Bước đầu xây dựng quy trình phát hiện EBNA 1 ở các mẫu mô thu nhận từ bệnh nhân ung thư vòm họng

84

KHOA HQC CONG NGHe

BUtiC DAU XAY DUNG QUY TRiNH PHAT HOT EBNA-1 O CAC
MAU MO THU NHAN 're 13tNH NHAN UNG THU' VOM HONG
NO), nhan bai: 16/06/2015
Ngay nh4n loi: 11/07/2015
Ngay duy4t clang: 26/10/2015

Lao Dar Thutini
Nguyen Hoeing Anh Tuan2
Nguyen Thi Yein Tuyit3
Nguyen Him Diing4
Le Huy'dn Ai Thay5

TOM TAT
TO Viet Nam, ung tint vont hong la clang ung tint pha bd'n nit& trong cac benh ung tint
thuac vitng deiu va c6. Nhieu nghien thu da charzg minh rang su xam nhiem caa Epstein — Barr
virus (EBV) co mai quan he mat thiet va la mat trong nhimg nguyen nhein gay benh ung tint yam
hong. Nham intang tai viec xely dung quy trinh than clown sant benh ung tint vOm hong, chting
toi lua chon gen EBNA-1 (Epstein — Barr nuclear antigen 1), mat gen can thief cho su xam
nhilm diet EBV, lam gen muc tieu cho viec xeiy dung quy trinh phat hien su hien dien cita EBV.
Trong nghien cau buoy dau, chting toi tien hanh thong ke tan so trung binh trong so ve su hien
dien cart EBV trong nhieu loaf mau benh pheim khac nhau. Dong thai, thong toi da xely dung
thank tong quy trinh PCR phat , hien gen EBNA-1 tren cac man mo benh ung tint vorn hong thu
nhein tit Benh Vin Cho. Ray. Ket qua nghien thu &tor dew nay fa tien de cho viec pheit trien cac
pingang phap than clown khong xam lan nham than clown sem: benh ung tint yam hong duct tren
Icy thuat sinh hoc phein to tren claf tztong cac gen lien quan den su xam nhiem adz EBV.
Tir khoa: EBV, EBNA-1, ung tint vOm hong.
ABSTRACT

In Viet Nam, Nasopharyngeal carcinoma (NPC), originating in the nasopharynx, is the most
common cancer of the head and neck cancers. The association of NPC with Epstein — Barr virus
(EBV) firmly demonstrated that EBV played an important role in nasopharynx carcinogenesis.
For the aim to establish the method for early diagnosis and prognosis of NPC, EBNA-1 (Epstein
— Barr nuclear antigen 1) which is necessary for infection of EBV, was chosen as target gene to
establish the method for detection of EBV. In this study, we systematically analyzed the mean of
percentage weighted of the presence of EBNA-1 in various sources of sample. Moreover, we
initially succeed to establish the PCR method for detection of EBNA-1 in biopsy tissues collected
from nasopharyngeal cancer patients in Cho Ray hospital. These results indicated that, in the
future, it will be useful in developing a non-invasive method for early diagnosis and prognosis of
NPC based on molecular techniques in detection of concerning genes which were EBV infection
required.
Keywords: EBNA-1, EBV, Nasopharyngeal carcinoma.
' ThS, Trutmg Dai hoc Ma TP.HCM.
Tnrtmg Dai hoc Ma TP.HCM.
3
Trutmg Dai hoc Ma TP.HCM.
4 TS. BS, B'e'nh vi4n Chq Ray TP.HCM.
5 PGS.TS, Truting Dai hoc Ma TP.HCM. Email:
2


TAP CHI KHOA HOC TRUONG DAI HOC MO TP.HCM - 56 6 (45) 2015
1. Giiri thieu
Ung thu yam hong (Nasopharyngeal
carinoma) là mot khoi u dc tinh xuat phat tir
lap bleu mo cua yam hong. DOng thai, day la
loai ung thu pH) bien nhat trong cac benh 15,
vimg dAu (Phiic va Ong su, 2008; Yang va
cong su, 2014). TS, le mac benh ung thu yam

hong c6 su khdc biet dia ly gicra cdc. khu vuc
khdc nhau. Trong do, Viet Nam norm
khu vuc c6 tS/ le mac benh cao, cu the
the theo
thOng ke norm 2012 (WHO), thAy tj, le mac
benh ung thu vom hong va tir vong Ian lugt
len den 3,9% va 3,3% dan so (World Health
Organization, 2012).
EBV (Epstein - Barr virus) la, virus co
tinh nhiem pho rung trong cong dOng dugc
phat hien a 90% ngtthi tnrang thanh, thuOc
nh6m Herpes virus type 4 va dugc nghien cim
rat sam tir norm 1980 (Philc va Ong su, 2008;
Niedobitek, 2000). Su xam nhiem cua EBV la
mot trong ba nguyen nhan chinh bao gem: ,(1)
su xam nhiem
EBV; (2) moi truong song
thOi quen sinh hoot; (3) bien doi di truyen dAn
den hinh thinh ung thu vom hong (Frappier,
2012; Tao va cong su, 2007). Tren the giai,
rat nhieu cong trinh nghien ciru dor chimg
minh su hien dien cua phan tir DNA, RNA,
cac protein cua virus bieu hien a cac to bao
ung thu yam hong va cac to bAo bf nhiem
EBV có nguy ca tie'n trien thanh ung thu vom
hong. Chang Ilan, nghien ciru dm Chang va
Ong su (1990) thuc hien ten 50 mh ,m13 yam
hong khong biet hoa, ket qua cho thay EBVDNA hien dien trong cac mh mo vai lugng
ban sao rat cao (Thompson va cong su, 2004).
EBNA-1 (Epstein - Barr nuclear antigen 1) la

mot gen thuOc ho EBNAs ram trong bO gen
cua EBV, co vai tro quan trong trong quA trinh
)(am nhiem, nhan len va duy tri bO gen EBV
trong mo ung thu vom hong. [Xing thai,
EBNA-1 con clang vai tre ‘quan trong trong
viec duy tri trong thdi tiem tan cua EBV
(Chang va cong su, 1990; Frappier, 2012; Tao
va cong su, 2007). Cu the horn, EBNA-1 ma
hoa cho protein EBNA-1, EBNA-.1 tuong tdc
vai vi tri OriP, diem tri khai dau sao chdp,
khoi phat su nhan len cua EBV-DNA

85

(Frappier, 2012; Thompson va cong su,
2004). Hon nita, EBNA-1 con c6 vai tro trong
viec kich hoot su bieu hien dm cac gen khdc
trong giai doan tiem tan nhu LMP-1,LMP-2,
BARTs,... (Brooks va cong su, 1992;
Frappier, 2012; Thompson va cong su, 2004).
Do do,,gensEBNA-I va san phAm cu n6 &Km
tim thy hau het cac khoi u vom hong lien
quan den EBV (Brooks va cong su, 1992;
Phiic va cong su, 2008; Zhang va cong
2012). Chinh dAc diem nay dor ma ra mot
khuynh huang sir dung EBV-DNA hay cac
gen lien quan nham phdt trien mot ky thuAt
chan dodn sam khong xam Ian benh ung thu
yam hong, tir do nang cao hieu qud dien tri va
khA nang song sot cua benh nhan.

Trong nghien ciru nay, chimg toi he thong
hoa 14i cac du lieu khoa hoc nghien dm lien
quan ten doi tugng ung thu yam hong va gen
EBNA-1 trong cac loaf benh pham khdc nhau.
Dong thai, bulk dau xay dung quy trinh PCR
phat hien su hien dien cua gen EBNA-1 trong
cac mo sinh thiet tir benh nhan yam hong
dugc Itru gift va bao quan trong hai dung moi
khac nhau là Formalin va PBS.
2. V4t lieu va phtrang phi')
Mau nghien cuv ,
6 mh mo sinh thiet tir benh nhan dugc
chan doan mac benh ung thu vom hong thu
nhAn tir benh vien Chg RAy Thanh phO HO
Chi Minh. Cdc mh mo dugc Itru gift bao
quan a nhiet dO 4 °C trong hai loci dung moi
khac nhau: formalin va PBS (Phosphate
buffer saline).
Khai thac dir lieu
Cdc bai bdo du.' lieu khoa hoc lien quan
dugc khai thac tren ca sor dft lieu NCBI
(www.ncbi.nlm.nih.gov) bang cac tir kh6a
nhu: nasopharyngeal carcinoma (ung thu vein
hong), EBNA-1, EBV (Epstein - Barr virus),
non-invasion method (phucmg phdp khong
xam ran), v.v... Dit lieu khai thdc duce sir
dung de thOng ke, xac dinh tan so trung binh
co trong so cua su hien dien cua gen EBNA-1
tren nhieu loci mau benh pham ung thu khdc
nhau nhu mau dich phet, mo sinh thiet, mdu,

v.v... bang cac ky thuAt sinh hoc phan tir khac


86

KHOA HOC CONG NGHe

nhau nhu PCR, Real-time PCR, v.v...
'rich chiet DNA
Cac mh benh pham dtroc tach chiet
theo phuong phap Phenol/Chloroform theo
quy trinh tach chiet cua Chomczynski va
Sacchi (1987) c6 su cai Nen Phenol tir pH=4
thanh pH=8 (Chomczynski, 1987). NOng dO
va dO tinh sach DNA tir cac mh benh pham
sau tach chiet &roc xac dinh bang phuong
phap do mat dO quang thong qua ti le

A260/A280 nm. Sau khi tach chiet, DNA mh
&roc luu
trong TE (Tris-EDTA) va bao
quan a nhiet dO -20 °C cho den khi sir dung
sau nay.
Phin irng PCR khuech did gen EBNA-1
Trinh to cap moi sir dung trong phan
PCR khuech dai gen EBNA-1 duoc tham khao
tir cong trinh nghien ciru cita Adam va cOng
su (2011). Trinh to cap moi cluoc the hien O.
Bang 1.

Bing 1. Trinh tir cep moi sir dung trong nghien cirti
Ki hieu

Trinh to

EBNA-F (M6i xtoi)

TGAATACCACCAAGAAGGTG

EBNA-R (MOi ngugc)

AGTTCCTTCGTCGGTAGTC

Chieu dai san pham
223 by

Chu trinh phan *rig PCR &roc thiet ke
nhu sau: 1 chu ky yen 95°C-5 phut, 40 chu kS,
14p lai yeti 95°C-30 giay, 42,5°C-30 gifiy,
72°C-30 giay va 1 chu kS' vai 72°C-5 phitt.
Tiing the tich phan ung la 15 bao gOm: 7,5
Mastermix 2X, 1 1.t1 moi (0,5 IA moi xuoi,
0,5 IA moi ngugc), 5,5 IA ddH2O, 1 1.11 DNA.
San pham phan ung PCR dugc kiem tra bang
phuong phap dien di teen gel agarose 1,5% va
gui giai trinh to tai cong ty Nam Khoa. Ket
qua giai trinh to dugc tier' hanh hieu chinh vi

kiem tra mirc dO tucmg dOng tren co sa dt lieu
Genbank (NCBI).

3. Ket qui vi thio luin
Khai thic dir lieu
Cho den nay, chimg toi khai thac dtroc 13
ding, trinh nghien ciru ten the giai lien quan
de tin hanh thong Ice danh gia ve loai mAussir
dung, ca mh, phuong phap sir dung nham
phat hien su hien dien cita gen EBNA7 I tren
berth ung thu vOm hong. Ket qua thong ke
dtrac the hien ei Bang 2.

Bing 2. ThOng la loai mau vi phirorng phip dime sir dung trong phit hien EBNA-1
MO sinh
•
thiet

,
Dich phet

Mau

Mo (tic
parraffin

TOng Ong
n(%)

Multiplex Methylation Specific
PCR

1

1

-

-

2 (15,4)

Nucleic Acid Sequence — Based
Amplification

1

-

-

-

1 (7,7)

1

-

1

2 (15,4)

Real — Time PCR
PCR

2

1

1

-

4 (30,7)

Nested PCR

-

-

1

-

1 (7,7)


87

TAP CHI KHOA HQC TRLIONG DAI HOC M6 TP.HCM - S6 6 (45) 2015

MO sinh
,
thiet

,
Dich phet

Mau

MO Mc
pang&

T6ng cOng
n(%)

Nested PCR and Sequencing

1

-

-

-

1 (7,7)

Quantitative PCR

1

1

-

-

2 (15,4)

T6ng Ong

6 (46,1)

4 (30,8)

2 (15,4)

1 (7,7)

13 (100,0)

Ghi chic - khong co sti clung trong nghien thu
Ket qua th6ng ke cho thy phucrng phap
sir dung chit yeu trong viec phat hien EBNA-1
trong cac nghien ciru tren the Oen la phucmg
phap PCR (chiem 30,7%). Ben canh do, ve
loai mh sir dung, chit yeu la mo sinh thiet

(46,1%) va dich phet (30,8%). ,Tir cor o.sdir
lieu thu thap dugc, chang Vol tien hanh thong

ke tan s6 trung binh phat hien gen EBNA-1
trong nhieu loai mh mo khac nhau dugc the
hien a Hinh 1.

Tin so
phiit hien
EBV (%)
100
0 n=<30

90
80

0

30
70

O

50
60
50
40

CD icio>n
Trung binh trong so

30
20
10
0
Te bac) binh Te bio Dich phet
thuong UTVH

M6

Mau

Hinh 1.ThAng ke sty hien dien ctia EBV trong to bao binh thtrirng, to bat) ung thy vont hong
(UTVH), trong cac loai mau bing thong ke trung binh co thong so
Chu thich: tam throng trim the hien tan so clic" cac bai bao nghien ctiu tren gen EBNA-1 nham phat hien
EBV, kick
throng Iron the hien so luvrng mciu trong cac nghien
&rang thing nom ngang the
hien tan so
s6 zueit hien EBV trung binh co trong so.
Ket qua th6ng ke tin so trung binh trong
so phat hien gen EBNA-1 a to bao ung thu vern
hong, dich phet, mo va mau tan luot la 99,24%,
99,70%, 98,55% va 68,85%. Ngoai ra, tan so
trung binh trong s6 a cac mh to bao binh
thugng la 0%. Ket qua cho thdy c6 su khac biet
rat lin ctia su hien dien gen EBNA-1 a cac mh

ung thu so voi to bao binh thlremg. Dieu nay
cho thdy, su hien dien dm gen EBNA-1 la dau
hieu d4c trung cho benh nhan ung thu vom

hong va c6 the htrang tai vi'ecsir dung nhu mot
dau chimg sinh hoc trong chin doan sgm ung
thu yam hong. Ngoai ra, chitng toi nhan thay
tan so trung binh trong , 6. mau dich phet va


88

KHOA HQC CONG NGHE

mo rat cao va c6 gia tri tan s6 gan bang nhau.
Dong thai, theo ket qua thong ke loai mau sir
dung thi mau dich phet va mau ma la hai loci
mau dugc sir dung nhieu nhat. Do dO, viec sir
dung hai loci mau nay la thich hop cho viec
phat hien su hien dien cua gen EBNA-1 bang
ky thuat PCR. Tuy nhien, trong giai han
nghien tuft nay, chimg toi lua chon sir dung
loci mau mo sinh thiet lam doi tugng nghien
dm de phat hien gen EBNA-I bang ky thuat
PCR. M6t van de can luu y la khi thu nhan
mau mo sinh thiet can phai dugc luu gift va
bao quan bang dung dich nao thich hap. Theo
cac nghien ciru tren the giori, hai loaf dung dich
dugc sir dung trong bao quan nhieu nhat la
dung dich Formalin va PBS. Do do, chimg toi
tien hanh khao sat tren cac mau mo sinh thiet
L

223 by

(-)

A1

A2

A3

A4

AS

dugc Itru gift bang hai dung dich nay.
Khuech dai EBNA-1
Trong nghien cim nay, chitng toi tien
hanh khao sat viec khuech dai gen EBNA-1 tir
cac mAti, m6 sinh thiet thu nhan tir benh nhan
dugc than down ung thu yam hong dugc luu
Oa a hai dung mai bao quan la PBS (14 hieu
tir Al den A6) va Formalin (ky hieu tir B1 den
B6). DNA sau khi tach chi& dugc tien hanh
kiem tra mirc d6 tinh sach va ruing do DNA
bang phucmg pilaf) do mat d6 quang a busk
song 260 nm va 280 nm. Nhin chung, cac mau
tach chiet deu co ty so A260/A280 nam trong
khoang chap nhan dugc, co the sir dung de
thuc hien phan ung PCR khuech dai gen muc
tieu (DO- lieu kung trinh bay).

A6

GINO MID

100b p

Hinh 2. Ket qui di?n di san phim PCR khuech d3i gen EBNA-1 cac mitt mo sinh thiet
ung thu vom hong
Ghi cha: L: Chang DNA 100 bp; (-): mau cluing am; , Al cl&I A6: 6 mau m6 sinh thief dwac
dugc btu girt
trong dung dich PBS; BI den B6:6 mow m6 sinh thiet dwo.c luu girt' trong dung moi Formalin.
Dua ten ket qua dien di, tai cac mau
dugc luu girt bang dung dich PBS xuat hien
mot bang vai kich thtrac khoang 223 by ar cac
mau Al, A2, A4 NIA A6. Kich thuac san pham
nay phi' hop veil kich thuac gen EBNA-I dugc
khuech dai vai cap moi EBNA-F va EBNA-R.
cac gieng A3, A5 kung ghi nhan dugc bat
kS, mot bang nao. Doi veil cac mau dugc hru
gift bang dung dich Formalin, a cac gieng fir
B1 den B6 khong ghi nhan dugc bat 14 mot
bang nao, chi ghi nhan dugc cac bang ky sinh
co kich thtroc dual 100 bp, cac bang nay dugc
du down la cac clang cau trac thin cap dm moi.
Tir ket qua PCR ghi nhan rang viec bao quail
m6 sinh thiet bang dung dich Formalin anh
huang den chit lugng cua DNA-EBV. Dieu

nay c6 the giai thich rang: cac mau mo sinh
thiet luu girt trong dung dich Formalin trong

mot khoang thai gian dai hcm 12 — 24 tieng,
ket qua dln den su phan huy dm DNA thong
qua viec be gay cac lien ket cua phan tit DNA.
Dieu nay hoan toan
hap vol nhan dinh
dm Campos va Gilbert (2012) (Campos va
tong su, 2012). Do do, chimg toi lua chon
dung dich PBS lam dung dich luu girt, bao
quan mau m6 sinh thiet ung thu yam hong a
nhiet d6 4 °C.
Nham khang dinh cac bang c6 kich thuac
223 by la gen EBNA-1, chting toi Wen, hanh
giri giai trinh
mot mau dai dien. Ket qua
giai trinh to dugc the hien a Hinh 3.


TAP CHI KHOA HOC TRUONG DAI HOC MO TP.HCM - SO 6 (45) 2015

T

6

89

2 2 8

tr

;

Hinh 3. Ket qui giii trinh tly san phim PCR: (A) mach xuoi; (B) mach ngulyc

Dua teen ket qua giai trinh tu cho thAy
cac peak dm san pham deus rat sri3 rang chi
trir mot so nucleotide tai phan dau cua trinh
tu. Dieu nay ducirc giai thich, tai vi tri mei
bat cap, khi giai trinh tu tin hieu viing nay
khong Ira rang. Wing nay se dugc loai be khi
tien hanh hieu chinh trinh tu. Doan trinh tu
gicra rOs rang duoc kiem tra mire dO
Bong bang chuong trinh BLAST. Ket
Ket qua
BLAST cho thay, trinh tu thu nhan dirge co
dO tucmg (long cao vei trinh tu gen EBNA-1
caa human herpes virus 4 (Ma so truy cap:
HQ827839) vai cac gia tri dO tucmg Tong
98%, gia tri e-value gan bang 0 (Ident =
98%, E-value = 4e-101). Nhu vay, chung toi
da khuech dai thanh cong gen EBNA-1
mau mo sinh thiet thu nhan tir benh nhan ung

thu vem hong chrorc luu giir bao quan bang
dung dich PBS.
4. Ket lua'n
Tir ket qua khai thac da lieu, cluing toi
ghi nhan rang su hien dien dm gen EBNA-1 la
mot dau chung dac trung cho benh nhan ung
thu yam hong. Ve thuc nghiem, chung toi da
buck dau xay dung thanh cong quy trinh phat

hien gen EBNA-1 bang phuang phap PCR tren
doi tuang mh mo sinh thiet &roc luu
trong dung dich PBS a nhiet dO 4 °C. Cac ket
qua btrac dau nay cho phep chting toi huang
tai viec khao sat su hien dien dm gen EBNA-1
tren so lucmg mh benh pharn Ian horn de
hureng tai viec ,irng dung xay dung mot
phuong phap chan down sem ung thu yam
hong or nguCri Viet Nam.

TAI LIEU THAM KHAO

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Sudanese patients and its Association with epstein-Barr Virus: A Report from a single
center in Khartou, Advances in Tumor Virology, 2, 1-6.
Brooks, L., Yao, Q. Y., Rickinson, A. B., &Young, L. S. (1992). Epstein-Barr virus latent gene
transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP I, and
LMP2 transcripts, J Virol., 66(5), 2689-2697.


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