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Chk1反义寡核苷酸影响胶质瘤放疗敏感性的研究【摘要】目的：观察转染chk1反义寡核苷酸(ason)后，对照射后u251细胞株中chk1表达、细胞周期及细胞凋亡的影响。
方法：采用脂质体转染法，chkl的正义、反义寡核苷酸对u251细胞株进行chkl转染。
以放射线照射后，测定其细胞周期和凋亡率变化，比较chkl转染正义链和反义链对细胞放射敏感性的不同。
用western blot法检测chk1蛋白，real time pcr检测chk1 mrna表达。
结果：转染chk1反义寡核苷酸后，能明显下调chk1蛋白和mrna的表达，显著增强放射线诱导的肿瘤细胞凋亡，并消除细胞周期阻滞。
结论：反义核酸技术灭活chk1基因显著增强放疗诱导的u251细胞凋亡，为增敏胶质瘤的放射治疗提供了理论依据。
【关键词】胶质瘤；细胞周期检测点激酶；反义寡核苷酸；放射敏感性transfection of chk1 antisense oligonucleotide to glioma increases the apoptotic sensitivity to irradiation/li yong，lai run-long， tan dian-hui，et al.//medical innovation of china，2012，9(13)：003-005【abstract】 objective：effect on expression of chk1 and changes of cell cycle after radiation in u251 cell line with antisense oligodeoxynucleotide (ason) wereobserved.methods：the u251 cell line was transfected with chk1 sense and antisense chain-lipofectamine plus complex. thenirradiated it and measured the changes of the cell cycle and apoptosis in order to compare the diference between the chk1 sense and antisense chain. the expression of chk1 was measured by western blot， and chk1 mrna was measured by real time pcr.results：the expression of chk1 protein and mrna markedly decreased after ransfecting u251 cell line with chk1 antisense oligonucleotide.transfecting u251 cell line with chk1 antisense oligonucleotide could increase apoptosis significantly and decrease cell cycle arrest markedly induced by irradiation.conclusion：the sensitivity to apoptosis is excessively increased after treated with irradiation by transfection of chk1 antisense oligonucleotide to glioma. this can be the theoretical basis for increasing the apoptotic sensitivity to irradiation of glioma.【key words】 glioma; checkpoint kinase; antisense oligonucleotide; irradiationfirst-author’s address：first affiliated hospital of shantou university medical college，shantou 515041，china doi：10.3969/j.issn.1674-4985.2012.13.002目前研究认为，细胞周期检测点信号传导通路主要由atm/atr-chk1/2-cdc25a、cdc25b、cdc25c-cdk轴组成[1]，chk1是细胞周期检测点信号中最关键的效应蛋白激酶[2]。
细胞周期调控和DNA损伤的关系及其作用机制细胞是构成生命的基本单元，它们通过不断的分裂来增加数量或完成重要的功能。
然而，细胞分裂过程中在DNA复制或配对时可能发生错误，或受到外部因素的损伤，导致DNA序列改变或断裂。
这些DNA损伤若不得到及时的修复，会严重影响细胞的稳定性、生长和分裂，造成疾病甚至致死。
因此，细胞如何及时发现和修复DNA损伤，并保障细胞正常的周期调节，是细胞生物学的重要课题之一。
1. 细胞周期调控基础知识在介绍细胞周期调控和DNA损伤关系及其作用机制之前，先介绍一些基本的知识：细胞周期包括G1、S、G2和M四个不同的阶段，每个阶段具有特定的比例和作用。
在G1期，细胞通过产生新蛋白质和RNA等以准备DNA的DNA复制，同时还需要完成各种新细胞的物质合成和释放。
在S期，细胞进行DNA复制的过程，确保每个新细胞都拥有完整的复制物。
在G2期，细胞继续生长并合成必要的蛋白质和RNA，为下一个细胞分裂做好准备。
最后，在M期，细胞分裂成两个同样大小的细胞，即产生了新的细胞。
细胞周期调控是一种高级形式的基因表达调控，它通过多个信号途径和分子机制来确保细胞在适当的时刻进入下一个周期，并避免过度分裂或紊乱。
细胞周期调控可以分为内部和外部两种调节方式。
内部调控是由细胞内信号传导通路和蛋白激酶等分子机制来控制的。
外部调控则是受细胞外的生长因素和讯息（比如细胞凋亡）的影响，从而调节细胞周期的进程。
2. DNA损伤的类型和影响DNA损伤是指DNA序列的改变或断裂，这种损伤可以发生在DNA分子的位置（单各核苷酸变异、缺失或增加）或结构（单链或双链断裂）。
环境因素或内源性生理原因都可能会导致DNA损伤，包括直接暴露于毒物、辐射和化学物质等。
另外，DNA损伤可能由一些生理性过程引起，包括细胞呼吸和新陈代谢等。
DNA损伤如果没有被及时修复，会导致染色体缺失、嵌合、倒位等恶性事件。
如果细胞无法检测到或修复这些问题，就会引发DNA复制错误、增加癌症的风险，以及导致细胞凋亡或死亡。
细胞信号通路在DNA损伤修复中的作用DNA是细胞内生物传递遗传信息的载体，一个组成人体的细胞中包含有数十亿的基因组成的DNA链。
然而，DNA也会受到各种内外因素的损伤，例如辐射、化学物质和紫外线等，这些因素能够引起DNA单链或双链断裂甚至基因重组。
一般情况下，细胞会根据不同的损伤类型启动不同的DNA修复机制，同时与之相关的是细胞内修复机制的信号传递通路网络。
本文主要探讨细胞信号通路在DNA损伤修复中的作用。
DNA双链断裂（DSB）和细胞凋亡DNA双链断裂是导致基因重组和染色体畸变的主要原因之一。
DSB的修复受到细胞信号通路的控制，通过激活DNA损伤应答通路来维持序列完整性和稳定性。
在DNA断裂的边缘，随之产生的一些因子和酶会导致其存在两个末端，这为细胞感应DNA损伤的存在提供了典型示例。
有关因子集合指向这些端点，以维持其不会被无法可逆性地瓦解或递减。
这个过程中主要由ATM、CHK2和p53这样的信号传导因子完成。
这里ATM和CHK2是通过减少DNA双链断裂恢复功能、改变染色体构象、促进细胞周期推进等来注释其基本功能的。
在ATM和CHK2的作用下，DNA才能够得到有效的DNA修复。
在DSB的处理中，特别值得注意的是细胞凋亡的作用。
细胞凋亡是一种程序性的细胞死亡，主要有这些方面：膜破坏、细胞核碎片的形成、色素颗粒的聚集和具体的凋亡程序等。
在DSB程序修复过程中，细胞凋亡是一个重要的负反馈控制点，当DNA损伤太严重而细胞无法进行修复时，将启动细胞凋亡程序，以此保证它们不会进一步损伤。
而如果在细胞进行凋亡之前，修复已经完成了，细胞将恢复其正常的功能。
ATM/ATR信号通路ATM和ATR信号通路是体细胞中最知名的两个DNA损伤敏感信号通路。
这两个通路是协同工作的，ATR上游是ATM和DNA-PKcs，最初被使用于DNA双链断裂的处理。
ATM和ATR的活性可以被许多不同的DNA损伤诱导，但他们对于不同损伤源的选择性还不清楚。
BRCA1蛋白的结构与功能研究作者：谌星武丹丹原荣荣来源：《绿色科技》2013年第05期摘要：指出了BRCA1蛋白是一种结构复杂，功能多样的大分子，有多种结合位点，两端的RING和BRCT结构域能与多种相关蛋白结合。
它在核内产生，但是当DNA损伤时，会发生出核现象，并且和多种磷酸化蛋白共同作用维持基因组的完整性。
发现在多种肿瘤细胞中BRCA1的表达有变化或有突变，至于这些变化是否引起肿瘤尚不确定，因此对BRCA1蛋白功能的研究将有利于阐明肿瘤的发生机理。
回顾和总结了BRCA1在DNA损伤修复、细胞周期、转录、细胞凋亡等细胞过程中的作用。
关键词：BRCA1；DNA损伤修复；细胞周期；转录；细胞凋亡1引言BRCA1是一种肿瘤抑制基因，它和多种肿瘤抑制基因共同作用，抑制肿瘤的生长。
肿瘤的发生是因为局部组织的细胞在基因水平上失去了对其生长的正常调控，导致细胞异常生长，一旦控制细胞增殖及生长的基因（如生长因子）发生突变或过量表达，肿瘤疾病极有可能发生。
早期研究发现，BRCA1是乳腺癌易感基因，BRCA1蛋白和多种相关蛋白共同作用维持基因组的稳定，并且受到转录控制、磷酸化和蛋白相互作用等机制的调节，最近研究表明，其蛋白功能可能还受到BRCA1亚细胞分布的功能调节[1]。
而事实证明BRCA1蛋白在DNA损伤修复、细胞周期、基因转录调节、细胞凋亡等机制中发挥着重大的生物学功能。
2BRCA1基因及蛋白的结构2.1BRCA1基因的结构BRCA1基因定位于人类染色体17q21，由24个外显子组成，包括22个编码外显子，2个非编码外显子，全长约为100Kb，转录产物mRNA为7.8Kb[2]，其中第11外显子为核心外显子，编码60%以上的氨基酸序列。
该基因有两个不同的启动子promoter1和promoter2，，共同调节BRCA1的转录活性，且两者活性保持一定比例，并以promoter1为主[3]。
2.2BRCA1蛋白的结构BRCA1蛋白是由1863个氨基酸组成的生物大分子，其N端有一个RING结构域，C端有两个纵串排列的BRCT结构，中部含有核内信号定位区（NLS1和NLS2），参与蛋白质的核内转运[4]，与其他任何已知蛋白没有同源性[5]。
ATMAtaxia telangiectasia mutated (ATM) is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks. It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Several of these targets, including p53, CHK2 and H2AX are tumor suppressors.The protein is named for the disorder Ataxia telangiectasia caused by mutations of ATM.[1]Contents1 Introduction2 Structure3 Function4 Regulation5 Role in cancer6 Interactions7 See also8 References9 Further reading10 External linksIntroduction[edit]Throughout the cell cycle the DNA is monitored for damage. Damages result from errors during replication, by-products of metabolism, general toxic drugs or ionizing radiation. The cell cycle has different DNA damage checkpoints, which inhibit the next or maintain the current cell cycle step. There are two main checkpoints, the G1/S and the G2/M, during the cell cycle, which preserve correct progression. ATM plays a role in cell cycle delay after DNA damage, especially after double-strand breaks (DSBs).[2] ATM together with NBS1 act as primary DSB sensor proteins. Different mediators, such as Mre11 and MDC1, acquire post-translational modifications which are generated by the sensor proteins. These modified mediator proteins then amplify the DNA damage signal, and transduce the signals to downstream effectors such as CHK2 and p53.Structure[edit]The ATM gene codes for a 350 kDa protein consisting of 3056 amino acids.[3] ATM belongs to the superfamily of Phosphatidylinositol 3-kinase-related kinases (PIKKs). The PIKK superfamily comprises six Ser/Thr-protein kinases that show a sequence similarity to phosphatidylinositol 3-kinases (PI3Ks). This protein kinase family includes amongst others ATR (ATM- and RAD3-related), DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and mTOR (mammalian target of rapamycin). Characteristic for ATM are five domains. These are from N-Terminus to C-Terminus the HEAT repeat domain, the FRAP-ATM-TRRAP (FAT) domain, the kinase domain (KD), the PIKK-regulatory domain (PRD) and the FAT-C-terminal (FATC) domain. TheHEAT repeats directly bind to the C-terminus of NBS1. The FAT domain interacts with ATM's kinase domain to stabilize the C-terminus region of ATM itself. The KD domain resumes kinase activity, while the PRD and the FATC domain regulate it. Although no structure for ATM has been solved, the overall shape of ATM is very similar to DNA-PKcs and is composed of a head and a long arm that is thought to wrap around double-stranded DNA after a conformational change. The entire N-terminal domain together with the FAT domain are predicted to adopt an α-helical structure, which was found by sequence analysis. T his α-helical structure is believed to form a tertiary structure, which has a curved, tubular shape present for example in the Huntingtin protein, which also contains HEAT repeats. FATC is the C-terminal domain with a length of about 30 amino acids. It is highly conserved and consists of an α-helix followed by a sharp turn, which is stabilized by a disulfide bond.[4]Function[edit]A complex of the three proteins Mre11, RAD50 and NBS1 (Xrs2 in yeast), called the MRN complex in humans, recruits ATM to double strand breaks (DSBs) and holds the two ends together. ATM directly interacts with the NBS1 subunit and phosphorylates the histone variant H2AX on Ser139.[5] This phosphorylation generates binding sites for adaptor proteins with a BRCT domain. These adaptor proteins then recruit different factors including the effector protein kinase CHK2 and the tumor suppressor p53. The ATM-mediated DNA damage response consists of a rapid and a delayed response. The effector kinase CHK2 is phosphorylated and thereby activated by ATM. Activated CHK2 phosphorylates phosphatase CDC25A, which is degraded thereupon and can no longer dephosphorylate CDK2-Cyclin, resulting in cell-cycle arrest. If the DSB can not be repaired during this rapid response, ATM additionally phosphorylates MDM2 and p53 at Ser15.[6] p53 is also phosphorylated by the effector kinase CHK2. These phosphorylation events lead to stabilization and activation of p53 and subsequent transcription of numerous p53 target genes including Cdk inhibitor p21 which lead to long-term cell-cycle arrest or even apoptosis.[7]ATM-mediated two-step response to DNA double strand breaks. In the rapid response activated ATM phosphorylates effector kinase CHK2 which phophphorylates CDC25A, targeting it for ubiquitination and degradation. Therefore phosphorylated CDK2-Cyclin accumulates and progression through the cell cycle is blocked. In the delayed response ATM phosphorylates the inhibitor of p53, MDM2, and p53, which is also phosphorylated by Chk2. The resulting activat ion and stabilization of p53 leads to an increased expression of Cdk inhibitor p21, which further helps to keep Cdk activity low and to maintain long-term cell cycle arrest.[7]The protein kinase ATM may also be involved in mitochondrial homeostasis, as a regulator of mitochondrial autophagy (mitophagy) whereby old, dysfunctional mitochondria are removed.[8] Regulation[edit]A functional MRN complex is required for ATM activation after double strand breaks (DSBs). The complex functions upstream of ATM in mammalian cells and induces conformational changes that facilitate an increase in the affinity of ATM towards its substrates, such as CHK2 and p53.[2] Inactive ATM is present in the cells without DSBs as dimers or multimers. Upon DNA damage, ATMautophosphorylates on residue Ser1981. This phosphorylation provokes dissociation of ATM dimers, which is followed by the release of active ATM monomers.[9] Further autophosphorylation (of residues Ser367 and Ser1893) is required for normal activity of the ATM kinase. Activation of ATM by the MRN complex is preceded by at least two steps, i.e. recruitment of ATM to DSB ends by the mediator of DNA damage checkpoint protein 1 (MDC1) which binds to MRE11, and the subsequent stimulation of kinase activity with the NBS1 C-terminus. The three domains FAT, PRD and F ATC are all involved in regulating the activity of the KD kinase domain. The FAT domain interacts with ATM's KD domain to stabilize the C-terminus region of ATM itself. The FATC domain is critical for kinase activity and highly sensitive to mutagenesis. It mediates protein-protein interaction for example with the histone acetyltransferase TIP60 (HIV-1 Tat interacting protein 60 kDa), which acetylates ATM on residue Lys3016. The acetylation occurs in the C-terminal half of the PRD domain and is required for ATM kinase activation and for its conversion into monomers. While deletion of the entire PRD domain abolishes the kinase activity of ATM, specific small deletions show no effect.[4]Role in cancer[edit]Ataxia telangiectasia (AT) is a rare human disease characterized by cerebellar degeneration, extreme cellular sensitivity to radiation and a predisposition to cancer. All AT patients contain mutations in the ATM gene (ATM). Most other AT-like disorders are defective in genes encoding the MRN protein complex. One feature of the ATM protein is its rapid increase in kinase activity immediately following double-strand break formation.[10][11] The phenotypic manifestation of AT is due to the broad range of substrates for the ATM kinase, involving DNA repair, apoptosis, G1/S, intra-S checkpoint and G2/M checkpoints, gene regulation, translation initiation, and telomere maintenance.[12] Therefore a defect in ATM has severe consequences in repairing certain types of damage to DNA, and cancer may result from improper repair. AT patients have an increased risk for breast cancer that has been ascribed to ATM's interaction and phosphorylation of BRCA1 and its associated proteins following DNA damage.[13] Certain kinds of leukemias and lymphomas, including Mantle cell lymphoma, T-ALL, atypical B cell chronic lymphocytic leukemia, and T-PLL are also associated with ATM defects.[14]Interactions[edit]Ataxia telangiectasia mutated has been shown to interact with RAD17,[15][16] RBBP8,[15][17] RAD51,[18] DNA-PKcs,[15][19] RRM2B,[20] FANCD2,[21][22] Nibrin,[15][23] TERF1,[24] BRCA1,[13][15][23][25][26][27][28] Abl gene,[18][24][29] TP53BP1,[30][31] MRE11A,[15][23] P53,[15][32][33][34][35] Bloom syndrome protein,[23][36] SMC1A[37] and RHEB.[38]199条建筑设计知识1. 公共建筑通常以交通、使用、辅助三种空间组成2. 美国著名建筑师沙利文提出的名言‘形式由功能而来’3. 密斯.凡.德.罗设计的巴塞罗那博览会德国馆采用的是‘自由灵活的空间组合’开创了流动空间的新概念4. 美国纽约赖特设计的古根海姆美术馆的展厅空间布置采用形式是串联式5. 电影放映院不需采光6. 点式住宅可设天井或平面凹凸布置可增加外墙面，有利于每层户数较多时的采光和通风7. 对结构形式有规定性的有大小和容量、物理环境、形状的规定性8. 功能与流线分析是现代建筑设计最常用的手段9. 垂直方向高的建筑需要考虑透视变形的矫正10. 橙色是暖色，而紫色含有蓝色的成分，所以偏冷；青色比黄色冷、红色比黄色暖、蓝色比绿色冷11. 同样大小冷色调较暖色调给人的感觉要大12. 同样距离，暖色较冷色给人以靠近感13. 为保持室内空间稳定感，房间的低处宜采用低明度色彩14. 冷色调给人以幽雅宁静的气氛15. 色相、明度、彩度是色彩的三要素；三元色为红、黄、蓝16. 尺度的概念是建筑物整体或局部给人的视角印象大小和其实际大小的关系17. 美的比例，必然正确的体现材料的力学特征18. 不同文化形成独特的比例形式19. 西方古典建筑高度与开间的比例，愈高大愈狭长，愈低矮愈宽阔20. ‘稳定’所涉及的要素是上与下之间的相对轻重关系的处理21. 人眼观赏规律H 18°～45°局部、细部2H 18°～27°整体3H ＜18°整体及环境22. 黄金分隔比例为1：1.61823. 通风屋面只能隔离太阳辐射不能保温，适宜于南方24. 总图布置要因地制宜，建筑物与周围环境之间关系紧凑，节约因地；适当处理个体与群体，空间与体形，绿化和小品的关系；合理解决采光、通风、朝向、交通与人流的组织25. 热水系统舒适稳定适用于居住建筑和托幼蒸汽系统加热快，适用于间歇采暖建筑如会堂、剧场26. 渐变具有韵律感27. 要使一座建筑显得富有活力，形式生动，在构图中应采用对比的手法对比的手法有轴线对比、体量对比、方向对比、虚实对比、色彩对比28. 要使柱子看起来显得细一些，可以采用暗色和冷色29. 巴西国会大厅在体型组合中采用了对比与协调的手法30. 展览建筑应使用穿套式的空间组合形式31. 室外空间的构成，主要依赖于建筑和建筑群体组合32. 在意大利威尼斯的圣马可广场的布局中，采用了强调了各种空间之间的对比33. 当坡地坡度较缓时，应采用平行等高线布置34. 建筑的有效面积=建筑面积-结构面积35. 加大开窗面积的方法来解决采光和通风问题较易办到36. 中国古代木结构大致可分为抬梁式、穿斗式和井干式三种37. 建筑构图原理的基本范畴有主从与重点、对比与呼应、均衡与稳定、节奏与韵律和比例与尺度38. 建筑构图的基本规律是多样统一39. 超过8层的建筑中，电梯就成为主要的交通工具了40. 建筑的模数分为基本模数、扩大模数和分模数41. 建筑楼梯梯段的最大坡度不宜超过38°42. 住宅起居室、卧室、厨房应直接采光，窗地比为1/7，其他为1/1243. 住宅套内楼梯梯段的最小净宽两边墙的0.9M，一边临空的0.75M住宅室内楼梯踏步宽不应小于0.22M，踏步高度不应小大0.20M44. 住宅底层严禁布置火灾危险性甲乙类物质的商店，不应布置产生噪声的娱乐场所45. 地下室、贮藏室等房间的最低净高不应低于2.0米46. 室内坡道水平投影长度超过15米时，宜设休息平台47. 外墙内保温所占面积不计入使用面积烟道、风道、管道井不计入使用面积阳台面积不计入使用面积壁柜应计入使用面积48. 旋转楼梯两级的平面角度不大于10度，且每级离内侧扶手中心0.25处的踏步宽度要大于0.22米49. 两个安全出口之间的净距不应小于5米50. 楼梯正面门扇开足时宜保持0.6米平台净宽，侧墙门口距踏步不宜小于0.4米，其门扇开足时不应减少梯段的净宽35. 加大开窗面积的方法来解决采光和通风问题较易办到36. 中国古代木结构大致可分为抬梁式、穿斗式和井干式三种37. 建筑构图原理的基本范畴有主从与重点、对比与呼应、均衡与稳定、节奏与韵律和比例与尺度38. 建筑构图的基本规律是多样统一39. 超过8层的建筑中，电梯就成为主要的交通工具了40. 建筑的模数分为基本模数、扩大模数和分模数41. 建筑楼梯梯段的最大坡度不宜超过38°42. 住宅起居室、卧室、厨房应直接采光，窗地比为1/7，其他为1/1243. 住宅套内楼梯梯段的最小净宽两边墙的0.9M，一边临空的0.75M住宅室内楼梯踏步宽不应小于0.22M，踏步高度不应小大0.20M44. 住宅底层严禁布置火灾危险性甲乙类物质的商店，不应布置产生噪声的娱乐场所45. 地下室、贮藏室等房间的最低净高不应低于2.0米46. 室内坡道水平投影长度超过15米时，宜设休息平台47. 外墙内保温所占面积不计入使用面积烟道、风道、管道井不计入使用面积阳台面积不计入使用面积壁柜应计入使用面积48. 旋转楼梯两级的平面角度不大于10度，且每级离内侧扶手中心0.25处的踏步宽度要大于0.22米49. 两个安全出口之间的净距不应小于5米50. 楼梯正面门扇开足时宜保持0.6米平台净宽，侧墙门口距踏步不宜小于0.4米，其门扇开足时不应减少梯段的净宽35. 加大开窗面积的方法来解决采光和通风问题较易办到36. 中国古代木结构大致可分为抬梁式、穿斗式和井干式三种37. 建筑构图原理的基本范畴有主从与重点、对比与呼应、均衡与稳定、节奏与韵律和比例与尺度38. 建筑构图的基本规律是多样统一39. 超过8层的建筑中，电梯就成为主要的交通工具了40. 建筑的模数分为基本模数、扩大模数和分模数41. 建筑楼梯梯段的最大坡度不宜超过38°42. 住宅起居室、卧室、厨房应直接采光，窗地比为1/7，其他为1/1243. 住宅套内楼梯梯段的最小净宽两边墙的0.9M，一边临空的0.75M住宅室内楼梯踏步宽不应小于0.22M，踏步高度不应小大0.20M44. 住宅底层严禁布置火灾危险性甲乙类物质的商店，不应布置产生噪声的娱乐场所45. 地下室、贮藏室等房间的最低净高不应低于2.0米46. 室内坡道水平投影长度超过15米时，宜设休息平台47. 外墙内保温所占面积不计入使用面积烟道、风道、管道井不计入使用面积阳台面积不计入使用面积壁柜应计入使用面积48. 旋转楼梯两级的平面角度不大于10度，且每级离内侧扶手中心0.25处的踏步宽度要大于0.22米49. 两个安全出口之间的净距不应小于5米50. 楼梯正面门扇开足时宜保持0.6米平台净宽，侧墙门口距踏步不宜小于0.4米，其门扇开足时不应减少梯段的净宽51. 入地下车库的坡道端部宜设挡水反坡和横向通长雨水篦子52. 室内台阶宜150*300；室外台阶宽宜350左右，高宽比不宜大于1：2.553. 住宅公用楼梯踏步宽不应小于0.26M，踏步高度不应大于0.175M54. 梯段宽度不应小于1.1M（6层及以下一边设栏杆的可为1.0M），净空高度2.2M55. 休息平台宽度应大于梯段宽度，且不应小于1.2M，净空高度2.0M56. 梯扶手高度0.9M，水平段栏杆长度大于0.5M时应为1.05M57. 楼梯垂直杆件净空不应大于0.11M，梯井净空宽大于0.11M时应采取防护措施58. 门洞共用外门宽1.2M，户门卧室起居室0.9M，厨房0.8M，卫生间及阳台门0.7M，所有门洞高为2.0M59. 住宅层高不宜高于2.8M60. 卧室起居室净高≥2.4M，其局部净高≥2.1M（且其不应大于使用面积的1/3）61. 利用坡顶作起居室卧室的，一半面积净高不应低于2.1M利用坡顶空间时，净高低于1.2M处不计使用面积；1.2--2.1M计一半使用面积；高于2.1M全计使用面积62. 放家具墙面长3M，无直接采光的厅面积不应大于10M263. 厨房面积Ⅰ、Ⅱ≥4M2；Ⅲ、Ⅳ≥5M264. 厨房净宽单面设备不应小于1.5M；双面布置设备间净距不应小于0.9M65. 对于大套住宅，其使用面积必须满足45平方米66. 住宅套型共分四类使用面积分别为34、45、56、68M267. 单人卧室≥6M2；双人卧室≥10M2；兼起居室卧室≥12M2；68. 卫生间面积三件3M2；二件2--2.5M2；一件1.1M269. 厨房、卫生间净高2.2M70. 住宅楼梯窗台距楼地面净高度低于0.9米时，不论窗开启与否，均应有防护措施71. 阳台栏杆净高1.05M；中高层为1.1M(但要＜1.2)；杆件净距0.1172. 无外窗的卫生间应设置防回流构造的排气通风道、预留排气机械的位置、门下设进风百叶窗或与地面间留出一定缝隙73. 每套应设阳台或平台、应设置晾衣设施、顶层应设雨罩；阳台、雨罩均应作有组织排水；阳台宜做防水；雨罩应做防水74. 寒冷、夏热冬冷和夏热冬暖地区的住宅，西面应采取遮阳措施75. 严寒地区的住宅出入口，各种朝向均应设防寒门斗或保温门76. 住宅建筑中不宜设置的附属公共用房有锅炉房、变压器室、易燃易爆化学物品商店但有厨房的饮食店可设77. 住宅设计应考虑防触电、防盗、防坠落78. 跃层指套内空间跨跃两楼层及以上的住宅79. 在坡地上建住宅，当建筑物与等高线垂直时，采用跌落方式较为经济80. 住宅建筑工程评估指标体系表中有一级和二级指标81. 7层及以上（16米）住宅必须设电梯82. 宿舍最高居住层的楼地面距入口层地面的高度大于20米时，应设电梯83. 医院病房楼，设有空调的多层旅馆，超过5层的公建室内疏散楼梯，均应设置封闭楼梯间（包括首层扩大封闭楼梯间）设歌舞厅放映厅且超过3层的地上建筑，应设封闭楼梯间。
去泛素化酶研究进展刘文斌【摘要】泛素化和去泛素化是两种广泛存在的蛋白质翻译后修饰方式。
阐述了其影响了目标蛋白在细胞内的定位、稳定性和功能。
其中,去泛素化过程涉及组蛋白的修饰,细胞周期,细胞分化,细胞凋亡,内吞和自噬的调控及DNA损伤修复,也参与机体的肿瘤发生,肌肉萎缩,组织发育及抗病毒感染。
【期刊名称】《武汉轻工大学学报》【年(卷),期】2016(035)003【总页数】12页(P1-12)【关键词】泛素;去泛素化;酶【作者】刘文斌【作者单位】武汉轻工大学医学技术与护理学院,湖北武汉430023【正文语种】中文【中图分类】Q7泛素(ubiquitin)是一个由76个氨基酸组成的小分子蛋白质，广泛存在于真核细胞中，它能够与细胞内的许多蛋白质共价结合，从而使之“泛素化”，由此影响蛋白质的行为及细胞的功能，如生长、分裂、运动、分化、凋亡等[1-2]。
泛素化是一种蛋白质翻译后修饰方式，也是一种细胞内信号。
泛素化由一系列酶促反应来完成，如泛素活化酶E1、泛素耦合蛋白E2及泛素连接酶E3催化的反应。
E1在ATP的帮助下催化泛素末端的甘氨酸，与自身的一个半胱氨酸之间形成一个硫酯键。
通过转酰基作用，活化的泛素再转移到泛素耦合蛋白E2的一个半胱氨酸上，形成E2-Ub；然后在泛素连接酶E3的作用下，将E2-Ub上的Ub连接到需要降解蛋白的赖氨酸上。
E3连接酶具有底物特异性。
这样，可以有单个或多个泛素被连接到目标蛋白质上，从而形成泛素多聚体。
各个泛素单体之间主要是通过第48位的赖氨酸(K48)来连接。
泛素化的目标蛋白在细胞质内被蛋白酶体(proteosome)降解。
E3分为两大类，包括RING结构域E3(可以直接将E2-Ub连接到底物赖氨酸上)和HECT结构域E3(与E6AP的C端)或RBR结构域E3(RING-between-RING)，这种E3含有一个活性的巯基，会额外形成一个E3-Ub硫酯中间物，然后再催化Ub和底物的连接[1，3]。
EG【摘要】新近发现的基因EG-1在乳腺癌中有高表达，其与内皮细胞及上皮细胞的增生激活状态相关。
Her-2、p53、c-myc、bcl-2在乳腺癌中的研究较多，其在细胞信号转导通路中的作用已有一定认识。
全文就5个基因的研究概况作一综述。
【关键词】乳腺肿瘤癌基因抑癌基因从乳腺癌的病因学入手，期望能找到乳腺癌发病的关键环节。
近几十年随着细胞分子生物学研究的深入，特别是人类基因组计划的完成、基因芯片技术的广泛应用，癌症分子水平发病机制越来越多被致力于这方面研究的科学家揭示。
乳腺癌也不例外，已经发现了很多在乳腺癌发生发展中起作用或相关的原癌基因及抑癌基因，在此总结一下乳腺癌的分子生物水平机制及相关基因EG-1、Her-2、p53、c-myc、bcl-2的研究概况。
1 乳腺癌的分子生物学机制概论随着分子生物学研究的深入，人体生命活动可以看成是细胞内信号转导的结果。
各种细胞因子的表达、生成、活化、细胞周期的进行等都是通过复杂的信号转导体系进行的。
目前发现的通路有很多，这些通路上的因子不是孤立作用的而是相互交织形成复杂的信号网络，而参与细胞周期调节及细胞转化的通路主要涉及到MAPK及PI-3K通路，每条通路上都有很多因子发挥着其正性或负性作用，而且相互作用、互相贯通。
乳腺癌研究中，生长因子、雌激素等促进乳腺细胞异常增生转化的机制就是通过这两条或EG-1Zhang L等于2002年在研究肿瘤条件培养液培养下的脐静脉内皮细胞对照正常情况下的脐静脉内皮细胞，发现了一种具有显着性差异的基因，他们将之命名为EG-1，该基因定位于4号染色体，编码一个178-aa，的蛋白质，其与内皮细胞、上皮细胞的增生激活状态相关，可能在肿瘤血管发生中起重要作用。
继而他们研究EG-1与实体肿瘤的相关性，表明其与乳腺癌、结直肠癌、前列腺癌有相关性，不过都是小样本，需要临床进一步的大样本证实。
他们随之继续研究EG-1可能的细胞内分子生物机制，发现其与ERK1、2，c-jun-NH2-kinase，p38的激活有关，与scr明显相关，因此考虑其可能参与了scr-MAPK途径而引起的细胞增殖效应[10]。
细胞核内的染色质复制和修复机制细胞核内的染色质复制和修复机制是细胞生物学中一个重要的研究领域。
在细胞分裂和DNA修复过程中，染色质发挥着关键的作用。
本文将重点探讨细胞核内的染色质复制和修复机制，并详细介绍相关的研究进展。
一、染色质复制机制染色质复制是细胞分裂过程中的一个关键步骤。
在有丝分裂中，染色体在细胞分裂之前需要进行DNA复制，从而使得每个子细胞都能获得完整的遗传信息。
染色质复制机制包括以下几个主要步骤：1. DNA解旋和DNA复制染色质复制开始时，DNA分子解旋成两条单链。
每条单链作为模板，由DNA聚合酶酶家族催化下的DNA复制酶进行复制。
这些酶使得新的DNA链与模板链互补配对，最终形成两条完整的双链DNA分子。
2. DNA合成和酶催化在DNA复制过程中，DNA聚合酶酶家族催化下的DNA合成酶在模板链上逐个添加互补碱基，从而合成新的DNA链。
此过程需要其他辅助蛋白质的参与，如DNA聚合酶结构域和DNA催化结构域等。
3. DNA连接和修复在DNA复制过程中，一些错误的碱基可能会被嵌入到新合成的DNA链中。
为了确保每个子细胞都获得正确的遗传信息，在复制结束后，一系列DNA修复机制被触发。
这些修复机制包括DNA体外修复和DNA内部修复等方式，可以修复复制过程中的错误。
二、染色质修复机制染色质的DNA在细胞生命周期中不仅需要复制，还需要在遭受损伤后进行修复，以维持基因组的完整性。
染色质修复机制可以分为以下几个方面：1. DNA损伤检测和信号传导当DNA受到损伤，细胞内的一系列检测系统会被触发，以检测和信号传导损伤信号。
这些检测系统包括ATM和ATR等DNA损伤信号传感蛋白激酶。
一旦损伤信号被检测到，细胞会启动DNA修复机制。
2. DNA修复机制染色质的DNA损伤可通过多种方式进行修复，其中最常见的包括以下几种：- 直接修复：对于一些较小的结构损伤，细胞可以通过直接修复的方式恢复DNA的完整性。
例如，光刺激酶可以修复光引起的损伤。
碱基切除修复（base excision rep本人r，BER）是细胞内一种重要的DNA修复机制，用于纠正DNA中的碱基损伤或修复碱基。
在这个过程中，细胞内的一系列酶和蛋白质协同作用，完成了DNA碱基的切除和修复，维护了细胞的遗传信息。
碱基切除修复主要包括以下几个步骤：1. 损伤识别：当DNA中的某个碱基受到损伤时，损伤识别酶（如DNA glycosylases）会识别并结合到受损碱基上。
损伤识别酶能够识别不同类型的损伤，如碱基的氧化、甲基化等。
2. 碱基切除：一旦损伤识别酶结合到受损碱基上，它会通过切除酶的作用，切除掉受损的碱基。
切除酶一般是内切酶（AP endonuclease），它能够在损伤碱基周围切割DNA链。
3. 修复酶的介入：在碱基切除之后，DNA链上会留下一个空位，这时修复酶（如DNA聚合酶）会介入到空位上，并根据模板DNA的信息，合成新的DNA链。
4. 连接过程：修复过的DNA链与原始DNA链进行连接，形成完整的DNA分子。
通过这样的一系列步骤，碱基切除修复机制能够及时、准确地修复DNA中的碱基损伤，保持DNA的稳定性和完整性。
这一机制在维护细胞遗传信息、预防细胞突变和肿瘤的发生等方面起着至关重要的作用。
以人为例，碱基切除修复机制在人体内发挥着重要的作用。
人体的DNA受到很多内外因素的损伤，如紫外线、化学物质、放射线等，都可能导致DNA中的碱基损伤。
如果没有及时修复，这些碱基损伤将会导致细胞的突变或逝去，甚至引发癌症等疾病。
然而，人体内的碱基切除修复机制能够及时发现并修复DNA中的碱基损伤，保证了DNA的稳定性和完整性。
在细胞分裂和增殖过程中，这一修复机制能够确保新生的细胞拥有稳定的遗传信息，避免了细胞的突变和异常发育。
无论是在个体的生长发育过程中，还是在维持成年人体健康状态中，碱基切除修复机制都起着至关重要的作用。
碱基切除修复机制是维护DNA稳定性和完整性的重要保障，在人体内有着重要的生物学意义。
ATMAtaxia telangiectasia mutated (ATM) is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks. It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Several of these targets, including p53, CHK2 and H2AX are tumor suppressors.The protein is named for the disorder Ataxia telangiectasia caused by mutations of ATM.[1]Contents1 Introduction2 Structure3 Function4 Regulation5 Role in cancer6 Interactions7 See also8 References9 Further reading10 External linksIntroduction[edit]Throughout the cell cycle the DNA is monitored for damage. Damages result from errors during replication, by-products of metabolism, general toxic drugs or ionizing radiation. The cell cycle has different DNA damage checkpoints, which inhibit the next or maintain the current cell cycle step. There are two main checkpoints, the G1/S and the G2/M, during the cell cycle, which preserve correct progression. ATM plays a role in cell cycle delay after DNA damage, especially after double-strand breaks (DSBs).[2] ATM together with NBS1 act as primary DSB sensor proteins. Different mediators, such as Mre11 and MDC1, acquire post-translational modifications which are generated by the sensor proteins. These modified mediator proteins then amplify the DNA damage signal, and transduce the signals to downstream effectors such as CHK2 and p53.Structure[edit]The ATM gene codes for a 350 kDa protein consisting of 3056 amino acids.[3] ATM belongs to the superfamily of Phosphatidylinositol 3-kinase-related kinases (PIKKs). The PIKK superfamily comprises six Ser/Thr-protein kinases that show a sequence similarity to phosphatidylinositol 3-kinases (PI3Ks). This protein kinase family includes amongst others ATR (ATM- and RAD3-related), DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and mTOR (mammalian target of rapamycin). Characteristic for ATM are five domains. These are from N-Terminus to C-Terminus the HEAT repeat domain, the FRAP-ATM-TRRAP (FAT) domain, the kinase domain (KD), the PIKK-regulatory domain (PRD) and the FAT-C-terminal (FATC) domain. TheHEAT repeats directly bind to the C-terminus of NBS1. The FAT domain interacts with ATM's kinase domain to stabilize the C-terminus region of ATM itself. The KD domain resumes kinase activity, while the PRD and the FATC domain regulate it. Although no structure for ATM has been solved, the overall shape of ATM is very similar to DNA-PKcs and is composed of a head and a long arm that is thought to wrap around double-stranded DNA after a conformational change. The entire N-terminal domain together with the FAT domain are predict ed to adopt an α-helical structure, which was found by sequence analysis. This α-helical structure is believed to form a tertiary structure, which has a curved, tubular shape present for example in the Huntingtin protein, which also contains HEAT repeats. FATC is the C-terminal domain with a length of about 30 amino acids. It is highly conserved and consists of an α-helix followed by a sharp turn, which is stabilized by a disulfide bond.[4]Function[edit]A complex of the three proteins Mre11, RAD50 and NBS1 (Xrs2 in yeast), called the MRN complex in humans, recruits ATM to double strand breaks (DSBs) and holds the two ends together. ATM directly interacts with the NBS1 subunit and phosphorylates the histone variant H2AX on Ser139.[5] This phosphorylation generates binding sites for adaptor proteins with a BRCT domain. These adaptor proteins then recruit different factors including the effector protein kinase CHK2 and the tumor suppressor p53. The ATM-mediated DNA damage response consists of a rapid and a delayed response. The effector kinase CHK2 is phosphorylated and thereby activated by ATM. Activated CHK2 phosphorylates phosphatase CDC25A, which is degraded thereupon and can no longer dephosphorylate CDK2-Cyclin, resulting in cell-cycle arrest. If the DSB can not be repaired during this rapid response, ATM additionally phosphorylates MDM2 and p53 at Ser15.[6] p53 is also phosphorylated by the effector kinase CHK2. These phosphorylation events lead to stabilization and activation of p53 and subsequent transcription of numerous p53 target genes including Cdk inhibitor p21 which lead to long-term cell-cycle arrest or even apoptosis.[7]ATM-mediated two-step response to DNA double strand breaks. In the rapid response activated ATM phosphorylates effector kinase CHK2 which phophphorylates CDC25A, targeting it for ubiquitination and degradation. Therefore phosphorylated CDK2-Cyclin accumulates and progression through the cell cycle is blocked. In the delayed response ATM phosphorylates the inhibitor of p53, MDM2, and p53, which is also phosphorylated by Chk2. The resulting activation and stabilization of p53 leads to an increased expression of Cdk inhibitor p21, which further helps to keep Cdk activity low and to maintain long-term cell cycle arrest.[7]The protein kinase ATM may also be involved in mitochondrial homeostasis, as a regulator of mitochondrial autophagy (mitophagy) whereby old, dysfunctional mitochondria are removed.[8] Regulation[edit]A functional MRN complex is required for ATM activation after double strand breaks (DSBs). The complex functions upstream of ATM in mammalian cells and induces conformational changes that facilitate an increase in the affinity of ATM towards its substrates, such as CHK2 and p53.[2] Inactive ATM is present in the cells without DSBs as dimers or multimers. Upon DNA damage, ATMautophosphorylates on residue Ser1981. This phosphorylation provokes dissociation of ATM dimers, which is followed by the release of active ATM monomers.[9] Further autophosphorylation (of residues Ser367 and Ser1893) is required for normal activity of the ATM kinase. Activation of ATM by the MRN complex is preceded by at least two steps, i.e. recruitment of ATM to DSB ends by the mediator of DNA damage checkpoint protein 1 (MDC1) which binds to MRE11, and the subsequent stimulation of kinase activity with the NBS1 C-terminus. The three domains FAT, PRD and FATC are all involved in regulating the activity of the KD kinase domain. The FAT domain interacts with ATM's KD domain to stabilize the C-terminus region of ATM itself. The FATC domain is critical for kinase activity and highly sensitive to mutagenesis. It mediates protein-protein interaction for example with the histone acetyltransferase TIP60 (HIV-1 Tat interacting protein 60 kDa), which acetylates ATM on residue Lys3016. The acetylation occurs in the C-terminal half of the PRD domain and is required for ATM kinase activation and for its conversion into monomers. While deletion of the entire PRD domain abolishes the kinase activity of ATM, specific small deletions show no effect.[4]Role in cancer[edit]Ataxia telangiectasia (AT) is a rare human disease characterized by cerebellar degeneration, extreme cellular sensitivity to radiation and a predisposition to cancer. All AT patients contain mutations in the ATM gene (ATM). Most other AT-like disorders are defective in genes encoding the MRN protein complex. One feature of the ATM protein is its rapid increase in kinase activity immediately following double-strand break formation.[10][11] The phenotypic manifestation of AT is due to the broad range of substrates for the ATM kinase, involving DNA repair, apoptosis, G1/S, intra-S checkpoint and G2/M checkpoints, gene regulation, translation initiation, and telomere maintenance.[12] Therefore a defect in ATM has severe consequences in repairing certain types of damage to DNA, and cancer may result from improper repair. AT patients have an increased risk for breast cancer that has been ascribed to ATM's interaction and phosphorylation of BRCA1 and its associated proteins following DNA damage.[13] Certain kinds of leukemias and lymphomas, including Mantle cell lymphoma, T-ALL, atypical B cell chronic lymphocytic leukemia, and T-PLL are also associated with ATM defects.[14]Interactions[edit]Ataxia telangiectasia mutated has been shown to interact with RAD17,[15][16] RBBP8,[15][17] RAD51,[18] DNA-PKcs,[15][19] RRM2B,[20] FANCD2,[21][22] Nibrin,[15][23] TERF1,[24] BRCA1,[13][15][23][25][26][27][28] Abl gene,[18][24][29] TP53BP1,[30][31] MRE11A,[15][23] P53,[15][32][33][34][35] Bloom syndrome protein,[23][36] SMC1A[37] and RHEB.[38]。
