation. Protein levels of ITGB1 and key molecules in the relevant pathways were measured. Mobility changes in SW480 were observed through Transwell assay after ITGB1 knockdown or STAT3 inhibition. Results Prolonged serum starvation significantly inhibited the proliferation of SW480 and DLD-1 cells, and DLD-1 mobility, while enhanced SW480 migration. Transcriptome analysis revealed that prolonged serum deprivation caused the upregulation of 3016 genes, among which 283 were involved in cell migration. Metascape analysis identified the correlations among potential core genes ITGB1, CD44, TNS1, STAT3, etc. Prolonged serum deprivation increased the mRNA levels of VTN, TNS1, VEGF A, STAT3, and ITGB1 while also increasing the protein levels of ITGB1 and MMP2 and the phosphorylation levels of JAK2 and STAT3. Mobility reduction in prolonged serum-starved SW480 cells was achieved through ITGB1 knockdown or a STAT3 inhibitor. Conclusion Colorectal cancer cells can endure chronic starvation stress which enhances migration capability by upregulating ITGB1 expression.Key words: Starvation; Colorectal Cancer; Cell Movement; Integrin beta1; STAT3 Transcription FactorFunding: General Program of the National Natural Science Foundation of China (No. 81972227)Competing interests: The authors declare that they have no competing interests.摘 要:目的 探讨慢性饥饿应激对结直肠癌细胞增殖、迁移能力的影响及其机制。
化学交换饱和转移技术在中枢神经系统疾病的研究进展
·177CHINESE JOURNAL OF CT AND MRI, JUN. 2023, Vol.21, No.06 Total No.164【第一作者】刘 悦,女,主治医师,主要研究方向:中枢神经系统影像诊断。
E-mail:**********************【通讯作者】于爱红,女,主任医师,主要研究方向:中枢神经系统影像诊断,骨骼及肌肉系统影像诊断。
E-mail:********************Advances in Chemical Exchange Saturation Copyright ©博看网. All Rights Reserved.178·中国CT和MRI杂志 2023年06月 第21卷 第06期 总第164期(下转第188页)在这些应用中显示了一些前景,但仍需要进一步的开发和更多的研究来验证将APT成像转化为临床的实践应用。
2.1.2 脑肿瘤 APT成像的技术不仅用于诊断、监测和治疗脑缺血,而且是一种研究脑肿瘤的方法。
酰胺质子的浓度越低,脑肿瘤恶性程度越低,APT图像的肿瘤信号强度也越低,反之越高。
APT成像可以区分高、低级别肿瘤[22],Zhou等人[23]首次研究了APT成像在3TMRI评估胶质瘤级别中的可行性。
结果表明,在经病理组织学证实为高级别的脑肿瘤中,实性部分的APT信号明显高于水肿或坏死区。
而在低级别脑肿瘤中,APT成像与周围组织信号相当。
基于这些评估效果,APT成像也被广泛应用于不同级别脑肿瘤的评估,包括将恶性肿瘤组织与其他病理成分相区别,如水肿、放射性坏死、转移,并监测临床治疗后恶性胶质瘤的病变进展情况[24]。
APT成像也可以更好地区别脑肿瘤性肿块与感染性肿块[25]。
Debnath等人对APT成像的研究结合4型归一化和病变感兴趣区参数直方图的方法鉴别肿瘤性和感染性肿块以及颅内不同肿块的区别。
2.1.3 神经系统退行性疾病 APT成像技术在帕金森病(Parkinson's disease, PD)中,可以检测不同大脑区域的神经元减少和蛋白质积累[26],PD患者苍白球、壳核和尾状核在3.5ppm的APT信号显著增加。
C- 反应蛋白对结直肠癌的临床价值
ZHOU Jia-hui, WEI Wen-e, ZHANG Li-tu*
(Department of Experimental Research, Cancer Hospital Affiliated to Guangxi Medical University, ning Guangxi 530021)
世界最新医学信息文摘 2021 年第 21 卷第 17 期
1
·论著·
C- 反应蛋白对结直肠癌的临床价值
周嘉慧,韦文娥,张力图 *
( 广西医科大学附属肿瘤医院实验研究部,广西 南宁 530021)
摘要:目的 越来越多的证据表明,炎性反应因子被认为是可以改善结直肠癌 (CRC) 预测准确性的生物标志物。我们旨在评估 C 反应蛋白 (CRP) 在 CRC 中的临床价值。方法 纳入 2013 年 10 月至 2019 年 9 月的 307 例 I-III 期 CRC 患者和 72 例结直肠 癌肝转移 (CRLM) 患者。分析 CRP 和临床病理特征之间的关系。构建 ROC 曲线以评估 CRP 对 CRLM 的诊断价值。KaplanMeier 分析和 log-rank 检验评估 CRP 高低两组之间的生存曲线差异。用 Cox 单变量与多变量分析评估 CRC 和 CRLM 潜在的 预后因素。结果 (1)CRP 对 CRLM 具有一定的诊断价值,CRP 结合 CEA 具有最高的诊断效能 (AUC=0.822)。(2) 无论对于 I-III 期 CRC 患者还是 CRLM 患者,Kaplan-Meier 分析和 log-rank 检验均表明 CRP 高的患者的 OS 较差。(3) 多因素分析表明,I-III 期患者的性别,肿瘤位置,分化程度,CEA,CRP 是 OS 的独立预后因素,CRLM 患者中 CRP 是 OS 的独立预后因素。结论 CRP 可以作为诊断 CRLM 的传统标志物的有效补充。在Ⅰ -III 期 CRC 患者和 CRLM 患者中具有独特的预后价值。 关键词:结直肠癌;转移;C 反应蛋白;诊断;预后 中图分类号:R73 文献标识码:A DOI:10.3969/j.issn.1671-3141.2021.17.001 本文引用格式:周嘉慧 , 韦文娥 , 张力图 . C- 反应蛋白对结直肠癌的临床价值 [J]. 世界最新医学信息文摘 ,2021,21(17):1-3,6.
crc的法规依据 -回复
crc的法规依据-回复Title: Regulatory Compliance for CRC: Understanding the Legislative FrameworkIntroductionThe rapid advancements in technology and the widespread use of data in today's digital era have led to the emergence of numerous challenges related to data privacy and security. In response to increasing concerns, governments around the world have enacted legislation to protect individuals' personal information and promote responsible handling of data. This article aims to provide a comprehensive understanding of the regulatory compliance framework for CRC, focusing on the legislative basis that governs its operations.Section 1: Definition and Importance of CRC1.1 Definition of CRCCRC, also known as Customer Relationship Management, refers to the strategies, practices, and technologies used by businesses to manage and analyze customer interactions and data throughoutthe customer lifecycle.1.2 Importance of CRCCRC plays a crucial role in enhancing customer satisfaction, strengthening customer loyalty, and driving business growth. It allows organizations to streamline their marketing efforts, better understand customer needs, and make data-driven decisions.Section 2: Legal Considerations in CRC2.1 Personal Data Protection2.1.1 Introduction to Personal Data Protection LawsData protection laws aim to safeguard individuals' information by imposing obligations on organizations to protect and process personal data lawfully.2.1.2 Key Legislation on Personal Data ProtectionDiscuss relevant legislation such as the General Data Protection Regulation (GDPR) in the European Union, California Consumer Privacy Act (CCPA) in the United States, and Personal Data Protection Act (PDPA) in Singapore.2.2 Consent and Data Collection2.2.1 Consent RequirementsExplain the importance of obtaining valid and informed consent when collecting customer data. Discuss explicit consent requirements for sensitive data.2.2.2 Data MinimizationHighlight the principle of data minimization, which states that organizations should collect only the necessary information required to fulfill the intended purpose.2.3 Data Security and Breach Notification2.3.1 Security MeasuresDiscuss the obligations of CRC systems to implement strict security measures to protect customer data from unauthorized access, disclosure, or alteration.2.3.2 Breach NotificationElaborate on the requirement to promptly notify authorities and affected individuals in the event of a data breach, including relevant timeframes and procedures.Section 3: CRC Compliance Strategies3.1 Privacy Impact Assessments (PIA)Explain the importance of conducting PIAs to identify and mitigate privacy risks associated with CRC activities. Discuss the procedure for conducting a PIA and its role in maintaining regulatory compliance.3.2 Appropriate Technical and Organizational MeasuresDiscuss the need to implement technical and organizational measures, such as encryption, access controls, and staff training, to ensure the security and confidentiality of customer data.3.3 Data Protection Officer (DPO)Highlight the requirement for organizations to appoint a Data Protection Officer responsible for overseeing CRC processes, ensuring compliance with relevant regulations, and acting as a point of contact for data subjects and authorities.Section 4: International Data Transfers4.1 Cross-Border Data TransfersDiscuss the challenges and legal considerations involved in transferring customer data across international borders, including the importance of data transfer agreements and the mechanisms provided by specific regulations.4.2 EU-US Privacy Shield and Standard Contractual Clauses Explain the relevance of the EU-US Privacy Shield framework and Standard Contractual Clauses in facilitating lawful transfers of personal data between the European Union and the United States.ConclusionIn conclusion, CRC is a vital tool for businesses in managing customer relationships effectively. However, its implementation requires compliance with various legal obligations to ensure the protection of personal data. By adhering to the regulatory compliance framework discussed in this article, organizations can build trust, maintain customer privacy, and avoid potential legal consequences associated with improper handling of customer data.。
DTX2通过Notch2
结直肠癌(CRC )是常见的消化道恶性肿瘤之一,且CRC 发病率呈逐年上升、年轻化的趋势。
2018年中国癌症流行病学调查显示,我国结直肠癌的发病率和死亡率在全部恶性肿瘤中分别位于第3位和第5位[1,2]。
CRC 已成为导致癌症死亡的主要原因之一[3],其中最主要原因为患者诊疗时多为进展期,甚至为伴发肝肺转移的晚期病人,15%~25%的CRC 患者初诊时即合并肝转移,这严重影响患者的生活质量及预后生存[4,5]。
因此,进一步揭示CRC 进展的作用机制,寻找助于抗CRC 诊疗的干预靶点,对提高CRC 的早诊率及预后生存具有重要意义。
近年来,随着对CRC 的分子基础[6]、表观遗传学的深入探究,发现泛素-蛋白酶体降解系统[7]在肿瘤生物学中的功能作用逐渐被重视,而泛素连接酶在该系统中起着关键性的作用,DTX2(deltex-2)为一种E3泛素连接酶,目前关于DTX2在肿瘤中的生物学作用的研究甚少及机制也尚不清楚[8]。
研究提示DTX2可能参与DTX2overexpression promotes migration and invasion of colorectal cancer cells through the Notch2/Akt axisMA Zhennan,ZHAO Xuefeng,ZHANG Xiaowei,XU Guangda,LIU FuquanDepartment of Anorectal Surgery,Dalian University Affiliated Xinhua Hospital,Dalian 116021,China摘要:目的探讨DTX2对结直肠癌(CRC )细胞迁移侵袭的影响及作用机制。
方法利用基因工具干预CRC 细胞,分为敲低组(DTX2-shRNA )、敲低空载组(neg-shRNA )、未转染组(con )、过表达空载组(pcDNA )及过表达组(pcDNA-DTX2),利用qRT-PCR 及Western blotting 法检测转染效率。
crc临床试验流程
crc临床试验流程英文回答:Clinical trial process for CRC:Clinical trials for colorectal cancer (CRC) follow a standardized process to evaluate the safety and efficacy of new treatments. The following is a general outline of the steps involved in a typical CRC clinical trial:1. Study Design: The clinical trial is designed by researchers and experts in the field. They determine the objectives, eligibility criteria for participants, treatment interventions, and outcome measures.2. Ethical Approval: Before the trial can begin, it must receive approval from an ethics committee or institutional review board (IRB). This ensures that thetrial meets ethical standards and protects the rights and welfare of the participants.3. Participant Recruitment: Researchers activelyrecruit eligible participants who meet the specificcriteria defined in the study design. Participants are informed about the trial, its potential benefits and risks, and are given the opportunity to provide informed consent.4. Randomization and Blinding: Participants are randomly assigned to different treatment groups. This helps to minimize bias and ensures that the results are reliable. In some trials, blinding is also implemented, where neither the participants nor the researchers know who is receiving the experimental treatment.5. Treatment Intervention: Participants receive the assigned treatment according to the study protocol. This could involve a new drug, a combination of drugs, or a different treatment approach. The treatment is closely monitored, and any adverse events or side effects are documented.6. Data Collection and Analysis: Throughout the trial,various data points are collected, including participant demographics, treatment response, and any adverse events. This data is carefully analyzed to assess the safety and efficacy of the treatment.7. Endpoint Evaluation: The trial has predefined endpoints, such as overall survival, progression-free survival, or response rate. These endpoints are evaluated to determine if the treatment is effective in achieving the desired outcomes.8. Statistical Analysis: The collected data is statistically analyzed to determine the significance of the treatment's effects. This analysis helps to draw conclusions about the treatment's efficacy and safety.9. Publication and Reporting: The trial results are typically published in scientific journals to share the findings with the medical community. The results may also be presented at conferences or shared with regulatory authorities for potential drug approval.10. Follow-up and Post-Trial Care: After the trial, participants may be followed up to monitor their long-term outcomes and provide any necessary post-trial care. This helps to assess the treatment's long-term effects and gather additional data for further analysis.中文回答:CRC临床试验流程:CRC(结直肠癌)的临床试验遵循一套标准化的流程,以评估新治疗方法的安全性和疗效。
临床crc的工作流程
临床crc的工作流程英文回答:Clinical research coordinators (CRCs) play a crucial role in the clinical trial process. As a CRC, my main responsibility is to ensure the smooth and efficient running of clinical trials. This involves a variety of tasks, from study initiation to study closeout.The workflow of a clinical CRC can be divided into several stages: pre-study, study initiation, study conduct, and study closeout.During the pre-study phase, I work closely with the study team to plan and prepare for the trial. This includes reviewing the study protocol, developing study-specific documents, and obtaining necessary approvals. For example, I may assist in drafting the informed consent form and submitting it to the Institutional Review Board (IRB) for approval. I also collaborate with the study team toidentify potential study participants and ensure that the study site is adequately equipped and staffed.Once the study is approved and ready to start, thestudy initiation phase begins. This involves recruiting and screening eligible participants, obtaining informed consent, and collecting baseline data. As a CRC, I may conduct study visits, administer study interventions or treatments, and collect and record data. I also ensure that the study is conducted in compliance with Good Clinical Practice (GCP) guidelines and applicable regulations.During the study conduct phase, I continue to monitor and manage the progress of the trial. This includes scheduling and coordinating study visits, monitoring participant adherence to the study protocol, and addressing any issues or concerns that may arise. For example, if a participant experiences an adverse event, I would promptly report it to the sponsor and take appropriate actions asper the protocol. I also maintain accurate and complete study documentation, such as case report forms and source documents.Finally, when the study is completed or terminated, the study closeout phase begins. This involves ensuring thatall study-related activities are properly concluded. As a CRC, I may assist in data analysis and reporting, prepare study closure reports, and archive study documents. I also coordinate with the study team to ensure that participants are appropriately informed about the study results and any follow-up care that may be required.中文回答:临床研究协调员(CRC)在临床试验过程中扮演着至关重要的角色。
crc面试工作总结
crc面试工作总结英文回答:I recently had an interview with CRC for a job position, and I must say it was quite an interesting experience. The interview process was thorough and challenging, but I felt like I was able to showcase my skills and expertise effectively.During the interview, I was asked a variety ofquestions related to my previous work experience, my knowledge of the industry, and my problem-solving abilities. One question that stood out to me was when I was asked to provide an example of a time when I had to deal with a difficult client and how I handled the situation. I shareda specific instance from my previous job where I had towork with a client who was unhappy with the services provided. I explained how I actively listened to their concerns, empathized with their situation, and ultimately found a solution that satisfied both the client and thecompany.Additionally, I was given a case study to analyze and present my findings. This was a great opportunity for me to demonstrate my analytical skills and ability to think critically. I was able to walk the interviewers through my thought process, discuss potential solutions, andarticulate my recommendations effectively.Overall, I felt that the interview was a positive experience. The interviewers were engaging and made me feel comfortable throughout the process. I appreciated the opportunity to showcase my skills and experiences, and I believe that I left a strong impression.中文回答:最近我参加了CRC的面试,我必须说这是一次非常有趣的经历。
结直肠癌患者血清白细胞介素6水平及与肿瘤疗效关系
结直肠癌患者血清白细胞介素6水平及与肿瘤疗效关系陈岩松;陈燕;陈娜娟【摘要】目的探讨人白细胞介素6(interleukin-6,IL-6)在结直肠癌患者中的水平及意义.方法收集132例首诊结直肠癌患者(CRC)、35例炎症性肠病(IBD)、31例肠道良性疾病患者(CBD)及84例健康体检者(HC)血清,采用电化学发光双抗体夹心免疫分析法(ECLIA)检测血清中白细胞介素6(IL-6)及癌胚抗原(CEA)水平,分析IL-6水平与结直肠癌患者临床病理特征的相关性;应用受试者工作特性曲线(ROC)和二元Logistic法回归分析IL-6和CEA两指标对结直肠癌的诊断价值;对随访资料完整的120例中晚期结直肠癌患者动态观察治疗前后血清IL-6和CEA水平,分析两指标与肿瘤疗效的关系.结果结直肠癌患者血清IL-6水平显著高于炎症性肠病组(P <0.05)、肠道良性疾病组(P<0.01)和健康对照组(P<0.01),结直肠癌患者血清CEA水平显著高于炎症性肠病组(P<0.05)、肠道良性疾病组(P<0.05)和健康对照组(P<0.01),差异均有统计学意义.CRC患者血清IL-6水平与肿瘤直径、分化程度、组织类型、淋巴结转移、远处转移、TNM分期均显著相关(P<0.05),而与年龄、性别及肿瘤发生部位无明显相关.IL-6诊断结直肠癌的灵敏度(72.7%)和准确性(78.6%)均高于CEA(分别为68.2%和77.9%),特异性(85.2%)低于CEA(88.9%),两指标联合检测能够提高灵敏度(97.2%)和准确性(85.6%).结直肠癌肿瘤控制组(CR +PR+SD)治疗后两指标均较治疗前有显著下降(P<0.05),差异有统计学意义,而肿瘤进展组(PD)治疗后两指标均未显著下降(P>0.05).结论 IL-6和CEA两指标联合检测有助于结直肠癌的诊断和疗效观察.%Objective To investigate the expression of serum interleukin-6(IL-6) in colorectal cancer patients andthe relationship between the IL-6 level and the tumor curativeeffect.Methods The electrochemiluminescence immunoassay was used toexamine the serum level of IL-6 and carcino-embryonic antigen (CEA) in 132 colorectal cancer patients (CRC),35 inflammatory bowel disease (IBD),31 colorectal benign disease(CBD) and 84 healthy donors as healthy control group (HC).The relationship between IL-6 and clinicopathological features of colorectal cancer was analyzed,and ROC curve and binary losistic regression were applied to compare the diagnostic value of the IL-6 and CEA.The dynamic levels of serum IL-6 and CEA before and after treatment in 120 advanced CRC patients with complete case data was observed,and the relationship between IL-6 and CEA and the cruative effect of the tumor was analyzed.Results Both the serum IL-6 and CEA levels of the CRC were higher than IBD (P < 0.05,P <0.05,respectively),CBD (P < 0.01,P < 0.05,respectively),and HC groups (P < 0.01,P < 0.01,respectively).Serum levels of the IL-6 was significantly correlated with tumor size,degree of tumor differentiation,pathological type,lymph node metastasis,distant metastasis and TNM stage (all the above,P < 0.05).The sensitivity and accuracy of IL-6 for CRC diagnosis were higher than CEA (72.7% vs 68.2%,78.6% vs 77.9%,respectively),while specifity was lower than CEA(85.2% vs 88.9%).The combined detection of IL-6 and CEA were highest in sensitivity (97.2%) and accuracy (85.6%).After treatment,both the serum IL-6 and CEA levels significantly decreased in CR + PR + SD group(both P <0.05),but not in the PD group (both P >0.05).Conclusion The combined detection of serum IL-6 and CEA could bea useful potential diagnostic and therapeutic evaluation method for colorectal cancer.【期刊名称】《标记免疫分析与临床》【年(卷),期】2017(024)011【总页数】6页(P1274-1279)【关键词】结直肠癌;白细胞介素6;癌胚抗原【作者】陈岩松;陈燕;陈娜娟【作者单位】福建医科大学附属肿瘤医院福建省肿瘤生物治疗重点实验室生物化学分子生物学研究室福建省肿瘤医院检验科,福建福州350014;福建医科大学附属肿瘤医院福建省肿瘤生物治疗重点实验室生物化学分子生物学研究室福建省肿瘤医院检验科,福建福州350014;福建医科大学附属肿瘤医院福建省肿瘤生物治疗重点实验室生物化学分子生物学研究室福建省肿瘤医院检验科,福建福州350014【正文语种】中文结直肠癌(colorectal cancer,CRC)是常见的恶性肿瘤之一,全球的结直肠癌的发病率一直在上升。
胃癌患者血清CCL2、ANXA2含量与癌细胞浸润性生长的相关性研究
中国细胞生物学学报Chinese Journal of Cell Biology2021,43(2): 404412DOI: 10.11844/cjcb.2021.02.0016胃癌患者血清CCL2、ANXA2含量与癌细胞浸润性生长的相关性研究王薇布力布•吉力斯汉许春蕾赛福丁•柯尤木*(新疆医科大学第三临床医学院(附属肿瘤医院)消化内科,乌鲁木齐830011)摘要 该文旨在探讨胃癌患者血清C C趋化因子配体2(C C L2)、膜联蛋白A2(A N X A2)含量与 癌细胞浸润性生长的相关性,并分析其与胃癌临床病理特征的关系。
选择187例胃癌患者为研究 对象,并选择153例正常人群为对照组,使用酶联免疫吸附试验(E L I S A)法检测所有研究对象血清C C L2、A N X A2含量,分析组间临床病理特征(癌细胞分化程度、浸润程度和是否发生淋巴结转移等)及C C L2、A N X A2水平差异。
采用q R T-P C R技术检测胃癌组织及癌旁组织中癌细胞浸润性生长相失I西 Cripto-I、ZEB1、Snail'Vimentin、Piwil2、STOML2、BCL2、Smo、XL4P^$J t。
分析组间血清C C L2、A N X A2水平差异,以及不同C C L2、A N X A2水平下夕卜泌体E x o s o m e s蛋白及Z£W、S«az7、尸/w"2、S7V9M L2、5C L入5>«〇、A X4尸基因的表达差异。
使用免疫组织 化学S P染色法检测全部研究对象中C C L2、A N X A2含量,分析各组间差异。
胃癌组血清C C L2、A N X A2水平显著高于对照组(/><〇.〇5);患者的性别、年龄、胃癌分型等数据在C C L2、A N X A2水平上作对比,无显著差异(F>〇.〇5),患者的分化程度、浸润深度和淋巴结是否转移等数据在C C L2、A N X A2水平上作对比,具有显著性差异(P<〇.〇5);高水平C C L2组、高水平A N X A2组E x o s o m e s蛋白及上皮-间质转化(E M T)基因(C W p t a-/、Z£5八5>za//、油•《)、增殖基因〇P h w72、5T O M L2、如〇、J TL4/3)表达分别显著高于低水平C C L2组、低水平A N X A2组(P<0.05);胃癌组织中E M T基因八八、增殖基因(朽wi./2、5T O M Z J、S C Z J、5>«〇、尸)表达显著高于癌旁组织(P<〇.〇5)。
长链非编码RNA_H19与结直肠癌关系的再认识
㊃综述㊃D O I:10.3969/j.i s s n.1672-9455.2024.07.032长链非编码R N A H19与结直肠癌关系的再认识周福荣,李龑杼综述,刘勇敢ә审校广东省中山市人民医院肛肠科,广东中山528400摘要:结直肠癌作为消化系统的恶性肿瘤之一,其早期症状不典型,通常确诊时已是晚期,近年来,结直肠癌发病率和病死率呈现上升的趋势;寻找有效的早期诊断标志物及个性化治疗方法迫在眉睫㊂长链非编码R N A(l n c R N A)H19参与调控结直肠癌细胞增殖㊁侵袭㊁转移等过程,在结直肠癌的诊断㊁治疗及疗效监测等方面有着重要作用㊂该文主要概括l n c R N A H19作为结直肠癌诊断㊁预后评估生物标志物的现状,同时总结分析l n c R N A H19参与结直肠癌上皮-间质转化㊁细胞自噬㊁化疗耐药㊁癌症干细胞等过程中扮演的角色㊂随着研究的深入,l n c R N A H19在结直肠癌中潜在的临床应用价值有望逐一被挖掘,为结直肠癌的早期诊断及靶向干预提供更多的参考㊂关键词:长链非编码R N A; H19;结直肠癌;生物标志物;癌症干细胞;上皮-间质转化中图法分类号:R735.3+5文献标志码:A文章编号:1672-9455(2024)07-1008-05 R e c o g n i t i o n o f r e l a t i o n s h i p b e t w e e n l o n g n o n-c o d i n g R N A H19a n d c o l o r e c t a l c a n c e rZ H O U F u r o n g,L I Y a n z h u,L I U Y o n g g a nәD e p a r t m e n t o f P r o c t o l o g y,Z h o n g s h a n M u n i c i p a l P e o p l e's H o s p i t a l,Z h o n g s h a n,G u a n g d o n g528400,C h i n aA b s t r a c t:C o l o r e c t a l c a n c e r(C R C)i s o n e o f t h e m a l i g n a n t t u m o r s o f t h e d i g e s t i v e s y s t e m,w i t h o u t t y p i c a l s y m p t o m s i n e a r l y s t a g e,a n d i t i s a l r e a d y t h e l a t e s t a g e w h e n i t i s d i a g n o s e d.I n r e c e n t y e a r s,t h e i n c i d e n c e r a t e a n d m o r t a l i t y r a t e o f c o l o r e c t a l c a n c e r s h o w t h e i n c r e a s i n g t r e n d;F i n d i n g e f f e c t i v e e a r l y d i a g n o s t i c b i o m a r k e r s a n d p e r s o n a l i z e d t r e a t m e n t m e t h o d s i s u r g e n t.L o n g n o n c o d i n g R N A H19(l n c R N A H19)i s i n v o l v e d i n r e g u-l a t i n g t h e p r o c e s s o f p r o l i f e r a t i o n,i n v a s i o n a n d m e t a s t a s i s o f c o l o r e c t a l c a n c e r c e l l s,a n d i t p l a y s a n i m p o r t a n t r o l e i n t h e d i a g n o s i s,t r e a t m e n t a n d e f f i c a c y m o n i t o r i n g o f C R C.T h i s a r t i c l e m a i n l y s u mm a r i z e s t h e c u r r e n t s t a t u s o f l n c R N A H19a s a b i o m a r k e r f o r t h e d i a g n o s i s a n d p r o g n o s i s e v a l u a t i o n o f C R C,m e a n w h i l e a n a l y z e s t h e r o l e o f l n c R N A H19i n t h e p r o c e s s e s o f e p i t h e l i a l m e s e n c h y m a l t r a n s i t i o n(E MT),a u t o p h a g y,c h e m o t h e r a-p y r e s i s t a n c e,a n d c a n c e r s t e m c e l l s(C S C)i n c o l o r e c t a l c a n c e r.W i t h t h e d e e p e n i n g o f r e s e a r c h t r a n s f o r m a-t i o n,t h e p o t e n t i a l c l i n i c a l a p p l i c a t i o n v a l u e o f l n c R N A H19i n c o l o r e c t a l c a n c e r i s e x p e c t e d t o b e e x p l o r e d o n e b y o n e,p r o v i d i n g m o r e r e f e r e n c e s f o r e a r l y d i a g n o s i s a n d t a r g e t e d i n t e r v e n t i o n o f c o l o r e c t a l c a n c e r.K e y w o r d s:l o n g n o n-c o d i n g R N A; H19;c o l o r e c t a l c a n c e r; b i o m a r k e r s;c a n c e r s t e m c e l l s;e p i-t h e l i a l m e s e n c h y m a l t r a n s i t i o n2022年国家癌症中心发布最新癌症统计数据显示:2020年我国癌症新发病例数和死亡病例数位于全球第1;2020年我国结直肠癌新发病例56万,排在十大癌症中的第2位,发病率和病死率持续上升[1]㊂结直肠癌的防治负担日益加重,防控形势异常严峻㊂随着人口老龄化的加剧及结直肠癌发病的年轻化,其防治难度不言而喻,因此寻找高特异度㊁高灵敏度的生物标志物对结直肠癌进行早期诊断,利用有效的创新药物实施精准靶向干预显得极为重要㊂研究表明长链非编码(l n c R N A)H19(以下简称H19)在结直肠癌的发病机制中具有重要作用,H19参与结直肠癌细胞增殖㊁转移,化疗药物抵抗等诸多过程[2]㊂然而,H19在结直肠癌中的作用机制仍未完全阐明㊂本综述旨在归纳㊁分析H19在结直肠癌中的功能,并讨论H19在结直肠癌中面临的挑战和可能的应用策略㊂1H19概述H19的长度为2.3k b,是较早发现的l n c R N A之一,其基因位于染色体11p15.5,通常在胎儿组织中高表达,出生后表达量大幅度减少㊂研究发现H19参与人体多种疾病的病理过程,如炎症反应㊁细胞再生㊁血管生成㊁纤维化等;此外,H19过表达被认为与人体多系统中癌症的发生㊁发展有关,例如结直肠癌㊁肝癌㊁胃癌㊁肺癌㊁乳腺癌㊁前列腺癌㊁神经胶质瘤等[3]㊂H19在恶性肿瘤中异常表达发挥癌基因作用,其功能复杂㊁多样,一直是研究的热点㊂H19既可以通过调控其靶基因参与结直肠癌发生的复杂过程,又可以作为㊃8001㊃检验医学与临床2024年4月第21卷第7期 L a b M e d C l i n,A p r i l2024,V o l.21,N o.7ә通信作者,E-m a i l:1990389480@q q.c o m㊂被调控的靶点与其他l n c R N A㊁微小R N A(m i R N A)联合参与结直肠癌的演变㊂近期的研究发现H19可通过多种机制调节肠黏膜机械屏障,可加重小鼠溃疡性结肠炎(U C)肠道损伤过程,而U C是结直肠癌发生的独立危险因素[4-6]㊂因此,H19被认为是结直肠癌中高表达的直接致癌因子,同时也是肠道疾病向结直肠癌演变的诱因㊂2 H19作为结直肠癌的生物标志物2.1血清或血浆中的H19有研究报道,与健康对照者相比,结直肠癌患者血清H19的表达增加11.38倍,H19和HO X转录本反义基因R N A(HO T A I R)检测诊断结直肠癌的灵敏度和特异度分别为92.9%和100.0%,H19在埃及结直肠癌患者中表达水平上调,是一种高灵敏度和高特异度的非侵入性结直肠癌诊断生物标志物[7]㊂邓玲慧等[8]报道,结直肠癌组患者血清H19表达水平显著高于对照组,H19高表达组中位总生存时间33.6个月㊁无进展生存期22.9个月,显著低于H19低表达组的55.8㊁43.3个月,说明H19与结直肠癌患者预后㊁化疗的敏感性相关㊂N G 等[9]在18例结直肠癌患者的血清外泌体中检测到H19,发现晚期结直肠癌患者中H19表达水平显著高于早期患者,表明血清外泌体中的H19可能是结直肠癌预后分期的生物标志物㊂2.2组织中的H19 N A C A R K A H Y A等[10]对51例结直肠癌患者的肿瘤和周围正常组织标本进行检测,发现肿瘤组织中H19的表达比周围正常组织高2.11倍,H19表达量与肌酐之间存在相关性,进一步分析表明H19是结直肠癌的一种候选生物标志物,可以评估结直肠癌预后和抗肿瘤治疗反应㊂王会新等[11]的循证研究为H19作为消化系统恶性肿瘤预后标志物提供了较充分的证据,其研究显示H19低表达结直肠癌患者的总生存期(O S)长于H19高表达患者㊂结肠癌组织中H19表达增加与K R A S基因突变状态和结直肠癌晚期有关,是无复发生存期缩短的重要危险因素[12]㊂这表明H19可能是结直肠癌患者预后的预测标志物㊂2.3粪便中的H19粪便检查作为一种无创的肠癌筛查方式,患者依从性高,可居家留取标本,提高了结肠癌的筛查率㊂G H A R I B等[13]的研究显示,粪便中l n c R N A稳定存在并可重复检测,粪便中H19及其他l n c R N A组成的长链非编码R N A谱可识别和区分结直肠癌患者与健康个体或息肉患者,在鉴别诊断结直肠癌方面,与l n c R N A单项检测相比,癌症相关l n-c R N A的联合检测具有更高的灵敏度和特异度,粪便l n c R N A组鉴定可能是早期发现结直肠癌的潜在生物标志物㊂2.4l n c R N A-m i R N A-m R N A调控网竞争性内源性R N A(c e R N A)调控网络一直是各类癌症研究的热门话题㊂l n c R N A已被证明通过充当c e R N A来影响肿瘤发生和进展㊂Y A N G等[14]成功构建了包含H19作为c e R N A的预测调控模型,该模型可以作为结直肠癌的独立预后模型,该模型与风险评分及临床特征结合可更好地预测结直肠癌患者的生存率;G U O 等[15]建立包括H19㊁S N H G7㊁h s a-m i R-29b-3p等c e R N A调控网络,发现l n c R N A-m i R N A-m R N A调控网络模型结合肿瘤免疫细胞浸润可作为预测结直肠癌患者预后的标志物㊂这表明通过使用生物信息分析构建包含l n c R N A的内源性R N A网络可预测结直肠癌患者的存活率㊂2.5 N6腺苷(m6A)与H19调控网 m6A的甲基化作为最常见的R N A修饰类型,可影响l n c R N A的产生和作用,研究显示m6A相关l n c R N A和调节因子之间的相互作用对癌症l n c R N A修饰及精准肿瘤学具有重要的临床效应,是当前研究的热点㊂然而, m6A相关l n c R N A对结直肠癌的预后价值仍不清楚, Z H A N G等[16]在622例结直肠癌患者中筛选了24个m6A相关的l n c R N A,确定5个l n c R N A(H19㊁S L-C O4A1-A S1㊁M E L T F-A S1㊁S H3P X D2A-A S1㊁P C A T6)与结直肠癌患者无进展生存期相关,表明m6A相关H19是预测结直肠癌患者无进展生存期的有前景的生物标志物㊂2.6 H19基因变异一项探讨H19基因单核苷酸多态性(S N P)与结直肠癌风险关系研究显示,S N P r s2839698多态性与结直肠癌风险降低有关,此外, r s2839698多态性还与结直肠癌患者的淋巴结转移和肿瘤大小有关,H19基因r s2839698多态性可能是预测中国汉族人群结直肠癌易感性的重要生物标志物和诊断标志物[17]㊂Q I N等[18]通过D N A测序鉴定出H19基因启动子中的16个S N P,发现携带S N P r s4930101㊁r s2735970和r s1*******或携带一种以上的组合风险基因型显著增加了结直肠癌的患病风险,表明H19启动子的S N P,尤其是这些选定组合S N P 和已知风险因素的联合检测显示出更高的预测效能,是晚期结直肠癌预后的最佳预测模型㊂3 H19与结直肠癌的发生发展3.1 H19与结直肠癌细胞增殖 H19已被发现在包括结肠直肠癌在内的各种癌症中过度表达,HU O 等[19]研究显示,H19在结直肠癌组织和细胞中的表达水平高于癌旁正常组织和正常N C M460细胞,H19可通过调控结直肠癌细胞周期来调控结直肠癌细胞增殖㊂Y A N G等[20]研究发现,m i R-200a与H19竞争性结合并抑制其靶基因β-c a t e n i n表达,从而减少结直肠癌细胞增殖,说明H19可通过与m i R-200a竞争性结合来调节β-c a t e n i n的表达和活性㊂H19有助于促进结直肠肿瘤细胞的生长,加快肿瘤细胞转移;沉默H19会诱导结直肠癌细胞凋亡[21]㊂这为结直肠癌靶向治疗提供了潜在策略,进一步阐明H19调节结直肠癌进展的机制㊂㊃9001㊃检验医学与临床2024年4月第21卷第7期 L a b M e d C l i n,A p r i l2024,V o l.21,N o.73.2 H19与结直肠癌侵袭及转移转移是癌症相关死亡的主要原因,研究表明H19与肿瘤转移具有相关性,H19可通过促进侵袭和迁移这一转移前步骤来加快结直肠癌患者肿瘤转移,但H19促进结直肠癌转移的机制并未阐明㊂R A S/丝裂原活化蛋白激酶(MA P K)是重要的细胞内信号转导通路,R A S/ MA P K信号通路的激活是人类癌症中最常见的致癌原因㊂Y A N G等[22]发现H19的过表达能够促进结直肠癌细胞的迁移和侵袭,其机制可能是通过激活细胞内R A S/MA P K信号通路实现;C H E N等[23]发现H19的过表达与结直肠癌患者的无复发生存率和总生存率降低有关,H19增加结肠癌细胞的活力和迁移㊂Z H A N G等[24]的研究中,H19还可通过直接与异质核核糖核蛋白A2B1(h n R N P A2B1)结合来促进结直肠癌细胞的迁移和侵袭㊂此外,H19还可作为s T L R4/M D-2复合物的靶基因,s T L R4/M D-2复合物通过下调H19的表达,在体外和体内抑制结直肠癌细胞迁移和侵袭[25]㊂3.3H19与结直肠癌细胞上皮-间质转化(E MT)上皮细胞转化为间充质细胞过程的特点是分子重编程,导致细胞骨架重组㊁细胞连接破坏以及细胞迁移㊁侵入邻近组织的能力增加,研究表明E MT 和表观遗传机制是癌细胞转移机制的重要组成㊂H19被描述为结直肠癌中E MT过程的一种新型调节剂,其如何调节侵袭-转移级联反应和相关过程并未阐明㊂C HOWD HU R Y等[26]的研究显示,H19可通过充当c e R N A来参与E MT的多个基因的表达,促进结直肠癌细胞体内和体外的生长过程㊂HU等[27]的研究中,表观遗传调控因子组蛋白去乙酰化酶(H D A C)缺失或敲低可上调H19,从而诱导结直肠癌细胞的E MT和转移,表明H D A C2是结直肠癌转移抑制因子㊂H19可组成调节轴通过作用于W n t信号通路促进结直肠癌细胞E MT[28]㊂3.4 H19与结直肠癌化疗耐药5-氟尿嘧啶㊁奥沙利铂㊁长春新碱㊁羟基喜树碱㊁氨甲蝶呤㊁伊立替康㊁紫杉醇和(或)新一代靶向药物西妥昔单抗㊁帕尼单抗或贝伐单抗的应用显著提高了结直肠癌患者的生存率;然而,大多数结直肠癌患者最终会产生化疗耐药,导致预后不良[29]㊂研究表明,H19在结直肠癌化疗耐药的形成中起关键作用[30]㊂R E N等[31]认为肿瘤微环境中癌症相关成纤维细胞(C A F)和癌细胞之间的串扰促进了肿瘤的发展并导致了结直肠癌化疗耐药性,其研究中建立A OM/D S S诱导的结肠炎相关癌(C A C)小鼠模型,发现结直肠肿瘤基质的C A F及H19过表达有助于结直肠癌发展和化学抗性增强㊂WU等[32]研究表明,H19敲低使H T-29-R细胞中的氨甲蝶呤抗性敏感,而其过表达改善了亲代细胞中的氨甲蝶呤抗性,表明H19介导了氨甲蝶呤抗性,W n t/β-c a t e n i n信号在H T-29-R细胞中被激活,H19敲低抑制了亲本细胞中的这种信号,说明H19通过激活W n t/β-c a t e n i n信号传导介导氨甲蝶呤耐药,这有助于将H19发展为氨甲蝶呤耐药结直肠癌患者有潜力的治疗靶点㊂二甲双胍最近被批准作为抗结直肠癌的药物,然而对二甲双胍治疗的耐药性是一个挑战性的问题, A B D-R A B O U等[33]引入二甲双胍纳米封装来使耐药癌细胞敏感,发现包裹在卵磷脂和壳聚糖纳米颗粒中的二甲双胍通过下调l n c R N A(包括H19)表达来增加细胞毒性,从而抑制结直肠癌的增殖能力㊂S U N 等[34]研究中,H19是结直肠癌耐药过程的l n c R N A,可预测奥沙利铂或伊利替康治疗的敏感性㊂3.5 H19与结直肠癌细胞自噬自噬是生物医学研究的主要焦点,化学治疗后诱导的细胞自噬被证明是抗癌药物耐药机制之一,而H19是自噬导致的化学抗性相关的l n c R N A[35],H19在结直肠癌自噬和化疗耐药中的作用和机制尚未阐明㊂WA N G等[36]发现H19与结直肠癌中的5-氟尿嘧啶抗性相关,H19促进结直肠细胞5-氟尿嘧啶抗性,通过S I R T1触发自噬以诱导癌症化疗耐药,H19可能作为预测对5-氟尿嘧啶化学治疗反应的标志物㊂另一项研究中也表明,H19和人尿路上皮癌相关基因1(U C A1)表达的变化是预测直肠癌中基于5-氟尿嘧啶新辅助化疗(N A C)的反应敏感性的生物标志物[37]㊂在乳腺癌的研究中,二甲双胍和H19可以调节自噬和铁死亡,自噬诱导剂和H19可以逆转脂质活性氧的产生和二甲双胍诱导的自噬抑制,二甲双胍可能通过H19抑制自噬来诱导铁死亡[38];由于肿瘤异质性及H19功能的复杂性,其在结直肠癌中调节自噬和铁死亡的机制有待进一步研究㊂3.6 H19与癌症干细胞(C S C) C S C具有自我更新㊁血管生成㊁免疫逃避的能力,研究显示H19可调节C S C相关转录因子在结直肠癌中的表达,增强致癌潜能,使其对常见的抗癌药物具有高度抵抗力[39]㊂H19被证明可以作为一种c e R N A隔离m i R-138和m i R-200a,从而导致锌指同源框蛋白1(Z E B1)和锌指同源框蛋白2(Z E B2)表达抑制[40]㊂R E N等[31]发现H19与结直肠癌标本中结直肠癌C S C的干性有关,H19促进C S C的干性并增加了肿瘤起始细胞的频率, C A F通过转移外泌体H19促进结直肠癌的干性, H19通过海绵m i R-141激活β-c a t e n i n途径增强结直肠癌细胞的干性㊂4总结与展望研究已表明,H19高表达与结直肠癌O S较短㊁肿瘤分化差㊁临床分期较晚㊁淋巴结转移较早和远处转移较早密切相关[41]㊂血液及粪便中的H19作为循环标志物用于结直肠癌的诊断具有无创㊁易采集的优点,可提高结直肠癌的筛查率;另外,H19基因的多态性是结直肠癌易感性㊁疗效预测㊁药物治疗敏感性预测的重要指标;H19作为c e R N A亦显示出强大的预㊃0101㊃检验医学与临床2024年4月第21卷第7期 L a b M e d C l i n,A p r i l2024,V o l.21,N o.7测能力㊂由于结直肠癌的高度异质性,H19在结直肠癌中的功能似乎比在其他类型肿瘤中的功能复杂得多,涉及与遗传因素㊁环境因素㊁生活方式及相关基因改变和肠道炎症状况之间复杂的相互作用㊂H19与m i R N A或其他靶基因调节途径是多重的:一方面, H19能够充当 海绵 来隔离m i R N A或其他靶基因并阻碍它们的作用;另一方面,H19本身是m i R N A 或其他靶基因的前体,可直接调节靶标,从而影响结直肠肿瘤的微环境㊁E MT㊁结直肠癌干细胞等过程㊂结直肠癌中H19高表达与化疗耐药相关,沉默H19可使耐药细胞对各种药物和离子辐射敏感,表明H19抑制剂与当前药物的联合治疗可能是结直肠癌放化疗耐药的逆转方法;但是H19抑制剂的开发,还需考虑其他非编码R N A发挥协同功能的可能,同时,需要进一步确定H19在表观遗传修饰中调节结直肠癌放化疗耐药信号通路的作用㊂尽管H19可作为结直肠癌生物标志物,但是当前仍然没有H19相关检测试剂盒进入临床试验㊁申请注册上市,由于研究方法㊁对象㊁标本㊁地域㊁种族的限制性差异,H19的具体功能可能还需多中心㊁更大样本量的临床研究㊂另外,循环(血液㊁粪便)H19作为监测结直肠癌进展的生物标志物的上调机制是否与组织中H19相同,未来有待进一步研究,为结直肠癌的无创筛查㊁早期诊断㊁预防监测提供更有效的方法㊂H19作为结直肠癌治疗靶点的临床试验还很缺乏,有必要进行更多的临床研究,加快H19抑制剂的临床转化和结直肠癌患者用药的精准化进程㊂参考文献[1]Z H E N G R S,Z HA N G S W,Z E N G H M,e t a l.C a n c e r i n-c ide n c e a n d m o r t a l i t y i n C h i n a,2016[J].J N a 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[30]Z I C H I T T E L L A C,L O R I A M,C E L E S I A A,e t a l.L o n gn o n-c o d i n g R N A H19e n h a n c e s t h e p r o-a p o p t o t i c a c t i v i t y o f I T F2357(a h i s t o n e d e a c e t y l a s e i n h i b i t o r)i n c o l o r e c t a lc a n c e r c e l l s[J].F r o n t P h a r m a c o l,2023,14:1275833.[31]R E N J,D I N G L,Z HA N G D Y,e t a l.C a r c i n o m a-a s s o c i a t-e df i b r o b l a s t s p r o m o t e t h e s t e m n e s s a n d c h e m o r e s i s t a n c eo f c o l o r e c t a l c a n c e r b y t r a n s f e r r i n g e x o s o m a l l n c R N AH19[J].T h e r a n o s t i c s,2018,8(14):3932-3948.[32]WU K F,L I A N G W C,F E N G L,e t a l.H19m e d i a t e sm e t h o t r e x a t e r e s i s t a n c e i n c o l o r e c t a l c a n c e r t h r o u g h a c t i-v a t i n g W n t/β-c a t e n i n p a t h w a y[J].E x p C e l l R e s,2017, 350(2):312-317.[33]A B D-R A B O U A A,A B D E L A Z I Z A M,S HA K E R O G,e t a l.M e tf o r m i n-l o a d e d l e c i t h i n n a n o p a r t i c l e s i n d u c e c o l o-r e c t a l c a n c e r c y t o t o x i c i t y v i a e p ig e n e t i c m o d u l a t i o n o f n o n c o d i n g R N A s[J].M o l B i o l R e p,2021,48(10):6805-6820.[34]S U N F F,L I A N G W W,Q I A N J.T h e i d e n t i f i c a t i o n o fC R ND E,H19,U C A1a n d HO T A I R a s t h e k e y l n c R N A si n v o l v e d i n o x a l i p l a t i n o r i r i n o t e c a n r e s i s t a n c e i n t h ec h e m o t h e r a p y o f c o l o r e c t a l c a n c e r b a s ed o n i n te g r a t i v eb i o i n f o r m a t ic s a n a l y s i s[J].M o l M ed Re p,2019,20(4):3583-3596.[35]B E R MU D E Z M,A G U I L A R M M,L I Z A R R A G A V E,e ta l.L n c R N A s a s r e g u l a t o r s o f a u t o p h a g y a n d d r u g r e s i s t-a n c e i n c o l o r e c t a l c a n c e r[J].P r o n t O n c o l,2019,9:1008.[36]WA N G M,HA N D,Y U A N Z M,e t a l.L o n g n o n-c o d i n gR N A H19c o n f e r s5-F u r e s i s t a n c e i n c o l o r e c t a l c a n c e r b y p r o m o t i n g S I R T1-m e d i a t e d a u t o p h a g y[J].C e l l D e a t hD i s,2018,9(12):1149.[37]Y O K O Y A M A Y,S A K A T A N I T,W A D A R,e t a l.I n v i t r oa n d i n v i v o s t u d i e s o n t h e a s s o c i a t i o n o f l o n g n o n-c o d i n gR N A s H19a n d u r o t h e l i a l c a n c e r a s s o c i a t e d1w i t h t h e s u s c e p t i b i l i t y t o5-f l u o r o u r a c i l i n r e c t a l c a n c e r[J].I n t J O n c o l,2019,55(6):1361-1371.[38]C H E N J D,Q I N C,Z HO U Y L,e t a l.M e t f o r m i n m a y i n-d u cef e r r o p t o s i s b y i n h i b i t i ng a u t o ph a g y vi a l n c R N A H19 i n b r e a s t c a n c e r[J].F E B S O p e n B i o,2022,12(1):146-153.[39]L E C E R F C,P E P E R S T R A E T E E,L E B O U R H I S X,e ta l.P r o p a g a t i o n a n d m a i n t e n a n c e o f c a n c e r s t e m c e l l s:am a j o r i n f l u e n c e o f t h e l o n g n o n-c o d i n g R N A H19[J].C e l l s,2020,9(12):2613.[40]L I A N G W C,F U W M,WO N G C W,e t a l.T h e l n c R N AH19p r o m o t e s e p i t h e l i a l t o m e s e n c h y m a l t r a n s i t i o n b y f u n c t i o n i n g a s m i R N A s p o n g e s i n c o l o r e c t a l c a n c e r[J].O n c o t a r g e t,2015,6(26):22513-22525.[41]Y U H,L I S,WU S X,e t a l.T h e p r o g n o s t i c v a l u e o f l o n gn o n-c o d i n g R N A H19i n v a r i o u s c a n c e r s:a m e t a-a n a l y s i sb a s e d o n15s t u d i e s w i t h1584p a t i e n t s a n d t h e C a nc e rG e n o m e A t l a s d a t a[J].M e d i c i n e(B a l t i m o r e),2020,99(2):e18533.(收稿日期:2023-09-16修回日期:2023-12-29)㊃2101㊃检验医学与临床2024年4月第21卷第7期 L a b M e d C l i n,A p r i l2024,V o l.21,N o.7。
岩土工程可靠度的快速遗传衡量与计算
2023 年 6 月
Vol.37 No.3
Jun.
2023
粉煤灰综合利用
FLY ASH COMPREHENSIVE UTILIZATION
研究与应用
岩土工程可靠度的快速遗传衡量与计算 ∗
Fast Genetic Measurement and Calculation of Reliability of Geotechnical Engineering
于正态分布状态且问题架构呈线性状态, 再对可靠度值进行快速遗传计算。 通过算法对比分析与实例列举验证
此种算法的有效性, 证明了此算法的计算精度与稳定性更优, 并能实际应用于山坡挖掘工程的施工方案可靠度
分析。
关键词: 快速遗传算法; 岩土工程; 岩土工程分析; 可靠度; 可靠度指标
中图分类号: G642 文献标志码: A 文章编号: 1005-8249 (2023) 03-0019-07
DOI:10.19860 / j.cnki.issn1005-8249.2023.03.019
ZHANG Lei, LEI Yu, ZHANG Lu
( Xi’ an Eurasia University, Xi’ an 710065, China)
Abstract: In order to accurately calculate any feasible solution when the reliability objective function of geotechnical engineering reaches
张 磊, 雷 雨, 张 璐
( 西安欧亚学院, 陕西 西安 710065)
摘 要: 为了准确地计算出岩土工程可靠度目标函数达到最值时的任意一种可行解, 本研究分析如何通过
以抗坏血酸作还原滴定剂电位滴定法测定铁矿石中全铁
以抗坏血酸作还原滴定剂电位滴定法测定铁矿石中全铁戚淑芳;张杰;王莹;宁松【摘要】提出了以抗坏血酸作为还原滴定剂、全自动电位滴定仪判定滴定终点的测定铁矿石中全铁的方法.对滴定酸介质和酸度、温度、电位滴定仪参数进行了优化,对干扰元素的影响等进行了试验.结果表明,全铁可在铁矿石溶样的酸介质及酸度下滴定,无需进行酸度调节,滴定的温度范围为50~80℃.采用过氧化氢或过硫酸铵作预氧化剂,过量的预氧化剂煮沸数分钟即可除去,操作简便.铁矿石中大多数元素均不干扰测定,尤其是铜、钼、钒的允许量可达到5%(质量分数).测定结果相对标准偏差(RSD)小于0.2%.对两个铁矿石标准样品进行加标回收试验,得到加标回收率分别为99.6%和100.4%.%The determination method of total iron in iron ore using ascorbic acid as reducing titrant was development. The end point of titration was determined by full-automatic potential titrimeter. The titration acid medium and acidity, temperature, and potential titrimeter parameters were optimized.The influence of interference elements was tested. The results showed that titration of total iron could be carred out in the medium and acidity of dissolving iron ore samples without pH adjustment. The titration temperature range was 50 -80 ℃ . The hydrogen peroxide or ammonium persulfate was used as pre-oxidant. Since the excessive pre-oxidant could be removed by boiling for a few minutes, the operation was easy. The majority elements in iron ores did not intcrfcrc in the determination, especially the tolerant amount of copper, molybdenum and vanadium could be up to 5 % (mass fraction). The relative standard deviation (RSD) of determination results was less than 0.2 % , and the recoveries for two samples were 99. 6% and 100.4 % ,respectively.【期刊名称】《冶金分析》【年(卷),期】2011(031)005【总页数】4页(P63-66)【关键词】抗坏血酸;电位滴定;铁;铁矿石;预氧化;还原滴定【作者】戚淑芳;张杰;王莹;宁松【作者单位】鞍钢股份公司技术中心,辽宁鞍山114009;鞍钢股份公司技术中心,辽宁鞍山114009;鞍钢股份公司技术中心,辽宁鞍山114009;鞍钢股份公司技术中心,辽宁鞍山114009【正文语种】中文【中图分类】O655.29铁矿石中全铁的测定大多采用重铬酸钾滴定法,该法虽经多次改进,但无根本性变化。
科研细胞模型就足够寻找CRC药物作用靶点?看大牛蛋白质组学研究的新思路
科研细胞模型就足够寻找CRC药物作用靶点?看大牛蛋白质组学研究的新思路导读肿瘤细胞系是肿瘤学研究领域最常用的模型。
很多基于肿瘤细胞系的大型研究项目,如NCI-60、CCLE和GDSC,通常利用分子异质性的细胞系来鉴定标志物及后续的精准治疗。
但是细胞系是否能准确代表原发性肿瘤,仍具有争议。
基因组分析数据表明在很多肿瘤模型中,所建立的细胞系能在分子水平上体现原发性肿瘤的特征,但是转录组水平分析显示,肝细胞癌和结直肠癌细胞系模型的数据类似。
然而,乳腺癌的转录组数据则存在显著差异。
虽然已有很多针对肿瘤细胞系蛋白质组学分析的报道,但是,目前尚无比较细胞系与原发性肿瘤样本的大规模蛋白质组学研究的报道。
目前尚不清楚癌细胞系是否能在蛋白质组水平上代表原发性肿瘤,以及在体外,分子机理和蛋白质组学相关性程度有多深也是未知的。
本文对44例人结直肠癌(CRC)细胞系进行蛋白质组学和转录组学分析,这些细胞系前期已进行过基因组水平测定。
同时,综合且全面的针对44例细胞系样本及95例CRC肿瘤样本、60例正常组织样本进行分析,并进一步将细胞系蛋白基因组数据与药物作用敏感性继续整合,以评估不同的组学数据用于预测治疗反应的适用度。
论文ID原名:Colorectal cancer cell line proteomes are representative of primary tumors and predictdrug sensitivity译名:细胞模型就足够寻找CRC药物作用靶点?看大牛蛋白质组学研究的新思路期刊:GastroenterologyIF:18.392发表时间:2017年通信作者:Oliver M. Sieber通信作者单位:Systems Biology and Personalised Medicine Division, The Walter and ElizaHall Institute of Medial Research, Parkville, VIC 3052, Australia实验内容1、CRC细胞系蛋白质组学分析采用基于LC-MS/MS的shotgun蛋白质组学分析44例CRC细胞系,鉴定到10643条不同的肽段,组装成了7796个蛋白质。
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Analysis of CRC Methods and Potential Data Integrity ExploitsB. Maxwell, D.R. Thompson, G. Amerson, and L. JohnsonComputer Science and Computer Engineering DepartmentUniversity of Arkansas, Fayetteville, AR, U.S.A.AbstractCyclic redundancy checks (CRCs) are commonly used to detect errors from noise in networks. They also have been used to verify the integrity of files in a system to prevent tampering and suggested as a possible algorithm for manipulation detection codes. It has been known that a CRC will not detect all errors but with random noise it is unlikely. But a malevolent adversary can efficiently modify a file without changing its length to maintain the same CRC. In this paper, we present an efficient algorithm for modifying an individual byte of a file and other additional bytes to maintain the same CRC. A previous attack on CRC padded a file with additional bytes to maintain the same CRC. In this work, the original file length is maintained and it demonstrates that a CRC, although good for error detection, may not be the best choice for a hash function without additional modifications.1. IntroductionCyclic redundancy checks (CRCs) were originally used to detect errors in hardware and in communication links to detect bursts of errors [1]. But now they are being used to validate the integrity of data in software applications [2] and in the wired equivalent privacy (WEP) protocol for the 802.11 standard for wireless networks [3], because they appear to offer good error detection and are easy to implement. But, using a CRC for an integrity check instead of its intended use for detecting random errors can provide a false sense of security. In this paper, a technique is presented that can be used to modify an individual byte of a message and other additional bytes to maintain the same CRC as the original message while keeping the length of the message the same. It is known that padding a message with extra bytes can be used to maintain the same CRC but a method to maintain the same CRC with the same message length has not been found in the formal literature [4]. This is similar to the method found in [3] for modifying an existing message and its CRC so that the CRC remains valid.There are three common ways of implementing CRC. The first is using polynomial division, the second uses a pre-computed table to increase the speed of the algorithm, and the third is the direct lookup table or fast table method. These three methods are described briefly in the next section. Then, an algorithm will be described that reverses the CRC table method; illustrating that it is possible to find a sequence of bytes that will change the CRC to any chosen value. In other words, the CRC algorithm becomes useless in detecting that the message was changed, because the CRC will be the same although data integrity has been compromised.2. CRC methods2.1. Polynomial divisionComputing the CRC of a message uses polynomial division. The message is considered as a large binary number and the sender divides it by a predetermined polynomial divisor. This computation yields a remainder, which becomes the checksum of the message. This checksum is used to verify that the message has not been changed. The recipient of the message calculates the CRC using the same polynomial division and compares this checksum with the checksum associated with the original message.The actual implementation of computing checksums does not use the division scheme. It uses the notion of polynomial modulo-2 arithmetic. Binary numbers are added without carries. Another property is that addition and subtraction are equivalent to the exclusive-or (XOR) operation. For division to take place, the greatest magnitude of two numbers is determined by the number with the highest 1 bit position [5].CRC computation is polynomial division, but is equivalent to XORing particular values at shifted offsets. There is a divisor, which is called the generator polynomial. The message is divided by the generator polynomial. The quotient is irrelevant, but the remainder is relevant because it becomes the checksum. The width w of a polynomial is defined as N-1 for a polynomial of size N. There is always an assumed 1 at the beginning of the generator polynomial and is ignored in some of the algorithms. Before division, w zeros are appended to the message where N is the length of the polynomial. Thereare two ways to verify the CRC. First, the receiver of the message can compute the CRC and compare the checksumto the newly calculated CRC. Or the entire message including the appended checksum can be divided by the generator polynomial to determine if it equals zero [5].A problem arises quickly with any implementation of CRC because polynomial division is different than numerical division on processors. Also, processors don't have registers large enough to hold big dividends. So the CRC division is implemented using a register and shifting the message through it. Assume that the polynomial has width w and the division register also has width w. Therefore, the message will have w zeros appended to it before the CRC operation. The pseudo-code for this algorithm is shown in Fig. 1.Zero out CRC register.Append W zeros onto message.While more message is leftTest the top bit of the register,if equals 1thenShift message left 1 bitXOR polynomial with register and storecontents back in registerelseShift message left 1 bitregister now equals the remainder.Figure 1. Pseudo-code for Bit-Oriented MethodBy comparing this algorithm to the long division algorithm, it becomes clear that this algorithm is subtracting (XORing) shifted versions (powers) of the polynomial from the message. This is equivalent to the long division method. This method will be called the bit-oriented shift-register method in the rest of the paper.2.2. Table methodThe bit-oriented shift-register method is a good way to introduce the way the CRC algorithm works but is not efficient or practical for implementation. The algorithm must loop through each bit in a message, which can be very large, and is very difficult to code in most programming languages. The first improvement is to decrease the number of loops. The algorithm is modifiedto process bytes instead of bits. The following example will be using a 4-bit generator polynomial 1001 and take advantage of the fact that computers handle bits in groupsof bytes instead of a serial bit stream [6]. The register looks the same as in the bit-oriented shift-register method, except the register processes bytes instead of bits. Let the top 8 bits of the 32-bit register (byte 3) be:x7 x6 x5 x4 x3 x2 x1 x0and let the top 8 bits of the polynomial be: p7 p6 p5 p4 p3 p2 p1 p0On the next iteration of the algorithm, x7 is popped off and examined to determine if the next 8 bits (now the top 8 bits) should be XORed with the generator polynomial. If x7 is a one, it will be XORed, otherwise it will not. A better way to look at this is to always XOR the polynomial with the top 8 bits of the register; however the polynomial is multiplied by x7 first. If x7 is 0, then you'll be XORing the top 8 bits with 0, which doesn't change the top 8 bits. However, if it's a one, you'll do the XOR as expected [7].x6 x5 x4 x3 x2 x1 x0 ??+ x7 *(p7 p6 p5 p4 p3 p2 p1 p0) (+ is XOR) The new top bit will control what happens in the next iteration of the loop; that value is x6 + x7*p7. The important thing is that the information required in calculating the new top bit was present in the top two bits of the original top byte. Taking this a step further, the next top bit could be calculated from the top three bits. In general, the value of the top bit in the register after k iterations can be calculated from the top k bits. Here's a clear example of how the method executes (the bold value is the second byte). The polynomial 1001 is used in the example below and it is assumed that the most significant bit in the register was a one.0100010011101000 Register1001 XOR this0000 . XOR this. 1001 . . XOR this0000 . . . XORthis0000 . . . XOR this0000 . . . XOR this1001 . . . XOR this0000 . . . XOR this--------------------------1111001010101000What if instead of all doing all these individual XOR calculations, that a single value could be pre-calculated and then just perform one XOR and get the same result? Well this is exactly what the table method strives to achieve. It is able to look at the top byte and determine what the individual XORs should be and therefore create the individual value. The example above could be done in one XOR calculation as shown below:0100010011101000 Register10110110010 XOR this--------------------------1111001010101000With the information shown in this example, pseudo code can be derived. The new algorithm for calculating the CRC is shown in Fig. 2.While(more bytes to process in message )BeginCalculate the control byte from the top byteof the registerSum all the polynomials at various offsetsthat are to be XORed into the register inaccordance with the control byteShift the register left by one byte, readinga new message byte into the rightmost byteof the registerXOR the summed polynomials to the registerEndFigure 2 Pseudo-code for byte-oriented methodThe algorithm has been changed a little, but the improvement isn't really there yet, because the algorithmis still having to look at each individual bit for summing the polynomials at different levels. However, the calculation of summed polynomials can be pre-computed and placed in a lookup table. This leads to the algorithm shown in Fig. 3.While( more bytes to process in augmented message )BeginTop = top_byte(Register);Register = (Register << 8)| next_augmessage_byte;Register = Register XORprecomputed_table[Top]EndFigure 3 Table methodThis new algorithm only requires a shift, OR, and XOR for each byte. Fig. 4 is an actual implementation of this in C.theRegister = 0;while(messagelength-->0){byte temp = (theRegister >> 24) & 0xFF;theRegister = (theRegister << 8) | *augMessagePtr++;theRegister ^= table[temp];}Figure 4 Table method in C2.3. Fast table methodThis algorithm in section 2.2 is referred to as the table algorithm. The algorithm is efficient. However, there is some room for improvement. Adding on those zeros at the end seems is inefficient. Notice that w/4 of the zero bytes are pulled in, however they have no effect in this algorithm. The zeros do not change the values after an XOR. The only purpose of the zero bytes is to push the real bytes through. In addition, there is some wasted time initially because it takes four iterations to get the real data started processing. The first four iterations do not do anything, because they are just zeros. Even if it's not a zero, all that would happen is to have multiple XORs with different offsets of a constant value. The initial value of the register would just be what is left after four iterations of whatever the default value is for the register. If it started with a zero the final value would be a zero also. This leads to an improved algorithm called the direct table algorithm shown in Fig. 5.While( more bytes to process in message )BeginTop = top_byte(Register);Register = (Register << 8)| next_augmessage_byte;Byte tempValue = Top XOR message byte justORed into the register;Register = Register XORprecomputed_table[tempValue];EndFigure 5 Fast table algorithm pseudo-codeAn implementation of this CRC32 algorithm in C is shown in Fig. 6.register = 0;while(messagelength-->0){theRegister = (theRegister << 8) ^table[(theRegister >> 24) ^ *messageptr++];}Figure 6 Fast table algorithm in CMost modern implementations use this version, because it is faster than the two previous versions shown. It is called the direct lookup table or fast table method. According to [Barr03], the bit by bit CRC algorithm requires 185 instructions per byte of message data, whereas the faster byte by byte algorithm only requires 36 instructions per byte of message data.3. How to reverse engineer the CRCA CRC-16 will be used to illustrate the method to reverse engineer the CRC. Using CRC-16, there are a possibility of 216 = 65,536 different codes. One way to create messages that have the same CRC but different data is to change the data, and then find a sequence of bytes that will force the CRC to be equal to the CRC of the original data. It requires n bits to change a CRC to any value, where n is the size of the generator polynomial. So to change a CRC-16 value, two bytes or 16 bits are required. The basic steps to change a message but maintaining the same CRC are as follows [4]:1. Calculate the CRC of the original message. Save this value as (CRC_Before).2. Calculate the CRC of the new message. Save this value as (CRC_New)3. Reverse the CRC algorithm using CRC_Before & CRC_New to calculate the 2 bytes that will change CRC_New to CRC_Before.Using this method, two extra bytes will have to be appended to the end of the new message to make the new message have the same CRC as the old message. If only part of the message is different between the original and new message and there are two bytes that can be used that are before the end of the message, they can be modified to maintain the same CRC with the same message length. Below is an example on how to modify bytes to maintain the same CRC.3.1. PaddingCRC-16 will be used to illustrate how to calculate the modified bytes. To calculate the two bytes that change CRC_New to CRC_Before, suppose the two unknown bytes are X and Y. Using the direct CRC table method previously described, the bytes X and Y are processed with the CRC starting at CRC_New.3.1.1. Definitionsa = starting CRC (CRC_New) (16 bits)b = value from CRC table (16 bits)c = value from CRC table (16 bits)d = ending CRC (CRC_Before) (16 bits)aH = Most Significant Byte of aaL = Least Significant Byte of aThe same format is used for b, c, and d.table = the precomputed CRC lookup table asdescribed in the table CRC algorithmsindex = an index into the table, that istable[index] = precomputed valuea << 8 means a is shifted left by 8 bitsCurly braces are used to show the most significant bit on the left (a1) and the least significant bit on the right.a = {aH} {aL}3.1.2. MethodRemember the direct table lookup method for calculating CRCs is as follows:n = number of bytes in the CRCIterate for nStep 1: top = MSB(CRC)Step 2: index = top XOR message[i] Step 3: CRC = ( CRC << 8 ) XOR table[index]Now lets process two unknown bytes X and Y using the direct table method described above. The three steps of the method are done for each byte.Step 1: top = MSB(a) = aHStep 2: index = top XOR X = a1 XOR X = index(b) table[index] = bStep 3: CRC = (a<<8) XOR table[index] ={aL XOR bH} {00 XOR bL}Step 4: top = MSB(CRC) = (aL XOR bH)Step 5: index = top XOR Y = (aL XOR bH) XOR Y =(aL XOR bH XOR Y) = index(c)table[index] = cStep 6: CRC = {bL XOR cH} {00 XOR cL} = dNow solve the equations backwards from step 6 to step 1. From Step 6: cL = dLbL = dH XOR cHFrom Step 5: Y = aL XOR bH XOR index(c)From Step 2: X = aH XOR index(b)So d0 is the LSB(CRC_Before), and cL = dH. Now, index(c) and cH can be found by searching the table looking for the entry whose LSB = cL. Next, bL is found by solving the second equation. So, bL = dH XOR cH, which are both known. Again to find bH and index(b) are found by searching the table until the entry that has its LSB = bL. Now, Y is found by the equation Y = aL XOR bH XOR index(c). Finally, X is found by X = aH XOR index(b). So now that X and Y are known, if a message has a CRC = CRC_New and the bytes X and Y are added to the end of the message, the CRC will now be the same as CRC_Before.To be able to do the algorithm described above, the exact implementation of the CRC must be known. Some CRC implementations initialize the CRC register with a value other than zero. The polynomial used by the CRC must also be known. If everything is known about the algorithm, then the data of the message can be changed and still produce the same CRC value as long as there are n bytes after the significant data that can be set to correct the CRC value, where n is the width of the polynomial.3.2. Maintaining the same message lengthThe location of the modifier bytes does not have to be at the end of the message. The bytes can be placed anywhere after the last position where the message was changed and the end of the file. Therefore, if the modifier bytes are placed before the end of the file, the file length is maintained. In order to place the modifier bytes at a particular place in the message, the method shown in Fig.7 is performed.1. Calculate the CRC up to the position where the data will be changed, save as CRC_A.2. Continue calculating until the position where the modifier bytes will be placed, save as CRC_B.3. Start with CRC_A and calculate the CRC of the changed bytes up to but not including the position of the modifier bytes, save as CRC_C.4. Now calculate the modifier bytes as described before with the original CRC being CRC_B and the new CRC being CRC_C.Figure 7 Method to maintain message length Since many integrity and authentication methods verify that the length of the sent message is equal to the length of the received message, the method described above still permits an attacker to modify a specified byte and place the modifier bytes somewhere in the message to maintain the same CRC and message length. The method above was implemented and can easily change a specified byte and put the additional modifier bytes between the modified byte and the end of the file. The implemented code uses the popular CRC-32.4. SignificanceSo why is the ability to create different sets of data with the same CRC significant? Because an attacker could intentionally alter data and it would be undetectable if a CRC is used as the detection method. Many programs use a CRC to check for errors such as bios updates, pkzip (Compression Software), png (Portable Network Graphics) and other software available on the Internet. Also, CRC is commonly used for error detection in communication protocols, such as Ethernet and ATM [8]. The CRC is popular for hardware implementations, because it has good random error detection properties and is easily implemented [2]. An attacker could edit a program and then fix the CRC so it appears to be a valid archive and use it to spread computer viruses or Trojan programs that allow the attacker to break into computer systems. Suppose a bank uses a CRC to verify the integrity of their database transaction and the transaction below is executed.UPDATE Account SET Balance='100' WHERE Name=Customer AND ...;Suppose someone changes the 100 to 100000 as shown below, and then fixes the CRC.UPDATE Account SET Balance='100000' WHERE Name=Customer AND ...XY;As long as the extra bytes X and Y can be added to the message, the CRC will be the same. Thus, the use of a CRC with a known generator polynomial and initial value in the register is not suggested when an adversary can intercept and alter the data.5. ConclusionIt has been shown that cyclic redundancy checks with known generator polynomials should not be used to verifythe integrity when someone can alter the data between its source and destination and can obtain the original CRC values. Two solutions are to keep the polynomial used to create the CRC secret [9] or to incorporate some secret value into the CRC that is unknown to the adversary as in HMAC-SHA1 or HMAC-MD5 [10]. A hash function such as MD5 or SHA1 may be a better choice to verify integrity of data. One-way hashes are mathematical functions that produce a relatively unique value for a particular message. So unlike the CRC algorithm, the one-way hash algorithms are designed such they cannot be mathematically reversed. A brute force method would be needed to attempt to attack these hashes, so they are currently the better choice to ensure data integrity unless a secret value is incorporated into the CRC. Many Unix archives are now providing MD5 sums to validate the integrity of archives downloaded off of the Internet. But currently Windows machines do not include an MD5 program.6. AcknowledgmentsThe authors would like to thank Logical Dynamics, Inc.,for the opportunity to collaborate on the message authentication research. The material is based upon work supported by the National Science Foundation under Grant No. 0090596.7. References[1] S. Lin, D. J. Costello, Jr., Error Control Coding: Fundamentals and Applications, Prentice Hall, 1982.[2] D. C. Feldmeier, “Fast Software Implementation of Error Detection Codes”, IEEE/ACM Transactions on Networking, vol. 3, no. 6, December 1995, pp. 640-651.[3] N. Borisov, I. Goldberg, and D. Wagner, “Intercepting mobile communications: the insecurity of 802.11”, in Proceedings of the Seventh International Conference on Mobile Computing and Networking, pp. 180-189, Rome, Italy, 2001.[4] Anarchriz (anarchriz@), “CRC and howto reverse it,” April 30, 1999, http://its.mine.nu/html/re/essays/CRC.html.[5] R. Williams, Rocksoft Pty Ltd of Austrailia, “A Painless Guide To CRC Error Detection Algorithms”, August 19, 1993, ftp:///papers/crc_v3.txt.[6] Dilip V. Sarwate, “Computation of Cyclic Redundancy Checks via Table Look-Up”, Communications of the ACM, August 1988, vol. 31, no. 8, pp. 1008-1013.[7] T.V. Ramabadran, S.S. Gaitonde, “A Tutorial on CRC Computations”, Micro, IEEE, vol. 8, no. 4 , Aug 1988, pp. 62-75.[8] T. Henriksson, D. Liu, “Implementation of Fast CRC Calculation”, in Proceedings of Design Automation Conference, 2003, pp. 563-564. [9] H. Krawczyk, “LFSR-based hashing and authentication,” in Proceedings of CRYPTO ’94, Lecture notes in Computer Science, vol. 839, Aug. 1994, New York: Springer-Verlag, pp. 129-139.[10] H. Krawczyk M. Bellare, and R. Canetti, “HMAC: keyed-hashing for message authentication,” RFC 2104, Feb. 1997.。