辐照诱导的人正常肝细胞系HL-7702细胞延迟效应

利用X射线辐照人正常肝细胞系HL-7702细胞,运用胞质分离阻滞微核法实验检测细胞微核率,Annexin V-FITC细胞凋亡检测试剂盒检测细胞凋亡率,细胞微核率和凋亡率随着辐照剂量的增加而显著增加。X射线照射后细胞传代培养,第7代时不同剂量辐照后子代细胞微核率和凋亡率同未辐照细胞相比已无明显区别。对不同剂量辐照后传代7代的细胞再次照射2.5Gy的相同剂量,发现它们细胞微核率和凋亡率存在明显差异,即初次受辐照剂量高的细胞,再次以相同剂量辐照后的微核率和凋亡率也高。这些结果表明,X射线辐照导致了HL-7702细胞基因组不稳定性这一辐射延迟效应,再次辐照使得辐射的延迟效应得以明显的表现。

重离子辐照HL-7702细胞诱导的损伤修复效应研究

<正>重离子束在肿瘤治疗方面具有其它射线无法比拟的优势,使得重离子束被誉为迄今为止最理想的放射治疗用射线。然而重离子在具有对靶区癌细胞的致死损伤效应的同

辐照诱导的人正常肝细胞系HL-7702细胞远后效应

近年来，放射治疗在肿瘤治疗中的作用受到了越来越多的重视，放疗技术和手段的迅速发展为人们选择放疗创造了更多的机会[1]。放疗是利用电离辐射对细胞，特别是细胞中的遗传物质DNA的损伤作用，诱发病灶部位不正常细胞的凋亡和坏死来治疗肿瘤的。即使制定了周密的放疗方案和计划，放疗过程中难免也会对正常组织和细胞造成一定程度的损伤，而且辐射的远后效应和旁效应的发现，也使得放射治疗肿瘤的安全性受到了很大的关注。要想充分发挥放疗的优势，而又使其对人体正常组织的毒副作用尽可能降低，就必须搞清楚电离辐射对细胞造成的各种损伤的类型及其分子机理。自从电离辐射的远后效应和旁效应被发现以来就一直是辐射生物医学领域的研究热点，国内外的科研人员进行了大量的实验试图解释它们的本质，可是几十年来相关领域的研究一直局限于细胞学的水平，在向分子水平前进的道路上遇到了巨大的困难[2,3]。目前对于电离辐射远后效应和旁效应的研究是相对独立进行的，很少有探讨二者相互关系的论文发表。本文试图在总结前人科研成果并结合对自己所取得的实验数据进行分析的基础上对电离辐射的远后效应、旁效应、基因不稳定性与电离辐射所产生的活性氧自由基的关系进行初步的探讨。实验方法与结果： 1.用X射线辐照人正常肝细胞系HL-7702细胞，运用胞质分离阻滞微核实验检测细胞微核率，AnnexinV-FITC细胞凋亡检测试剂盒检测细胞凋亡率，细胞微核率和凋亡率随着辐照剂量的增加而显著增加。X射线照射后细胞继续传代培养，第七代时不同剂量辐照后子代细胞微核率和凋亡率同未辐照细胞的微核率和凋亡率相比已经没有明显区别。对不同剂量辐照后传代七代的细胞再次照射2.5Gy相同的剂量，发现受初次不同剂量辐照的细胞其微核率和凋亡率再次出现明显差异，初次辐照剂量高的细胞再次相同剂量辐照后的微核率和凋亡率也高。 2.对不同剂量X射线辐照后的细胞继续传代到第十五代时用H2O2浓度为1ul/ml的培养基处理15min，发现受初次不同剂量辐照的细胞其微核率再次出现比较明显差异，初次辐照剂量高的细胞H2O2处理后的微核率也高。 3.对受到不同剂量X射线辐照的人正常肝细胞存活后代进行二次辐照，分两组分别给予1.5GyX射线和1Gy碳离子束辐照，并检测二次辐照后细胞的微核率，发现重离子束二次辐照后并不像X射线一样可以诱发初次X射线辐照造成的损伤信息的表达，而只是表现出二次重离子辐照所造成的损伤。结论： 1.X射线辐照导致了HL-7702细胞基因组不稳定性这一辐射远后效应，X射线二次辐照辐照存活细胞的子代细胞可以诱发辐射的远后效应（如基因组不稳定性）明显的表现； 2.X射线辐照导致的HL-7702细胞后代基因组不稳定性，可以通过H2O2处理而得以诱发，揭示电离辐射过程中产生的氧自由基可能与辐射的远后效应存在密切的联系； 3.电离辐射产生的ROS在诱发细胞产生微核中具有重要的作用，但是并非唯一的影响因素。 4.X射线和12C6+重离子束辐照后存活细胞的后代中的细胞损伤类型可能存在着本质上的区别

~(12)C~(6+)重离子辐照对人肝细胞、肝癌细胞端粒酶活性表达的影响

为探索碳离子束辐照对细胞中端粒酶活性的变化,利用兰州近代物理研究所重离子研究装置(Heavy ion research facility in lanzhou,HIRFL)产生的碳离子(31MeV/μ12C6+),以人肝细胞HL-7702,肝癌细胞SMMC-7721为实验对象,用不同剂量1Gy、2Gy、3Gy、4Gy的重离子分别对两种细胞进行照射,用多聚酶链式反应-银染端粒重复序列扩增法(PCR-telomeric repeat amplification protocol,TRAP-PCR)银染端粒重复序列扩增法检测不同剂量下细胞端粒酶活性的变化。结果显示,人肝细胞HL-7702自身没有端粒酶活性,经1Gy辐照后也没有端粒酶活性,在2和3Gy处出现端粒酶活性,4Gy处端粒酶活性又消失。肝癌细胞SMMC-7721在1～3Gy处随着剂量的增大端粒酶活性升高,在4Gy处又开始下降;在1～3Gy处随着时间的推移端粒酶活性随着时间而加强(p<0.05)。分析得知,重离子辐射可以诱导人肝细胞产生端粒酶活性,也可以改变肝癌细胞的端粒酶活性。端粒酶参与细胞受辐照后DNA单链损伤的修复;辐照后DNA双链断裂导致端粒酶活性减弱。本实验使重离子在辐照治疗中的优势得以体现。

X射线辐照后人肝细胞、肝癌细胞存活与端粒酶活性关系的研究

利用X射线对体外培养的人肝癌细胞SMMC-7721和正常人肝细胞HL-7702进行辐照,以细胞克隆形成法测定细胞存活率,多聚酶链式反应银染端粒重复序列扩增法(Telomeric repeat amplification protocol,TRAP-PCR)检测细胞中端粒酶活性变化,探讨辐照后细胞存活与细胞中端粒酶活性变化的关系。结果表明,SMMC-7721和HL-7702细胞的存活率都随辐照剂量的增加而相应降低。在1-4Gy剂量范围内,两种细胞的端粒酶活性变化均呈剂量依赖性增强。但在1Gy、2Gy、3Gy剂量下,SMMC-7721其端粒酶活性较对照(0Gy)细胞低;当辐照剂量达4Gy时,端粒酶活性略高于对照细胞。在1-4Gy剂量范围内,HL-7702和SMMC-7721的细胞存活与端粒酶活性在变化趋势上呈负的关系。

12C6+重离子辐照对细胞端粒酶活性表达的影响

端粒是染色体末端由重复DNA序列和相关蛋白组成的一种特殊结构，具有稳定染色体结构及完整性的功能，会随染色体复制与细胞分裂而缩短。端粒酶是一种核糖核蛋白，能以自身RNA模板合成端粒DNA，催化合成TTAGGG重复序列，添加到染色体末端，维持端粒长度不变。端粒酶主要由hTR, TEPl和hTERT组成，一般认为hTERT是端粒酶激活的限速因素。大多数永生化细胞和恶性肿瘤细胞具有端粒酶活性，因而端粒酶目前已成为为细胞持续分裂提供遗传基础的原因。由于端粒酶与细胞衰老、肿瘤发生等关系如此密切，因此已成为肿瘤放射治疗研究热点。目的： 本文利用兰州近代物理研究所重离子研究装置（Heavy Ion Research Facility in Lanzhou,HIRFL）产生的碳离子（31MeV/u 12C6+）对人体细胞、癌细胞进行不同剂量的辐照，探索细胞中端粒酶活性的变化及与之相关的生物学信息。材料与方法： 以人肝细胞HL－7702，肝癌细胞SMMC－7721为实验对象，用不同剂量1Gy、2Gy、3Gy、4Gy的重离子分别对两种细胞进行照射，用多聚酶链式反应-银染端粒重复序列扩增法（PCR- telomeric repeat amplification protocol，TRAP-PCR）检测不同剂量下细胞端粒酶活性的变化。并提取不同剂量下的细胞转入培养皿中，培养10天，固定，染色。统计大于50个细胞的克隆数，绘制细胞存活曲线。结果与讨论： 结果显示，人肝细胞HL-7702自身没有端粒酶活性，经1Gy辐照后也没有端粒酶活性，在2、3Gy处出现端粒酶活性，4Gy处端粒酶活性又消失。肝癌细胞SMMC-7721在1－3Gy处随着剂量的增大端粒酶活性升高，在4Gy处又开始下降；在1－3Gy处随着时间的推移端粒酶活性随着时间而加强（p<0.05）。随着剂量的增大，细胞存活率呈剂量依赖型下降。分析得知，重离子辐射可以诱导人肝细胞产生端粒酶活性，也可以改变肝癌细胞的端粒酶活性。端粒酶参与细胞受辐照后DNA单链损伤的修复；辐照后DNA双链断裂导致端粒酶活性减弱。本实验与其他低LET射线相比，使重离子在辐照治疗中的优势得以体现

Crystallographic identification of a ‘stepped-cubane’ structure for the Cu4O4 core in [Cu4L2(bipy)4(µ3-OH)2][ClO4]4(HL = 5-hydroxy-6-methylpyridine-,4-dimethanol, bipy = 2,2′-bipyridine)

The structure of [Cu4L2(bipy)4(µ3-OH)2][ClO4]4 containing a Vitamin B6 ligand, pyridoxine (5-hydroxy-6-methylpyridine-3,4-dimethanol, HL), and 2,2′-bipyridine (bipy) has been determined by single-crystal X-ray analysis. This is the first report on a copper(II) complex having a ‘stepped-cubane’ structure. The compound crystallizes in the triclinic space group P[1 with combining macron](Z= 1) with a= 11.015(3), b= 11.902(1), c= 13.142(2)Å, α= 105.07(1), β= 102.22(1) and γ= 99.12(1)°; R= 0.054). The co-ordination geometry around each copper is trigonally distorted square pyramidal. Two of the basal sites are occupied by bipyridyl nitrogens in a bidentate fashion. The remaining basal positions for Cu(1) are filled by a phenolic oxygen and a 4-hydroxymethyl oxygen of the L moiety, whereas for Cu(2) they are occupied by two µ3-OH oxygens. The axial sites are occupied by a µ3-OH oxygen and the 4-hydroxymethyl oxygen of the same pyridoxine for Cu(1) and Cu(2), respectively. Both the bridging nature of the 4-hydroxymethyl oxygen of the L moiety and the unsymmetrical bridging nature of the µ3-OH groups with axial–equatorial bridging are novel features. The structure is discussed in relation to stepped-cubane structures reported in the literature. A comparative study is also made with µ3-hydroxo-bridged copper(II) complexes. Both the plasticity effect of CuII and the stacking interactions between the various rings appear to be important in stabilizing this unusual structure.

Stereochemical properties and x-ray structure of a water-soluble diastereomeric (arene)ruthenium(II) Schiff-base complex: [Ru(.eta.-MeC6H4Pri-p)(H2O)(L*)](ClO4), containing a weakly bound aqua ligand [HL* = (S)-(.alpha.-methylbenzyl)salicylaldimine]

An air-stable and water-soluble diastereomeric half-sandwich ruthenium(I1) complex, [Ru(s-MeCsH4Pr'-p)(H*O)-(L*)] (C104) (l), has been isolated and structurally characterized [HL* = (27)-(a methylbenzyl)salicylaldimine,2-HOC6H4CH-NCHMePhI. Complex 1, Czd-I3oNO&lRu, crystallizes in the noncentric triclinic space group P1 with a = 9.885(1) A, b = 10.185(1) A, c = 14.187(2) A, a = 110.32(1)', 6 = 102.17(1)', y = 102.41(1)O, V=1243( 1) A3, and 2 = 2. The X-ray structure shows the presence of two diastereomers in a 1:l ratio having RR,,,SCand SR,,,&c onfigurations. The Ru-OHz bond distances are considerably long, and the values for RR, - a~n d SRu-1isomers are 2.1 19(5) and 2.203(5) A, respectively. The aqua complex (1) exists as a single diastereomer in solution,and it forms stable adducts with P-, N-, and halide-donor ligands. The stereochemical changes associated with adduct-forming reactions follow an inversion order: PPhs >> P(OMe)3 > pyridine bases >> halides (I, Br, Cl) >H20.

Telomerase Activity and Microgravity-Dependent Apoptosis in Human Leukemia HL-60 Cells

Mammalian cells subjected to conditions of spaceflight and the microgravity environment ofspace; manifest a number of alterations in structure and function. Among the most notable changes incells flown on the Space Shuttle are reduced growth activation and decline in growth rate in the totalpopulation. Other changes include chromosomal aberrations, inhibited locomotion, alteredcytokine production, changes in PKC distribution, and increased apoptos.

Syntheses, crystal structures and properties of two Cu(II) coordination polymers: Cu(C3N2H4)(2)(HL)(2) andCU(C3N2H4)(2)L with C3N2H4 = imidazole, H2L = adipic acid

The reactions of freshly prepared Cu(OH)(2).xH(2)O and Cu(OH)(2-2y)(CO3)(y).zH(2)O precipitates with imidazole and adipic acid in CH3OH/H2O at pH = 5.4 yielded CU(C3N2H4)(2)(HL)(2) 1 and CU(C3N2H4)(2)L 2, respectively. Complex 1 consists of ribbon-like polymeric chains (1)(infinity)[CU(C3N2H4)(2)(HL)(4/2)], in which the octahedrally coordinated Cu atoms are doubly bridged by bis-monodentate hydrogen adipato ligands. The interchain N-H...O hydrogen bonding interactions are responsible for supramolecular assembly of the polymeric chains into open 3D frameworks and two-fold interpenetration of the resulting open frameworks completes the crystal structure of 1. Within complex 2, the Cu atoms are penta-coordinated to form CuN2O3 square pyramids and condensed into CU2N4O4 dimers, which are doubly bridged by twisted bis-monodentate adipato ligands into polymeric chains (1)(infinity)([CU(C3N2H4)(2)](2)L-4/2) with 4- and 18-membered rings progressing alternatively. The polymeric chains are assembled due to interchain N-H...O hydrogen bonding interactions. The thermal and magnetic behaviors of 1 and 2 is discussed.

Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100?µM) inhibited 44?±?0.05% (mean?±?s.e.m., n?=?11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15?µM at -150 ?mV to 148?µM at -75 ?mV in 120 ?mM external K(+). This current was insensitive to 10?µM glybenclamide. A component of whole-cell current was sensitive to 150?µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype.

pLR-HL: a novel amphibian Bowman-Birk-type trypsin inhibitor from the skin secretion of the broad-folded frog, Hylarana latouchii

In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR-HL, whose structure was deduced from its biosynthetic precursor-encoding cDNA cloned from the skin secretion-derived cDNA library of the broad-folded frog, Hylarana latouchii, by employing a "shotgun" cloning technique. It contains a disulphide loop between Cys5 and Cys15 which is consistent with Bowman-Birk-type protease inhibitors. The primary structure of pLR-HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse phase HPLC and subsequent analysis using MALDI-TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesised by solid-phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a Ki of 143 nM. The substitution of Lys-8 by Phe (Phe8 -pLR-HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a Ki of 2.141 μM. Additionally, both the disulphide loops of pLR-HL and Phe8 -pLR-HL were synthesised and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically-active moiety.

Impact du statut de différenciation des cellules promyélocytaires HL-60 sur l’efficacité anticancéreuse et antiinflammatoire de l’EGCG

L’altération de la barrière hématoencéphalique (BHE) par les cellules tumorales et les cellules immunes circulantes peut mener à la neuroinflammation. Les cellules leucémiques promyélocytaires HL-60 sont un excellent modèle pour étudier et comprendre les mécanismes de signalisation moléculaires qui caractérisent le développement tumoral et métastatique. La cancérogenèse peut s’accompagner de modulations de l’expression de biomarqueurs tels que la cyclooxygénase-2 et la métalloprotéase-9. Les recherches décrites dans ce mémoire relatent l’analyse des biomarqueurs inflammatoires et invasifs régulés lors de la différenciation induite par le PMA des cellules HL-60 en macrophages. Le statut de différenciation cellulaire pourrait avoir un impact sur les gènes cibles de la voie NF-κB. Nous émettons l’hypothèse que le PMA active la voie NF-κB et que cette signalisation peut être renversée par l’(-)-épigallocatéchine-gallate (EGCG). En effet, une régulation à la hausse de l’expression de plusieurs gènes combinée à la diminution de l’expression d’IκB mettent en évidence l’implication de la voie NF-κB dans l’activation des mécanismes pro-inflammatoires et pro-invasifs. Les mêmes observations sont faites dans les cellules différenciées appelées «macrophages-like». L’EGCG, un polyphénol dérivé du thé vert, a un potentiel chimiopréventif. Il est capable d’inhiber la signalisation moléculaire passant par la voie NF-κB dans les cellules HL-60 traitées simultanément par l’EGCG et le PMA, mais pas dans les cellules «macrophages-like». Cette différence peut s’expliquer par une modulation de l’expression du récepteur de surface cellulaire de l’EGCG, le récepteur à la laminine de 67 kDa, et de son précurseur de 37 kDa. Collectivement, nos résultats montrent que le statut de différenciation des cellules promyélocytaires HL-60 concorde avec l’activation des mécanismes favorisant le développement d’un cancer et des métastases. Cet effet peut être prévenu par l’utilisation d’agents naturels tel l’EGCG. Le ciblage de biomarqueurs liés au statut de différenciation des cellules tumorales impliquées dans la perturbation de la barrière hématoencéphalique qui cause la neuroinflammation permettrait l’avancement des connaissances dans la prévention de la cancérogenèse.