Rescueasd.docx
- 文档编号:4380314
- 上传时间:2022-12-01
- 格式:DOCX
- 页数:33
- 大小:474.85KB
Rescueasd.docx
《Rescueasd.docx》由会员分享,可在线阅读,更多相关《Rescueasd.docx(33页珍藏版)》请在冰豆网上搜索。
Rescueasd
硕士学位论文
科学学位
NU7441对肝癌HepG2细胞放射增敏效应及其作用机制的研究
研究生:
王全绪
导师:
杨川杰副教授
专业:
内科学
二级学院:
第二医院
2015年4月
目录
中文摘要…………………………………………………………8
英文摘要…………………………………………………………8
英文缩写…………………………………………………………10
研究论文NU7441对肝癌HepG2细胞放射增敏效应及其作用机制的研究
前言……………………………………………………………12
材料与方法……………………………………………………18
结果……………………………………………………………30
附图……………………………………………………………35
附表……………………………………………………………40
讨论……………………………………………………………45
结论……………………………………………………………50
参考文献………………………………………………………53
综述DNA依赖性蛋白激酶及其抑制剂与肿瘤治疗的研究进展
致谢…………………………………………………………………60
个人简历……………………………………………………………62
NU7441对肝癌HepG2细胞放疗增敏效应及其作用机制的研究
摘要
目的:
原发性肝癌(HCC)是一种恶性程度高、治愈率低的临床常见恶性肿瘤,它在全球恶性肿瘤中的发病率居第六位,而病死率居第三位。
在发展中国家,病因以乙型肝炎病毒感染为主,而在发达国家,主要与酒精性肝病、肥胖等有关。
原发性肝癌的病理类型主要包括肝细胞型、胆管细胞型及混合型,前者占多数。
临床上原发性肝癌的主要治疗方法包括手术治疗、化疗、放射治疗、肝动脉化疗栓塞、分子靶向治疗、中医治疗等方法。
目前针对肝癌的治疗是个体化的综合治疗。
由于早期肝癌的临床症状不明显,因此大部分患者就医时肝癌已处于晚期,失去了手术机会,并且随着肝癌精确放疗的发展,放射治疗已成为晚期肝癌的主要治疗方法之一。
放疗无异于电离辐射,
当肿瘤细胞受到射线照射后,会发生DNA损伤,细胞如果不能及时精确地修复,继而会诱发细胞的凋亡。
然而,肿瘤细胞具有高效的修复机制,在受到电离辐射时,会导致细胞DNA双链断裂,细胞随后启动修复机制,在真核细胞中主要存在两种修复方式:
非同源末端连接(NHEJ)和同源重组(HJ),在电离辐射引起的DNA损伤中,修复途径以非同源末端连接为主,因此抑制DNA的修复途径有利于促进肿瘤细胞的凋亡,提高肿瘤放射治疗的效果。
DNA依赖性蛋白激酶(DNA-PK)复合物是非同源末端连接途径的关键组分,且DNA-PKcs作为DNA-PK的催化亚基更是其中的关键蛋白,目前各种DNA-PK抑制剂已用于肿瘤放疗增敏的实验研究。
人工合成的小分子化合物NU7441作为DNA-PK的特异性抑制剂,在乳腺癌、肺癌、白血病、前列腺癌联合放疗的研究中表现出了放射增敏效应。
本实验拟应用NU7441联合放疗作用于肝癌HepG2细胞,对NU7441的放射增敏效应及其作用机制加以研究,为临床上肝癌放疗增敏剂的开发与应用提供理论指导。
方法:
1.通过CCK-8实验检测NU7441在不同浓度及作用时间下对HepG2细胞增殖的影响,并绘制相应的作用曲线。
2.通过Westernblot的方法检测①DNA-PKcs在正常肝细胞LO2、肝癌细胞HepG2细胞及4Gyγ射线照射后的HepG2细胞中的表达情况。
②NU7441对4Gyγ射线照射后的HepG2细胞中Ku70、Ku80、DNA-PKcs及pDNA-PKcs(S2056)蛋白表达的影响③NU7441和另一种放疗增敏剂NU7026在相同条件对pDNA-PKcs(S2056)蛋白表达的影响
3.通过单细胞凝胶电泳(SCGE)实验及免疫荧光实验检测在NU7441有无的情况下,4Gyγ射线照射后的HepG2细胞在不同时间点的DNA损伤修复情况
4.通过流式细胞术检测HepG2细胞在对照组(Control)、单纯照射组(IR)、单纯加药药组(NU7441)、加药联合放射组(NU7441+IR)中,作用24h后,比较各组细胞在细胞周期的各时相所占比例。
5.采用统计学软件17.0进行实验数据分析,实验结果用均数±标准差表示。
两独立样本采用t检验,多个独立样本的比较采One-WayANOVA的分析方法,p<0.05视为有统计学差异。
结果:
1.CCK-8增殖实验表明NU7441对HepG2细胞的增殖具有一定抑制作用,且具有浓度和时间依赖性,NU7441在浓度为5μmol/L及以上浓度下,这用抑制作用更加明显(P<0.05)。
2.Westernblot实验表明①DNA-PKcs在LO2细胞、HepG2细胞及4Gy照射后HepG2细胞中的表达依次增高②与对照组相比,在NU7441作用下,Ku70,Ku80及DNA-PKcs的蛋白表达量没有明显变化,而磷酸化pDNA-PKcs(S2056)的蛋白表达量明显降低。
NU7441的抑制作用主要是了影响DNA-PKcs的活性③在相同实验条件下,NU7441组比NU7026组pDNA-PKCS(S2056)的蛋白表达量下降明显,NU7441对DNA-PKcs的活性抑制作用强于NU7026。
3.免疫荧光实验表明:
HepG2细胞在受照射后,细胞中的γ-H2AX焦点数量在一定时间范围在先增加后减少,在加入NU7441作用以后,γ-H2AX焦点数量也是先增加后减少,但与单纯照射组相比,γ-H2AX焦点数量下降的速率比照射组缓慢(P<0.05)。
4.中性单细胞凝胶电泳实验表明:
HepG2细胞在经过4Gy射线处理后,DNA发生损伤,表现为照射组彗星细胞的尾矩明显大于对照组(P<0.05);经过4h的修复后,加入NU7441照射组的修复速度较单纯照射组减慢,表现为加药照射组比照射组的尾矩大(P<0.05)。
5.流式细胞术检测细胞周期的实验表明:
IR组与Control组相比,G2/M期细胞的比例明显增加(P<0.05),NU7441组没有统计学差异(P>0.05),(NU7441+IR)组的G2/M期细胞的比例较IR组明显增加(P<0.05),且细胞凋亡率与对照组Control相比明显增加(P<0.05)。
结论:
NU7441对HepG2细胞的增殖具有抑制作用,且这种作用具有时效和量效关系。
DNA-PKcs在肝癌细胞中高表达,可以作为一个较好的放疗增敏研究的靶点。
在相同条件下,NU7441对DNA-PKcs的活性抑制作用强于NU7026。
NU7441对HepG2细胞具有放射增敏效应,其主要机制为:
抑制DNA修复途径中DNA-PKcs的活性,引起细胞周期中G2/M期的阻滞,诱导细胞凋亡,最终达到放疗增敏作用。
关键词:
NU7441;肝癌;放疗增敏;非同源末端连接;DNA-PK;细胞周期
Studyonthemechanismofenhancingradiosensitizationby
NU7441onHepG2cell
Abstract
Objective:
Primaryhepatocellularcarcinoma (HCC) isakindofhighmalignantdegree, lowcurerate ofclinical commonmalignanttumor, itsincidenceinmalignanttumor raterankssixth, whichthemortality rateranksthird intheworld .Inthedevelopingcountries, themostcommoncauseofcanceris hepatitisBvirusinfection, whileindevelopedcountriesalcoholicliverdisease,obesity andsoonhavearelevantrole.Thepathologicaltypesoflivercancerarehepatocellularcarcinomawhichisthemajorhistologicaltype,bileduct cell typeandmixedtype.Themainmethodsof clinicaltreatment ofprimarylivercancer treatmentincludingoperation,chemotherpy,radiotherapy, liver arteryembolismchemotherapy, moleculartargetedtherapy, Chinesemedicine treatment.Individualcomprehensivetherapyisthe treatmentoflivercancer atpresent.Theclinicalsymptomsof earlyHCC isnotobvious, somost patients havebeeninadvancedstage HCCwhenlost thechanceofoperation, withthedevelopmentof preciseradiotherapyfor livercancer, radiationtherapyhasbecomeoneofthemain methodsforthetreatmentof advancedhepatocellularcarcinoma.Radiotherapy istantamountto ionizingradiation,whentumorcells wereexposedtoradiationwhich occurDNAinjury,whichifnotpromptlyand accuratelyrepaired,caninduce cell apoptosafterthisprocess.However, tumor cellshave efficientrepairmechanisms, by ionizingradiation, canleadto cell DNAdoublestrandbreaks, therearetwomain waysofrepairineukaryotesaftertherepairingmechanismof thecell:
nonhomologousendjoining (NHEJ) andhomologousrecombination (HJ),repairpathways in nonhomologousendjoining repairpathways primarilyinthe ionizingradiation DNAdamage, theinhibitionofDNA willimprove theeffectivenessofradiationtherapy oftumor,thatisbeneficialtopromotethe apoptosisoftumor cells.DNAdependentprotein kinase (DNA-PK) complex isakey groupof nonhomologousendjoining pathway.As thecatalyticsubunitofDNA-PK asthekey protein,various DNA-PK inhibitorshavebeen usedin experimentalstudyof tumorradiosensitizeratpresent.Syntheticsmallmolecule compoundNU7441 isaspecific inhibitorofDNA-PKshownradiosensitizingeffectduringthetreatmentofthebreastcancer, lungcancer, leukemia prostatecancer radiotherapy.ThisexperimentuseNU7441 combinedwithradiotherapyeffecton hepatocellularcarcinomaHepG2 cells, tostudy NU7441radiosensitization effectandits mechanismofaction, toprovidetheoreticalguidanceforthe development andapplicationofclinical livercancer radiotherapy sensitizer.
Method:
1Drawing thecorresponding function curvethroughCCK-8testNU7441 effectontheproliferationofHepG2cells indifferentconcentrations and actiontime.
2①DNA-PKcs’sexpressioninnormalliver cellLO2, livercancercellHepG2 and4Gy gammarayirradiation ofHepG2cells,②NU7441on Ku70, Ku80DNA-PKcsandpDNA-PKcs 4Gy afterirradiationby HepG2cells (S2056) ontheproteinexpression,③NU7441andNU7026 theradiosensitizerunderthesameconditionsonpDNA-PKcs (S2056) proteinbyusingthe methodsofWestern blot detection.
3 4Gy afterirradiationby HepG2 cellsinDNA damagerepairin differenttimepointwithorwithoutNU7441 throughthe neutral singlecellgelelectrophoresis andimmunefluorescenceassay.
4HepG2cellline(Control) inthecontrolgroup, theirradiationgroup (IR), simple medicineadding druggroup(NU7441), combinedwithradiation group (NU7441+IR), cellsinthe cellcycle werecompared ateachphase proportionafter24htreatmentdetectedby flowcytometry.
5Theanalysisofexperimentaldatausingstatisticalsoftware17.0.For theexperimentalresults withthe standarddeviation indicated. Twoindependentsamples ttestwasusedto comparethe analysis methodT,One -Way ANOVA ofseveralindependentsamples, p<0.05as astatisticaldifference.
Result:
1CCK-8 proliferationassaydemonstratedthat NU7441 hasacertain inhibitoryeffectHepG2cells’proliferation, withtimeandconcentrationdependent. NU7441 attheconcentrationof 5mol/Landabovethe concentrationhas moreobvious inhibitoryeffects(P<0.05).
2Western blotresults showedthat:
①ExpressionofDNA-PKcsin LO2cells, HepG2 cellsand4Gy HepG2cellsirradiated in increasing.②TheexpressionoftheKu70Ku80and DNA-PKcsdon’tchangeobviouslyduringthepresenceofNU7441,andthe phosphorylationofpDNA-PKcs (S2056) expressionsignificantlydecreased,comparedwiththecontrolgroup. ③ThegroupNU7441thanthegroup’sNU7026 (S2056) pDNA-PKCS expression significantlydecreased.NU7441’sactivity ofinhibitedDNA-PKcsisstrongerthanNU7026bythesameexperimentalconditions.
3Immunofluorescence results showedthat:
Afterirradiation,thefocus NUmberof γ-H2AXin HepG2 cellsreducingafter increasedfirstinacertaintime range.AfterjoiningtheNU7441in action, thefocusNUmberof γ -H2AX is firstincreasedandthendecreased.Butcomparedwith controlgroup, thedecreased rate ofγ-H2AX NUmberslowthanirradiatedgroup (P<0.05).
4 Neutralsinglecell gelelectrophoresis’resultshowedthat:
HepG2cells’DNAdamagingafter 4Gyray treatment, as tailmoment ofcomet cells irradiatedgroup wassignificantlyhigherthan thecontrolgroup (P<0.05).Afterthe4hrepairing, speedwith NU7441 group repairis slowerthanthecontrolgroup ,asdosing irradiationgroup tailmoment CF groupshownaslarger (P<0.05).
5Thedetection resultofcellcycle byflowcytometryshowedthat:
ComparedwithIR groupandControlgroup, thepercentageofcellsinG2/Mphase increasedsignificantly (P<0.05), nostatisticallysignificantdifferencesbetweenthe NU7441group (P>0.05),(NU7441+IR) groupofG2/M phase cellswassignificantly increased thanIRgroup (P<0.05), andthe cell apoptosisrateand Controlincreasedsignificantly comparedwith controlgroup (P<0.05).
Conclusion:
NU7441 hasinhibitoryeffectsduringtheproliferationofHepG2cells, andthiseffectwithdoseandtimeeffectrelationship.ThehighexpressionofDNA-PKcs inhepatomacells canbe usedasagood radiosensitizertarget point.Underthesameconditions, theactivityofNU7441 ofDNA-PKcs inhibitedisstrongerthan NU7026.NU7441has radiosensitizingeffect onHepG2cells,themain mechanismisthatas:
InhibitedtheDNA-PKcs activityin DNA repairpathway,caused cellcyclearrest in G2/Mphase, inducingapoptosis,increasingtheradiotherapysensitizationfinally.
Keyword:
NU7441;livercancer;radiosensitization;Nonhomologousendjoining;DNA-PK;cellcycle
英文缩写
HCC
Hepatocellularcarcinoma
肝细胞癌
IR
IonizingRadiation
电离辐射
DNA-PK
DNAdependentproteinkinase
DNA依赖性蛋白激酶,
DNA-PKcs
CatalyticsunbunitoftheDNA-PK
DNA依赖性蛋白激
酶催化亚单位
DSBs
Double-strandbreaks
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- Rescueasd
![提示](https://static.bdocx.com/images/bang_tan.gif)