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DOI:10.13350/j.cjpb.191112 ·!"
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】 #$ 23Y=>CagAv;¡Í¶;<Ü�ª¡/O=>�UV
。 %& MeCagAfa�½Ý¯?
pAdenoMCMVCagA3FlagIRES2EGFP(pAdenoCagA),hÄ HEK293;<
,@-�½Ý
,UÃÄEÃ
(Multiplicityof
infection,MOI)50ÃÄ;¡Í¶;<72h,A6�_�¶:
(犎犲犾犻犮狅犫犪犮狋犲狉狆狔犾狅狉犻,Hp)(^�GZ7(CagA+)
U MOI50
ÃÄ;¡Í¶;<24h,XY Westernblot2QCagAN!Ü�O2(HK2)、deÜhrDE1(GLUT1)、,4CO1
(ENO1)、ÿ���O1/2(PKM1/2)、Õ�&£OA(LDHA)Ü�ª¡/O�DE=>
,56T�;<m2QͶ;<
Xÿ�æ
。 '( ��Me¬CagAfa�½Ý¯?pAdenoCagA,6pAdenoCagA�½ÝN HpGZ7ÃÄ;¡Í
¶;<yÜ�ª¡/O HK2、ENO1、GLUT1�=>-p¯aù Hp4ÃÄaZ�Áb"�c
(犘<0.05)。-p¯aZ
�
,pAdenoCagAÃÄͶSGC7901;<24、48、72h,KG2/Mÿ;<õ�Zb"È´
(犘<0.05),ÃÄ72h�p¯
aNCagA�½ÝaG2/Mÿ�Z}�ÂJ16.90% N65.93%。 ') HpXYCagAZ;¡Í¶;<Ü�ª¡/
O=>�c
,�«-DNAÚ®Z;<·¸�G2/Mÿø�
。
【*+,
】 �_�¶:
;�½Ý
;CagA;Ͷ
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;;<Xÿ
【-./01
】 R378.911 【23456
】 A 【2781
】 16735234(2019)11129706
[犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔.2019Nov;14(11):1297-1302,1311.]
犈犳犳犲犮狋狊狅犳犆犪犵犃犵犲狀犲犲狓狆狉犲狊狊犻狅狀狅狀犲狓狆狉犲狊狊犻狅狀狅犳犵犾狔犮狅犾狔狋犻犮犲狀狕狔犿犲狊犻狀狆狉犻犿犪狉狔犵犪狊狋狉犻犮犮犪狀犮犲狉犮犲犾犾狊犫狔犪犱犲
狀狅狏犻狉狌狊
YANGLiping1,2,3,JIACencen1
,2,LONGNiya1,2,LUO Mei1
,2,YANGDan3,WANGJun3,XIE
Yuan1,2,ZHAOYan1
,2,WANGQinrong1,2,ZHOUJianjiang
1,2,4 (1.犓犲狔犔犪犫狅狉犪狋狅狉狔狅犳犈狀犱犲犿犻犮犪狀犱
犈狋犺狀犻犮犇犻狊犲犪狊犲狊,犕犻狀犻狊狋狉狔狅犳犈犱狌犮犪狋犻狅狀,犌狌犻狕犺狅狌犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔,犌狌犻狔犪狀犵,犌狌犻狕犺狅狌,犆犺犻狀犪550004;2.犓犲狔犔犪
犫狅狉犪狋狅狉狔狅犳犕狅犾犲犮狌犾犪狉犅犻狅犾狅犵狔,犌狌犻狕犺狅狌犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔;3.犆犾犻狀犻犮犪犾犚犲狊犲犪狉犮犺犆犲狀狋犲狉,犌狌犻狕犺狅狌犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔
犎狅狊狆犻狋犪犾,犌狌犻狔犪狀犵;4.犆犾犻狀犻犮犪犾犔犪犫狅狉犪狋狅狉狔,犌狌犻狕犺狅狌犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔犎狅狊狆犻狋犪犾,犌狌犻狔犪狀犵)
【犃犫狊狋狉犪犮狋】 犗犫犼犲犮狋犻狏犲狊 ToinvestigatetheeffectofCagAoverexpressionontheexpressionofglycolyticenzymesin
primarygastriccancercells. 犕犲狋犺狅犱狊 TherecombinantadenovirusvectorpAdenoMCMVCagA3FlagIRES2EGFP
(pAdenoCagA)wasconstructedandtransfectedintoHEK293cellstopackageadenovirus.Thisadenovirusand犎犲犾犻犮狅
犫犪犮狋犲狉狆狔犾狅狉犻GZ7(犎.狆狔犾狅狉犻GZ7,CagA+),anEastAsianstrainof犎.狆狔犾狅狉犻,wererespectivelyusedtoinfectprima
rygastriccancercellsfor72and24hoursatamultiplicityofinfection(MOI)of50.TheexpressionofCagA,hexokinase
2(HK2),glucosetransporter1(GLUT1),enolase1(ENO1),pyruvatekinase1/2(PKM1/2),andlactatedehydrogen
aseA(LDHA)proteinwereevaluatedusingWesternblotanalysis,andthecellcycleofinfectedcellswasdetectedusing
flowcytometry. 犚犲狊狌犾狋狊 TherecombinantvectorpAdenoCagAwassuccessfullyconstructed.Comparedtothecellsin
fectedwiththeemptyvectororcellsnotinfectedwith犎.狆狔犾狅狉犻,expressionofHK2,GLUT1,andENO1proteinwas
significantlydownregulated(犘<0.05)inprimarygastriccancercellsinfectedwithpAdenoCagAor犎.狆狔犾狅狉犻GZ7.
ThepercentageofcellsintheG2/MphaseincreasedsignificantlyingastriccancerSGC7901cellsinfectedwithpAdeno
CagAfor24,48,or72h(犘<0.05).TheratioofcellsintheG2/Mphasewas16.90%intheemptyvectorgroupand
65.93%inthepAdenoCagAgroupafter72h. 犆狅狀犮犾狌狊犻狅狀 犎.狆狔犾狅狉犻inducedthedownregulatedexpressionofglyco
lyticenzymesbyCagAinprimarygastriccancercells.ThisactionmayberelatedtoDNAdamagethatarrestscellsinthe
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犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔 2019û11ü ý14þý11ÿ
Nov.2019, Vol.14,No.11
【9:;#
】 !"#$+&0%&'
(No.31560326,31660031);ÏË)+4¾:+âa&¦&'
[2017]5652;ÏËÖ+4E�+
â~&'
[2017]5-16,ÏË)�Ë�+40%&'
(No.gzwjkj20151010)。
【<=>?
】 Xe�
,Email:jianjiangzhou@sina.cn
【>?@A
】 t£2
(1995-),-,ÏËÎaÂ
,�1/0
,ϸ]nͶ[e�23
,Email:1754177190@qq.com
G2/Mphase.
【犓犲狔狑狅狉犱狊】 犎犲犾犻犮狅犫犪犮狋犲狉狆狔犾狅狉犻;adenovirus;CagA;gastriccancer;glycolysis;cellcycle
Ͷ·º[¡ýð%ËÞÕ ÌÍNý�%uË
Þ¶Ð12;`
[1],K_.·�`ñXâe6�Lì
,
ϸ@>�_�¶:
(犎犲犾犻犮狅犫犪犮狋犲狉狆狔犾狅狉犻,Hp)ÃÄ
、
WÏ�`ñ¤OP`ñ
,Kb HpÃķͶf¸
�V_`ñ
[2]。;<Ýñ��0`ADE
(Cytotoxin
associatedgeneA,CagA)· Hpf¸�Ýô`n
,�
Hp0`abCagAÝôí
(cagpathogenicityisland,
cagPAI)4ab
。p Hpã¯�WÏÍ¡@;<y
,
XY;:Ⅳ¤�´@�sCagA@i;<
,�;<=
U��Cù4��C�[��P��WOXK
,õ-
Ͷ�_._¹
,QK¼¶õömn9N*o
[3]。Ca
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;<=DE_ße6�¶DE
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。
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,üO%�deÜ
,Z{
HË�¡/[��¥J"ø�Ü�ª"(aerobicglycol
ysis),R� WarburgëL
(warburgeffect)[4-5]。
de
Ün«#�XY;<ÿÛHäpLMwi;<
,�¸
½§;<ÿ¡�deÜhrDE
(glucosetransport
ers,GLUT)¦«�¢hr
。Ü�ªb�¡/Oø�
�O
(hexokinase,HK)、��ìÜ�O1(Phospho
fructokinase1,PFK1)、��½���O
(phospho
glyceratekinase,PGK))、,4CO
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(pyruvatekinase,PK)NÕ�&£O
(lac
tatedehydrogenase,LDH),Kb HK、PFK1、PK·
Ü�ªYab��¥O
,��Cde��CJ6
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,4ÿ��hC�ÿ���©n�6�¦6KL
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[6-9]。©23K
MepAdenoMCMVCagA3FlagIRES2EGFP�½
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(N¥JpAdenoCagA),-admax�½Ý@-
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(genebank�iO
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PKM1/2、LDHA;¦
/�PQ¨èª§«!CellSig
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âã|}~ª§�©TAKRA¬
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Invitrogen¬
。
2 %&
2.1 CagA ÄÅPCR �� DdpMD18T/CagA
GZ7HgDNAJÑÒ
,ÊËAøEcoRI、BamHIOµ
{¶�¡�ÌwV
,Z6PrimeSTAR�k£OÇÈ
CagA'�0`
(äV��3570bp)。PCR?@
:5×
PrimeSTAR? Buffer(Mg2+plus)10μl,dNTPMix
ture(�2.5mM)4μl,wV
:CagAF(10μM)1μl,
CagAR(10μM)1μl,PrimeSTAR?HSDNAPoly
merase(2.5U/μl)0.5μl,ÑÒDNA1ng,ÿ:!�
`"#ì50μl?@
;PCRKLaq
:DNA^
:98℃
,10s;ÓÔ
:55℃,5s;72℃;ÕÖ
;30FØO
。µÞ
âãBC PCR äV
。CagAF:5'AGCTTGGGCTG
CAGGAATTCGCCACCATGACTAACGAAACCA
TTAATCAAACAACAAC3';cagAR:5'CATCCTT
GTAGTCGGATCCAGATTTCTGGAAACCACTT
TTTGTAGTATTTTTGAC3'。
2.2 ª 6 ú @ A pAdenoMCMVCagA3Flag
IRES2EGFPJUV Z6@�PQ|}~¾¿äO
µy�� ¯?NCagA 0`
,hCÃS�犈.犮狅犾犻
DH5α,èdL PQDdHgDNA,6EcoRI、Bam
HI½ö =µOvL PQ�HgDNAwxäO
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犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔 2019û11ü ý14þý11ÿ
Nov.2019, Vol.14,No.11
µ
、: é PCR (
w V Forward:5'CTAC
GAAAAAGAATGCTTTGAAG3';Reverse:5'CCT
CACATTGCCAAAAGACG3')¤QqáR
。wVâ
�¤Qq�¡ÚNÅ.V4m¬9�
,áRÊð�
/NJpAdenoCagA。
2.3 QRª6úpAdenoCagAJ«¬
、��
、]^?
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denoCagA-admax@�í§Hg121∶1×hÄ
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。ãúÃÄ12、24h�;<DEH6
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E6BCA\QRDE��
,Westenblot2QCagA、
HK2、GLUT1、ENO1、PKM1/2、LDHA DE=>
。
d30~50μgDE¡Ç
,wxSDSPAGEßà
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;<
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1 u1ZÜg狆犃犱犲狀狅犆犪犵犃$ÀÝ�WX
6PCR[\]pMD18T/CagAGZ7HgbÇ
ÈÅ�CagA�N�qr
(J1A),Me�fa�½Ý
¯?�EcoRINBamHIäOµ
,'�I?%BJ3
570bp(J1B);:é PCRáRfa¯?MeÊð
(J1C);s�½Ý¯?QqAwxBLASTZv�
q
,-pMD18T/CagAGZ7bCagA qr9N¨¼
(J1D),�½Ý¯?pAdenoCagA Me��
。s
pAdenoCagA�½ÝÃÄ HEK293T;<
,6Flag
;�PQè?XY Westernblot2QH�;<bø
CagADE=>
(J1E)。spAdenoCagA�½ÝU
MOI50ÃÄ;¡Í¶;<
,56CagA;�PQè?
Wersternblot2Q;<bøCagADE=>
(J1F)。
2 vYZ犆犪犵犃¶Ø�wx¤¥ac$yz
;<�¢"��¤"���&·^· Hp(CagA+)
ÃÄ�GH ²K
。�Â6pAdenoCagA�½ÝU
MOI50ÃÄ;¡Í¶;<72h¤ HpGZ7U MOI
50ÃÄ;¡Í¶;<24h,-v2;<Z�ÃÄ;<
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L�«
。
·9921·
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犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔 2019û11ü ý14þý11ÿ
Nov.2019, Vol.14,No.11
A CagA0`PCRäVßàJ B CagA�½Ý¯?�EcoRINBamHIäOµäVßàJ C L PQ:éPCRäVßàJ D
pAdenoCagAHgê�qrZv E Westernblot2QHghÄ293T;<bFlag�=> F Westernblot2QpAdenoCagA�½ÝÃÄ;¡
Ͷ;<72hCagADE�=>
.1 u1ZÜg狆犃犱犲狀狅犆犪犵犃$WX
A ElectrophoresisofCagAgenePCRproducts B ElectrophoresisofCagAadenovirusvectorbyEcoRIandBamHIrestrictionenzymediges
tionproducts C ElectrophoresisofpositiveclonesPCRproducts D pAdenoCagAPartialsequencealignment E TheexpressionofFlagwas
detectedintransfected293TcellsbyWesternblot F TheexpressionofCagAproteinwasdetectedinprimarygastriccancercellsinfectedwithpAd
enoCagAadenovirusfor72hoursbyWesternblot
犉犻犵.1 犐犱犲狀狋犻犳犻犮犪狋犻狅狀狅犳狆犃犱犲狀狅犆犪犵犃犪犱犲狀狅狏犻狉狌狊狏犲犮狋狅狉
A pAdenoCagA�½ÝÃÄ;¡;<���^C B HpGZ7ÃÄ;¡;<���^C Mock pEv2a NC p¯a CagA pA
denoCagAa
.2 犆犪犵犃¶Ø�wx¤¥ac$yz
A MorphologicalchangesofprimarygastriccancercellsinfectedbypAdenoCagAadenovirus B Morphologicalchangesofprimarygastric
cancercellsinfectedbyHpGZ7 Mock Controlgroup NC Emptyvectorroup CagA pAdenoCagAgroup
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