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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<
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<
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<
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
apoptosafterthisprocess.However,
tumor
cellshave
efficientrepairmechanisms,
by
ionizingradiation,
canleadto
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
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
proteinbyusingthe
methodsofWestern
blot
detection.
3
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.
attheconcentrationof
5mol/Landabovethe
concentrationhas
moreobvious
inhibitoryeffects（P<
0.05）.
2Western
blotresults
showedthat:
①ExpressionofDNA-PKcsin
LO2cells,
cellsand4Gy
HepG2cellsirradiated
increasing.②TheexpressionoftheKu70Ku80and
DNA-PKcsdon’tchangeobviouslyduringthepresenceofNU7441,andthe
phosphorylationofpDNA-PKcs
expressionsignificantlydecreased,comparedwiththecontrolgroup.
③ThegroupNU7441thanthegroup’sNU7026
pDNA-PKCS
expression
significantlydecreased.NU7441’sactivity
ofinhibitedDNA-PKcsisstrongerthanNU7026bythesameexperimentalconditions.
3Immunofluorescence
results
showedthat:
Afterirradiation,thefocus
NUmberof
γ-H2AXin
cellsreducingafter
increasedfirstinacertaintime
range.AfterjoiningtheNU7441in
action,
thefocusNUmberof
γ
-H2AX
is
firstincreasedandthendecreased.Butcomparedwith
controlgroup,
thedecreased
rate
ofγ-H2AX
NUmberslowthanirradiatedgroup
（P<
4
Neutralsinglecell
gelelectrophoresis’resultshowedthat:
HepG2cells’DNAdamagingafter
4Gyray
treatment,
as
tailmoment
ofcomet
cells
irradiatedgroup
wassignificantlyhigherthan
thecontrolgroup
0.05）.Afterthe4hrepairing,
speedwith
repairis
slowerthanthecontrolgroup
asdosing
irradiationgroup
CF
groupshownaslarger
5Thedetection
resultofcellcycle
byflowcytometryshowedthat:
ComparedwithIR
groupandControlgroup,
thepercentageofcellsinG2/Mphase
increasedsignificantly
0.05）,
nostatisticallysignificantdifferencesbetweenthe
NU7441group
（P>
0.05）,（NU7441+IR）
groupofG2/M
phase
cellswassignificantly
increased
thanIRgroup
andthe
apoptosisrateand
Controlincreasedsignificantly
comparedwith
controlgroup
Conclusion:
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
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
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