ph6i cria t i ph6i chuQt

(1)

T4p chi Khoa hgc DHQGHN, Khoa hgc Tr.r nhi6n vA C6ng nghQ 26 (2070)77-82

ph6i cria t ⁱ ^ph6i ^chuQt

D?ng vdn Dricr'*, Nguy6n Lai Thenhr, Bni ViQt Anhr, Dd Doan Lgi2

'Khoa ^Sinhhqc, Trudng Dqi hqc Khoa hpc

Ttr

^ian,^DHQGHN,³³⁴Nguydn Trdi, Hd ^Npi,Vi€t Nam zTrudng

Dqi

^pc^YHd ^N1ri,

I

T6n Thdt Timg, Di5ng Da, Hd NOi

Nh4n ngdy 19 th6ng 01 nlm 2010

T6m

tit.

O Viet Nam, cdng nghQ t6 bdo g6c vAn cdn kh6 mdi md ilflc biet le ti5 Uao g6c ph6i (Embryonic stem cells

-

^eSCs;.Cac nghiOn cuu hiQn nay chri yiiu tQp trung vdo t6 bdo gdc trucrng tfraofr ua hAu hrit m6i chi thu dugc nh{ing thnnh c6ng bu6c dAu. Nghi€n cuu ndy cria chring t6i nhim thiiit lflp vd hodn thiQn phuong ph6p phdn lfp cflng nhu nu6i cAy l6u ddi ESCs tit nft ph6i

cria

tui

^dng^Swi

trung

v

^ldn^cAY

lo4i

t6

^dugc^th

mdOct3/4 _-

^Oct3l4).

dich

hulnh

^ioct3l4^cho^th6y^{c6c t6}^bdo^g6c^ph6i^chugt^(mouse

.

^-

mESCs)

^chuytin^v6i^gan:O^ngdy^nu6i^cAy^v6n

^git

^dugc^tl6c

di6m cria tO bdo g6c.

Ti kh6a: ti5 uao gi5c ph6i, tii bao goc ph6i chuQt, Alkaline phosphatase, oct3/4.

1. M& ttau

ESCs ld mQt dang tti bao gtic don ^nh6tthu nhfln tir

nit

ph6i (inner cell ^mass)cria tr"ri ph6i (blastocyst) elQng

vft

c6 vri. ^ESCsc6 khd nlng

t.u d6i m6i vd biet ^h6athdnh t6t cd chc dang ti5 bdo cta co th6 trudng thenh. Bing chung

li

khi nudi c6y in vitro, ^ESCstao n6n c5c qudn l4c ^ti5 bdo tlu-o. c ggi ln th€ ph6i (embryoid bodies), ^c6 nhting virng biQt h6a thanh c6c ^d4ng^tObio kh6c nhau c6 ngudn g5c

tt

^ba¹⁶ph6i [1-5]. Khi ti6m ESCs vdo chuOt thiiSu hut hQ th5ng mi6n dich ^sE

-ra.

g,u tien hQ. DT.: 84-4-35589653.

E-mail : [email protected]

t4o thdnh u qu6i mang c6c lo4i m6

h

d6n xu6t cria ba 16 phdi [6,7]. ^{rnESCs c6}

khi

ndng t4o n6n th6 khim vd ttic bigt ld t4o n6n co ^thiSkhem ddng sinh t8l. ^MQt^vdiddng mESC ^tlu-o.c chimg minh c6

khi

ndng t4o thdnh

cic

^bdo_{thai [9].}

Sau

khi

ddng mESC dAu

ti6n

clugc thu

nhin

ndm 1981 [10,11], c6c ddng ESC ^kh6c cfing dd ^du-o.c ph6n lflp

tir

nhiAu lodi dQng vflt kh6c nhau, dac

bipt t6

bno g6c

phdi

ngudi (human embryonic stem cells

-

^hESCs)^dd^tluo.^c

Thomson

tao ddng vdo nlm

1998 ll2-17].

ESCs dugc chimg minh ^{biQt h6a}in vitro thirnh c5c t6

bio

gi5ng t.5 bdo gtic t4o m6u [18], ^tt5^bdo thAn kinh

|g-22),

^t6^bao^co

^tim

^123,24),^c6c^t6

7l

(2)

72

^{D.V. Ditc}^ud^nnk.^/^T4pchi Khoahqc DHQGHN, Khoahgc Ty nhi€n ud C6ng nghi 25 (2010) 7L-82

bdo nQi m6 _125,26)vd c6c

tti

bao ei6o tpy tii5t insulin...[27-29]. DiAu ndy cho'th6y ESCs c6 tiAm ning biQt h6a

thinh

t6t cd nhfrng d4ng tiS

bdo di6n hinh c6 ngu6n gtic

tt

ba l5 ph6i. Chfnh

vi

ti6m ndng bipt h6a efac biet

cta

ESCs ldm cho chring trd thdnh mQt trong nhirng m6 hinh ti5t nh6t d€ nghiOn cr?u: phdi sinh hgc, c5c qu6

trinh biQt h6a, liQu ph6p thay thd t6 bao vd m6, phuong tipn cho liQu ph6p gen, trong nghi€n c{ru co ch6 c6c benh d ngudi, ph6t tri6n vd

tht

tlOc tinh dugc phAm. ..120,29-31).

C6c nghiOn

criu tr€n th6 gi6i dd

chung

minh dugc ring

mESCs bi'5u

hipn

enzyme Alkaline phosphatase.

N6

le mQt enzyme thriy phAn chiu tr6ch nghiQm c6t gtic phosphate ra kh6i r6t nhi€u c6c phAn

tu

nhu c6c nucleotide, protein

vd

c6c alkaloid,

do vfy, n6

<lugc sri dgng nhu ld mQt marker <l4c tnmg vd <lugc srl dsng ph6 biiin tl€ x6c <linh c6c ddng ESC chua bigt h6a [32,33]. Ngodi ra, mESCs bi€u hiQn manh cdc marker bd mat kh6ng nguydn ctac hi€u

giai

cloan

ph6i-1

(Stage-specific embryonic antigen-l

-

SSEA-I), khSng nguyOn

nhin

d4ng

ftrOi

u-OO

(Tumor

recognition antigen-60 ^- TRA-1-60)

vd

TRA-1-81 [34,35]. ESCs ^cfing bi6u hi€n m6t si5 gen

tl{c

hi€u,

tlitin hinh

ld Oct3l4, mQt nh6n t6 phi€n md c6 li€n quan di5n

qu6 trinh t.u

d6i m6i

cria ESCs [36-38]. ^MQt

nhAn t6 phi6n m6 tlii3n hinh kh6c

li

Nanog cfrng c6 vai trd trong qu6 trinh t.u d6i m6i cta t6 bao vd thudng duo. c sri dpng d6 x6c dlnh ESCs chua biQt h6a

[3941].

^Trong^nghiOn^cr?u^ndy,^chring

tdi lya

chgn

hai lo4i

marker

img ct vi€n li

Alkaline phosphatase

vd

Oct3/4 d6 ti6n hdnh nhfln bitit mESCs nu6i c6y in vitro c6 cdn birSu hiQn nhtng marker cta tidm ndng hay khdng.

2. Nguy€n liQu vir phuong ph6p 2.1. D1ngvdt

Otii

tuqng nghidn ciru chring

t6i

sri dgng trong thi ^nghiQm

li

^chuOtnhat trang ddng Swiss

(Mus musculus) dugc cung c6p

bdi

ViQn VQ

sinh Dich t6 Trung uong. ChuQt c5i tluoc kich thich si€u bdi nodn d6 thu phdi theo md td cria Nagy vd cQng sp [42]. ^ChuQt^thdurh^thgc^sinh sdn (2-3 _th6ng

tu6i, trgng

lugng

ft

^28-329)

tlugc nudi trong tli€u ki€n 14 gicr chi6u s6ng vd 10 gid t6i. Chudj c6i du-o. c ti€m hormone PMSG (l0lU/chuQt) vdo

gita

chu kj,s6ng, sau 4648 gid ti6m hCG (I0ru/chuQt)

vi

dusc ghdp tl6i v6i chuQt <luc.

l8h

^saukhi ti6m hCG ti6n hinh kiiim tra ^chuQtph6i. ChuQt c6i c6 d6u hi6u giao ph6i cluo. c nhlit rieng vd chim s6c di5n 3,5 ngdy cl6 thu phdi.

2.2. Chuiin bi lop te bdo nu6i

mESCs tlugc thu nhf,n theo md ta cria Nagy

vi

^cQngsg, tuy nhi€n, cfing c6 mdt

chit

^bia5n

ddi

_t421.Trong

thi

nghiQm

niy,

chring

t6i

sri dpng lop t6 bdo nu6i ld c6c nguy€n bdo soi phdi chuQt (mouse embryonic fibroblasts

-

^mEFs)

<lugc thu nhf,n tu thai chu6t 13,5 ngdy nhu m6 ta tru6c tl6y cria Klimanskaya

vi

^cQng_{sg [43].}

Chring

t6i

thuong

sri

dtrng mEFs

d 6n

c6y chuyiln thft

t

ho[c

thf

2 d6

hm

lop t6 bio nu6i bdi

vi

^chring

t6i

th5y

ring

mEFs d c6c lAn cAy chuy6n sau d6 (c6c lAn c6y chuy6n thri 3-5) de trd ndn gid h6a, do

viy

kh6ng h5 trq nhi6u cho ESCs tdng sinh kh6ng bipt h6a. mEFs tlugc b6t ho4t phAn bdo bing Mitomycin C (Sigma) n6ng ilQ lOpg/ml trong 2,5

- ³

^grd.^Sau

^khi ^xri ^lf

bing Mitomycin C, t6 bao dugc nia bing ^PBS, phdn trich

bing

trypsin vd duo. c c6y

d mft

tlQ khodng 4xl0a t6 bio/cm2 trong c6c tlia nu6i c6y 96 giting (Corning) da phri gelatin dO tit5n hnnh thi nghipm phdn lflp mESCs.

2.3.Phdn IQp vd nu6i ciiy tii bdo giic ph6i chuQt OC thu nhin tui phOi, chuQt c6i dd giao phtii duqc gi6t d khoing 3,5

ngiy

sau khi giao phtii vd tri cung cria chring ngay lQp tuc tlugc chuytin

(3)

D.V. Dttc ai nnk. / ^Tqpchi Khoahoc DHQGHN, Khoahgc Tq ^nhiAnai C6ng ngh€ 26 (2070)

71-82

73

sang PBS cld ^duo.c

lim

6m tru6c

t6i

37oC. Sau

khi loai hi5t mo b6m xung quanh, tft cung du-o. c chuy€n sang m6i trudng

M2

^(Sigma)

vi

titin hdnh

6y

^ph6i

ra khdi tt

^cung

^bing

^c6ch

^st

dpng born ti6m

lml

hlit m6i trudng born

vio

trl cung dti <tAy phdi ra ngodi nhu m6 td ^criaNary

vi

^cQng

^sg

[42]. ^Thaot6c

thu nhin tfi

^ph6i

clugc thuc hiQn du6i kinh hi6n

vi

soi n6i. C5c

phdi

ph6t

tri6n t6t tlugc

chuyiin

bing

^pipet

Pasteur dAu nh6 dudng

kfnh

xAp

xi

200 pm sang c6c

vi

gigt m6i truhng M2 tru6c khi clua

nu6i c6y tr6n l6p t6 bao nudi dd b6t ho4t ^phdn bdo clusc chuAn

bi

tru6c d6 24

-

48 gid. M6i trudng nu6i ^cAy^{ESCs bao}g6m FBS (20%

vlv) (Gibco), LIF (2000U/ml) (Sigma),

^Non-

essential

amino acid (l00pM) (Gibco),

^p-

mercaptoethanol (100pM) (Gibco),

^L-

Glutamine

(2mM) (Gibco),

HEPES ^(2mM)

(Gibco),

Penicillin/Streptomycin ^(100U/ml Penicilline, 100

pglml

Streptomycin) (Gibco)' Knockout DMEM (Gibco). Dia tO bdo ^nu6i.^d6, b6t hoat phAn

bio

^du-o.^c^thay^mdi^truong^bing m6i

trulng

nudi c6y ESCs

tru6c khi

dflt tui ph6i

l-3

gid. Trii ph6i ^s€tho6t khoi ming ^s6ng vd

bit

^dAu^b6m^dinh^xuiSng^lop

^tii

^bAo^nudi^sau

l-2

ngiry nu6i c6y. ^Saukhoing 5-6 ngdy nu6i c6y, kh6i ti5 bao nrit phdi hic ndy ^ddthuc

tulT

vd c6 hinh dpng cria ^{mQt quAn}l4c t6 bdo gdc rlflc tnmg. LAn phdn t6ch kh6i

tii

bAo

ntt

ph6i

ttiu ti6n dugc

tiaSn

hdnh nhu m6 td cta

Klimanskaya vd cQng sy [a3]. ^{KhOi t6}^bdo^nrit ph6i sinh trudng ^{ti5t sau}

khi

clugc tach ra kh6i

LC rnat dia nudi c6y bang mQt pipet Pasteur dAu nh6 s€ dugc chuy6n sang timg gi6ng cfia

ila

96 gii5ng.

vt5i

^mQtkh6i t6 ^bdonrit ph6i ^ttuo.c phdn t6ch bing trypsin thdnh nhirng cum tri

bio

nhd hon vd ^chuyi5n^sangl6p t6 bao nu6i m6i clugc chuAn bi tru6c d6

ft

24 tl6n 48 gid trong

&a96

giting. Qu6 trinh nu6i cAy

nhtng quin

l4c c6 hinh th6i gi5ng ESCs sau

lin

cAy chuy6n thft

nh6t

^du-o.

c ti6n ^hdnh ^theo m6 ta

^cta

Klimanskaya vd ^cQng^s1r[43].

Nhtng

gitSng c6

^ c6c quAn lac ttl bdo giting ESCs ^sE^du-o.c

xrl li

bing

^trypsin

vd

cAy chuy6n

toin b0

vdo dia nudi c6y c6 ^diQntich bA

m[t

l6n hon (nhu dia

24

gi)ngho{c clia nu6i c6y ^elubngkinh 35 cm) tl6 c6 sin ^{lop t6}bio nu6i.

2.4. Nhu)m h6o m6 marker

d(c tmg

^Alkaline

phosphatase

Phuong phSp nhuQm Alkaline phosphatase

su

dpng

kit

Leukocyte

Alkaline

Phosphatase (Sigma) tlu-o.

c

ti6n hdnh theo

chi din

^cria^nhd

sAnxu6t.

rii

^Uao^sinh^tnrong^duo.^{c c5}^dlnh^bing

dung dich cO ainn (Cinate solution

*

Acetone

*

Formandehide) trong ^1-2phrit. Sau khi c6 ainn rua t6 bdo

blng

nu6c c6t

2

^lAntrong

45

gi6y.

EO

ust

vd nhuOm

t6 bio bing kit

FRV ^hoac

FBB

15 phrit trong

t5i.

Sau 15 phrit rua lai t6 bdo 2 ^lAnbing nudc c6t trong 2 phirt vd c6 th€

quan s6t.

2.5. Nhu\m mi6n dich huj,nh quang protein dflc hi€u Oct3/4

t6

Uao sinh truong tr6n ^p6c^phi6nkinh trdn

rtugc cd dinh bing ^PBS chfa

^4%

Paraformaldehyde (Sigma)

trong l0 ^phfit

^d

nhiet dO phdng. Sau khi rua bing PBS 3 lAn, t6

bdo ^<lu-o.

c tao tinh

th6m

bing Triton

X-100 (Sigma) d nhiQt dQ phdng trong 5 phrit. Sau tl6, ti5 bao dugc rria

lai bing

PBS vd ri trong ^PBS chtra 2Yo BSA

trong l0

^phrit(Gibco). Kh6ng thC

tht

nh6t kh6ng Oct3l4 (l:150, Sigma) tlusc

ti v6i tti bio

trong 60 phrit. ^Sau

khi

rrla bing PBS,

t ^titip ^vdi

^khSng^the

^{thti 2}

(Invitrogen)

gin hujnh

quang trong 60 phfit. NhAn t6 Uao

tlugc nhuOm bing Hoeschst33342 (Invitrogen).

3. K6t

qui vi thio

lufln

3.1.

Kh

qud chudn bi lop ^tdbdo nu6i

Ndm 1981, Evans

vi

^{cQng sY}^c6ng^U6^ea

thi6t

np

dugc ddng mESC ^dAuti6n ddng tr€n

(4)

t4p chi Nature sri dgng mEFs

lim

lop t6 ^bdo nu6i

[0].

^Sau^d6,^ndm^1998,Thomson vd cQng

sp c6ng b5 tren t4p

chi

Science viQc ph6n

lfp

duo. c dong hESC dAu ti6n c6

khi

ning ting sinh kh6ng biet h6a sri dgng lop t6 bno nu6i

li

^mEFs

[6]. Dga tr€n nhfrng c6ng bd ct6 md r6t nhidu phdng

thi

nghiQm t6 Uao g5c tr6n thiS gi6i tiiSp

tuc sri dpng mEFs ldm lop

t6

bdo nu6i trong nghi€n criu ESCs. C6c t€ bdo nudi ndy sE girip

hO trq ^sgtdng sinh kh6ng biQt h6a vd ludn lu6n tU d6i

m6i

cria mESCs trong nu6i c6y. mEFs

tluo, c thu

nhin tu

thai chuQt 12,5

-

^{13,5 ngdy}

phii

ttuqc b6t ho4t phdn

bio

clti ngSn cdn sg tdng sinh cfia chring vd h5 trq sU teng sinh cho mESCs. K6t

qui

nu6i c6y mESCs sri dgng lorp ti5 bdo nuOi cria chring

t6i

cho th6y mEFs

d

lAn

nudi c5y nguyOn ph6t (Hinh

lA)

c6 khA nnng h5 trg sinh trudng vd sg

ting

sinh kh6ng bipt h6a

cho ESCs

t6t hon

mEFs qua nhi€u lAn c6y

3.2. KAt qua ktch thich chuQt siAu bdi nodn Si6u bdi nodn du-o.

c ti6n

hdnh

bing

^cdch

ti€m kich duc t6 A6 kicn thich

vi

ldrn t6ng ryng trung

tg

nhi6n. Kfch dpc t5 dugc

st

dpng phd bi6n

h

^PMSGtric ctQng gi6ng nhu kich dpc

tii n6i

sinh

cta

hormone

kich thich

nang tnmg FSH, sau

tl6

sri dpng

kich

dpc

tti

mdng clQm

nguoi hCG tac tlQng gi6ng hormone kich thi5

Ht"lr ^1.mEFs thu nh{n tu thai chuQt 13,5 ngdy.

(A): mEFs d lAn nu6i c6y nguyEn phrit sau 3 ngdy nuOi i6y (10x20).

(B): mEFs da b6t ho4t phdn bdo vdi mflt dQ t6t du-oc sri dung lam ldp t6 bao nu6i mESCs (lOxl0).

chuyGn bdi khi d6 mEFs de trd n€n gid hon vd m6t nhimg khd ndng ndy. mBFs duo. c b6t ho4t phdn bdo

bing

Mitomycin C ndng tlQ 10pg/ml tru6c khi tlugc sri dpng

lim

lop t6 bao nudi tri 24-48h. MAt d0 mEFs cfing inh huong r6t nhiCu ct6n sU sinh truong

vd tlng

sinh cria mESCs.

Mat d0 mEFs thich hqp sE h5

trq

mESCs ttit

ntr6t

luinn tn;.

Niiu mft

tlQ mEFs

cao s€ tic

ch6 sinh tru&ng cria mESCs, cdn n6u m$t ttQ mEFs th6p sE khdng c6 khd

ning

hd

trg

sinh truong cho mESCs. mEFs phdn

hp ^tu

^thai^chuQt^12-13,5

ngdy khi nudi c6y chring c6 d4ng hinh thoi, ddi, c6 tua thudn nhgn hai ctAu vd b6m dinh xu6ng bd mat clTa

nu6i

cdy. Cic nguyEn bdo sgi clang ph6t tritin mgnh c6 th6 itugc c6t

git

lanh trong vdng

6

th6ng d6

lim

ngudn

t6 bio

nu6i chri dQng trong nu6i c6y tii ^bdog6c.

vdng LH. Di6u quan trgng cria vipc

ti6m hormone kich thfch si6u bdi nodn ld

lim

tdne

I r.X ,l A , .r. :

gap nnreu lan so lugng tn?ng 4rng tl6i v6i m5i con c6i vd kii5m so6t dugc thoi gian ryng trimg tlQc lflp vdi chu trinh ^qunhi€n, qua cl6 lu6n ludn chri itQng du-o. 4l c vO ngu6n ph6i phpc vu mpc dich

thi

nghipm. Trong nhiing c6ng b5 nghi€n cr?u vA mESCs ddng tr€n

cic

t4p chi chuy€n ngdnh

(5)

D.V. Dirc od nnk. I Tap chi Khoahgc DHQGHN, Khoahgc Tt ^nhiAn^ztir^C6ng^nghQ^{25 (2070)}

77-82

75

thi ph6i cfing du-o. c thu tir nhirng chuQt c6i sieu

bei noen dugc kich thich bing

^hormone

133,43,44f. Chring

t6i

de ti6n

hinh 32

ldn

thi

nghiQm kich thich si€u bdi nodn tr€n 192 chuQt c5i vd srl dpng t6ng cQng

22

chuQt tlUc dO tiiin

hdnh giao ptrtSi vOi c6c chuQt c6i ndy. Ktit

qui

ld c6 tiSng cQng 106 chuQt c6 giao phtii (rtat

tj

^l€

giao ph5i khoang 55,2yo), hong 106 chuQt giao ptrOi. cO 92 chuQt c6 ph6i (<lat

ty

^lQa8%). T6ng c$ng sd ph6i thu dugc khi m6 chuQt giai do4n 3,5

ngiy

ld 2.024 ph6i, trong ^<16

c6

1.522

ttti

ph6i

(tj

lQ

ld

75,20%), 458

ph6i

ddu

(ty

l0

22,63yo),

26

ph6i

4 t.i

bdo

(t! le

1,28%), 18

ph6i 2 t6 bdo (r,i

lc

0,89%). Nhu vay

li

^c6^su

3.3. Phdn tQp vd nu6i ciiy ilon ddng mESCs

tt

khAi tii bdo nilt ph6i

cia

tili ph6i

O giai

tlogn 3,5 n$dy,

tui

ph6i ttugc ^bao quanh

bdi

mang s6ng. Mang s6ng c6

vai

trd trong thp tinh

d[c

hiQu

loii, bio

^vQn6 cdn c6 chric

n[ng

girip ph6i tn[nh

hm t6 d

5ng ^d6n trung. MQt ngiy tru6c khi ti6n hinh thi nghiQm ph6n

l{p

mESCs tii5n hdnh

thay m6i

tru}ng mESCs cho t6

bio d

^cfucfra nu6i c6y 96 gi6ng

xu6t hiQn phOi

d

c6c giai do4n ph6t trii5n kh6c nhau nhtmg rlai rta sti vAn

h

tui ph6i.

M[t

^kh6c,

chring

t6i

cfing tld

thu

nhfln

cic

phdi ddu d6

nu6i c6y

in vitro vi

ddu

nhfn

^ttuo.

c

c6c ph6i ph6t tri6n du-o. c d6n giai tlogn

tiri

phdi. Nhmg

trii

ph6i

losi

ndy cfing cho

ktit qui

phdn

lip

mESCs tuong t.u nhu tu trii ph6i thu tryc ti6p tu

trl

cung chuQt m9. MQt chuQt c6i si6u

bii

nodn etugc thu tinh sE cho trung binh khodng 22 ph6i, c6 nhtng chuQt cho

tt

40-50 ph6i,

thim

chi

li

nhiAu hon. Trong khi d6 chuQt thu tinh ttr nhiCn chi ^chotir 4-l

I

ph6i. C6c ph6i chuQt sau khi thu

tt ^tu

cung ^duo.

c git

trong m6i truong

M2

^O

37"C (Hinh 2).

c6 sin mEFs ttd b6t tro4t phdn bdo. C6c tui ph6i tlugc rua

34

Dn trong c6c

vi

gigt m6i trudmg

M2 d6 ldm s4ch ph6i. Sri dpng pipet Pasteur dd k6o dflt ph6i vdo trung t6m cria giling. ^Sau^tt6

cfuc gi6ng s€ tlugc ki6m tra sg c6 m{t cria ph6i (Hinh 3A). Sau 1-2 ngdy nu6i cAy ph6i tr€n lop t6 Uao nu6i cirng v6i m6i trudng nudi cAy thich hqp,

tui

phdi tho6t kh6i

ming

s6ng

vi

Uit AAu b5m xutSng l6p t6

bio

nu6i. Sau khi tho6t khoi mdng siing, lop dud,ng bdo xung quanh chuyiln Hinh 2. Phdi thu nh$n tL chuQt si€u bdi noiln sau khi thu tinh 3,5 ngdy.

(l):

Trii ^phdivdi nrit ph6i vd

x .Q):

^{tlang d}

giai

vd b€ m{t xir xt.

(3): Ph6ich6t vdi tr5 bdo

ch6tp mdu

^il6^cho

th6y

^{6n hriy.}^(4):^Ph6i

giai _{tlopn +}ti5 Uao vAn cdn

xu6t diAu

^{td ph6i}

ndy

^ph6t^tririn.

(6)

76

^{D.V. D{rc}^od^nnk.^/^Tap^chiKhoa hoc DHQGHN, I(hoa hoc Tu nhi€n ad C6ng ngh€ 25 (20L0) 7t -52

d4ng,

bit

ddu sinh trudng vd b6m xu6ng l6p tC

bdo nu6i phia du6i. Nrit ph6i c6 hinh th6i nhu mQt quAn lac b6m nhe l€n tr€n. _QuAnlac ph6t trii5n lcrn dAn,

v6i

nhirng t6 bdo c6 kich thu6c l6n. KhOi t6 beo nrit ph6i no ra, c6 th6 a6 aang

nhin

thAy cum t6 bdo

nft

phdi dang

t[ng

dAn

l6n v6 kich thubc (Hinh 3B). Sau 3 ngdy nu6i

ciy

^cilct6 bao du0ng

bio

sinh truong m4nh ra xung quanh. Trong khi d6 cum t6 bdo nrit ph6i nhin'r6 hon

vi

tdng nhanh vC kich thuoc. Lric ndy chring c6

hinh

thhi rfit ddc

hmg

cria c6c quAn l4c

tti

bao gtic elang trong qu6 trinh

ting

sinh.

Ph6n t6ch khdi t6 bdo nrit ph6i sinh trudng m4nh thing thoi tti6m c6 l€ ld yi5u t6 quytit dinh trong viQc nh6n nudi mESCs (thuong ld khoiqg 5-6 ngdy) (Hinh 3C). N6u nhu phdn t6ch s6m, kh6i te bdo nrit phdi s€ kh6ng

dt

t6 beo dd sinh trucmg vd c6c t6 bao sE ch6t. Ntiu nhu ^phdnt6ch qu6 mudn, ttrOi

tti

bdo

nrit ph6i

sE biQt h6a thdnh cdc dang t6 bdo kh6c nhau vd c6 thC thenh c6c t6 bdo chuy€n h6a. Nhu v4y sE khdng thu

du-o. c c6c quAn l4c gi5ng ESCs. Kh6i t6 beo

nft ph6i

ph5t

triiln

tt5t sE dugc phdn t6ch bing

enzyme thdnh nhirng cpm tii

bio

nh6 khodng 5-

l0

t6 beo. O ^lAnphdn t6ch kh6i t6 beo nrit ph6i ndy kh6ng n6n chia thdnh c6c t6 bio ri6ng r€.

Sau khi phAn t5ch csm t6 bdo nrit ph6i nu6i c6y, c6c t6 bdo sE sinh truong chQm hcrn.

t6

^bao

cdn phdi ctuoc thay m6i truhng vd kitim tra hdng ngdy. Chring ta n6n kii5m tra vd thay m6i trudng vdo m6t gid nh6t dinh trong ngdy. Lric ndy, c6 ba kh6 ndng c6 th6

xiy ra: (i)

xu6t hiQn c6c d4ng tt5 bdo hay

quin

l4c kh6ng giring ESCs,

(ii)

trong <lia nu6i c6y chri y6u ld c6c quAn l4c giiSng ESCs,

(iii) trong

<lTa

nu6i c|y c6

^cir

nhiing quAn l4c giSng ESCs vd c6c d4ng tti bdo khSc nhau.

Trong truong hgrp kh6ng th6y xudt hi6n c6c quAn l4c sau 8 ngdy kti

tt

ngdy phAn t6ch kh6i t6 bao

nft

ph6i

thi

cAn lo4i b6 rlia nu6i

ciy

^d6.

CLc ffia nu6i c6y xu6t hiQn c6c quAn l4c hay dang ti5 bdo kh6ng phdi ESCs cflng

bi

loai b6.

Nhiing <lla nu6i cdy c6 cd quAn l4c giSng ESCs vd c6c quAn l4c hay dgng tiS bao kh6ng gidng ESCs thi tii5n hdnh c6y chuy6n nhirng quAn lpc ctgp nhu aOi vOi phdn t6ch kh6i t6 bdo

nft

^phdi

b6m

d

b€n tr€n. Nhirng rlia nu6i c6y xuAt hiqn

chi y6u c6c

quAn

lpc gi6ng

ESCs

thi

c6y chuy6n theo nhirng quy trinh chuAn ct6i vdi nu6i c6y mESCs nhu m6 tn d ^phAnphuong ph6p.

Khi c6c quAn lac t6 bao dat d6n kich thudc c6 thri c6y chuy'Sn (cluong kinh ^quAnl4c khoing 400 pm) thi duoc c6y chuyiin nhu md

ti

o trdn.

mESCs sau

3'vi

4 ldn c6y chuyiSn (Hinh 3D

vi

Hinh

3E)

vin gift

tluoc nhirng <tdc cliiim ttdc tnmg v€ hinh th6i cria ta5 bdo g6c trong nudi c6y

in vitro d6

h

kha ndng tdng sinh vd hinh thdnh nhirng quAn l4c t6 bdo sau khi c6y chuyiin 2-3 ngdy. mESCs

d

lAn c6y chuy6n

tht

5 vAn t4b thdnh c6c quAn l4c khi nu6i c6y (Hinh 3F). Tuy nhi6n, xung quanh c5c

quin

lac ndy rtd xu6t hi€n r6t nhi6u c6c t.5 bdo l6n phdn trtn thdnh lop dcm c6 hinh trdn

v6i

nh6n l6n vd nhdn chi6m hAu h6t t6 bao ch6t. oiAu ndy cho thdy c6c t6 bdo ndy <td c6 d6u hi€u biQt h6a. Sang dt5n lan c6y chuy6n thu 6 de chi6m uu th6, chi cdn r6t it t6 Uao tao thdnh cum dang ESCs. Nguy€n nhdn gAy n6n hi6n tuqng ndy c6 thC ld ky thu4t nu6i c6y chua cao ilflc biet

h

vi6c nh6n dinh <luo. c

giai

clopn c6y chuytin thuc sg thich hqp. B€n c4nh

tl6

cdn

do su

kh5c nhau

gita

c6c ctgt

mEFs, c6c tlgt huy6t thanh kh6c nhau cfing t5c dQng di5n sU tAng

sinh

kh6ng biQt h6a cria mESCs. NgoAi ra cdn c6 mQt s6 nguy6n nhdn kh6c

din

d6n

tinh

tr4ng bigt h6a mESCs php thudc vdo c6c con tluong truydn tin hiQu...

(7)

D.V. Dnc od nnk. / T1p chi Khoa hoc DHQGHN, Khoa hoc Tt nhiAn od C6ng nghQ 26 (201.0) 77-82 77

Hinh 3. Ph6n lap vh nu6i c6y mESCs.' t€n) (l0xl0).

ptr6i tlang b6m xu6ng ld mdng sang. C6c t€ bdo td bdo ntt ph6i tlang nd r4 c6 th€ d6 dAng nhfln ).

.1

c6y chuyCn c6c qudn lpc ndy vin ^gitl^tlugchinh th6i tt{c l4c khi nu6i c6y (lOxl0). Tuy.nhi€n, xung.quanh cdc ^quAn 6ng vdi nhdn lon

vi

nhdn chi€m hdu h€t t€ bAo chdt. Di6u nAy cho th6y c6c tti bio ndy tl6 c6 d6u hiQu biQt h6a.

3.4. Nhdn bttit mESCs nu6i c$t qua

sr

bidu

hi€n

niry cho

thiy

^cilc^t.5^{bdo b6n}ngodi nhdn ttugc ft Alkaline

phosphatase

sU h5 trq. tusng titc gi1a cic t6 bdo hon n6n c6 nhfrng dliu hiQu biqt h6a (Hinh 4A). Di6u

niy

Alkaline phosphatase ld mQt enryme

thiy

cfing"thudng hay gap

phii khi

nu6i c6y ESCs phan chfu tr6ch nghiQm c6t g6c phosphatl

-ra ""tu" ^on.

^Theo^c6c^tdi^liQunghien criu tr€n kh6i r6t nhi6u c6c phAn

tu

nhu c5c

nucleotide,

_,t#

Ftiniruu ^tfro-g ^t-f

^O^iAn

jy

_chuy€n_cac

protein vd c6c alkaloid. Alkaline phosphatase

ld

_{tti Uio}_s€_trd_n6n_OnALn hon vd ^kh6ngc6 hi.n

mQt marker tlflc tnmg, ptrO Uien kh6ng nhirng

d il; .t,

"aV.

ei"g'rie. iiep

tuc

c6i

"huy3n c6c ddng

'ESC,

hESC md cdn tl{c tnmg

cho^cd

_pnAn_t6_bdob€n trong c6c quAn l4c tuong img nhfrngddngESC

c6,gd,lqn... K.itqudnhuQm

_{nOi pnAn}_b6t

_miu _do _ruoi

vOi inutic

.ilrO,i

Alkaline phosphatase cria chring

tdi

cho

thdy _ekaline

phosphatase, chring

t6i titip tpc

thu c6c quAn l4c

t6 bio

duong

tinh v6i Alkaline d"* ffi; ^q"a,

^,n6

^t6

^Ua'o^duong

^tinh

^v6i

phosphatase (c5c quAn lac tti

bio

c6 miu d6

dac _OfUffr.

Ofr"rpf,uo."

6

c1clAn c6y ciuyCn tii5p

trung) (Hinh 4). Tuy nhi6n, ^chct6 bdo bao

b6n ;;;G;;t

"nuyen ^thtt

4

-ninr, +s

"jra"

"av ngodi c6c quAn l4c kh6ng

bit

^mdu^et6tuoi,

di6u

"h*#;5

^-^{Hinh 4C).}

(8)

78 D.v. Dnc ad nnk. / ^Tapchi Khoahgc DHQGHN, IQrcahgc Try nhi\n od c6ng nghQ 2G (20L0) 71-92

r

f{inh

4.

S

(A): Kh6i tii beo nrit ph6i sau 5 bi€u hiQn Alkaline phosphatase.

Alkaline phosphatase. (C): SU biriu hiQn Alkaline phosph bi6u hiQn Alkaline phosphatase sau l6n c6y chuy6n thrl 5 mESCs rlang ddn biet h6a (10x20).

3.5. Nhdn

biiit

mESCs nu6i ct$t bEng marker dqc hi€u Oct3/4

Oct3/4 (hay cdn gei

li

^Pou5Fl)tlu-o.c biiSt ct6n nhu

li

^nhdn

tti

phi€n

mi git

vai trd quytit tlinh ttugc bi6u hipn

d{c

hiQu

d

mESCs, phdi giai do4n s6m cfing nhu c6c t6 bao sinh dpc vd n6

git

vai nd ^quantrgng trong viQc duy tri tinfi da tidm nlng cria mESCs. Do nhiing thu6c tinh ciaOct3/4 md n6 lu6n tlu-o. c lga chgn ld marker

hang dAu trong viQc nhfln birSt mESCs nudi c6y

l6u ddi in vitro. K6t qui

nhuQm mi6n dich huj,nh quang protein OcB/4

st

dpng hai lo4i kh6ng th6, kh6ng thd

thf

nh6t khdng Oct3l4 dang IgM dugc sdn xu6t tir th6 vd kh6ng the

thf

2khang IgM th6

gin

truyntr quang mdu dd cho th6y c6 srl bigu hi6n cria protein OcB/4

0

^c6c

quAn lac mESC sau

4

lAn c6y chuyOn (Hinh 5B). Nhan

tii

bdo tluo.

c

nhuQm

bing

Hoechst 33342 (Hinh sC).

Hinh 5. Su bi6u hiQn Oct3/4 cria mESCs sau 4 lAn c6y chuy6n d cirng mgt hi6n vi tnrlng.

(A): Quan lpc mESC o 6nh ^s6ngthudng. (B): _Quanhc mESC O anh srang tU ngoqi ph6t hiQn protein OcB/4 c6mdu tt6 (Cy3). (C): _Qudnlqc mESC d 6nh s6ng tu ngofi phSt hiQn nhdn c6

-a, ^**tt ^ia

^trOi

^ginfl)

^dugcnhuQm bdng Hoechst 33342 (10x20).

hiQn

Oc8/4

m4nh hon c6c ph6n xung ^quanh.

Di6u ndy c6 th6 tluo. c

lf

^gi6ireng c6c tt5 bdo b6n trong nhdn ilugc sg hd trq

cta

c6c tti bdo xung quanh nhidu nhdt n6n

it

biQt h6a ntr6t. Cac t6 bdo bao b6n ngodi

phii

titip xric vdi mdi truong n6n d6

cim img bdi m6i

truong vd biQt h6a nhanh horn.

K6t qu6 chup 6nh

bing

kfnh hi6n

vi

laser qu6t c{n canh mQt quan lpc mESCs tir tr6n dinh

Alkaline phosphatase, phAn b€n trong

gita

bi6u

(9)

D.V. Ditc od nnk. I Tqp chi Khoahgc DHQGHN, Khoahgc Try nhi€n t:d C6ng nghQ 26 Q010)

7L-82

79

Htnh 6. Antr cnpp cfln c6nh mQt phdn cria quAn l4c mESC d ldn c6y chuy6n

thf

5 bi6u hign OcB/4 bAng kinh hi€n vi laser qudt.

Thf tg

anh

tu A

d6n

F

clugc qu6t

tu

^tr6n

clinh cria quAn lgc d6n s6t cl6y ctia clTa nu6i c6y.

C6c br?c dnh cho th6y mfrc d0 bi6u hiQn

crii

Oct3l4 tdng ^dAn

tir

tr€n bA mat quAn l4c vdo trong

gita

vd

giim

^dAn

tri

trong gifia quAn l4c

*u6ng phia du6i ctia quAn l4c, phAn nim s6t cl6y

tlia nu6i cAy. Cfing ^gitSngnhu kiSt

qui

^nhuQm

Alkaline

phosphatase, phAn

trong gifia

bi6u hiQn OcB/4 m4nh hon c6c phAn xung ^quanh.

Didu ndy c6 th6 clugc

lf giii

ring c6c t6 bdo b6n trong nhdn clugc su h5 trq cira c6c t6 bdo xung quanh nhi0u nh6t n6n

it

biQt h6a ntr6t. cac t6 bdo bao b6n ngoii phdi tiiSp xric vdi mdi truhng n€n dE cdm img

bdi m6i

trudng

vd

bi€t h6a nhanh hon (10x63).

4.

Kiit luin

V6i nhtng ktit qui

tr6n chring

t6i rut

ra dugc nhirng k6t lufn sau:

- Dd ph6n

lfp, nu6i

cdy,

bin quin vi

^phpc

trdi etugc ddng mEFs srl dpng ldm lop

tti

bao nudi trong nudi c6y mESCs.

- Xdy dpg vd

hoan thien dugc

k!

^thu4t

kich thich ^chuQtc6i si6u bdi nodn vdi hiQu qud cao, qua tl6 lu6n ludn

cht

dOng ngudn ph6i

st

dpng d6 phdn lap mESCs.

- Ap

dpng thdnh c6ng c6c

k! ^thuit

^thu,

.,lil

chuy€n vd nu6i cdy ph6i chuQtinvitro.

- PhAn l4p, nudi c6y vd duy

tri

mESCs tdng sinh trong ^gAn30 ngiy vdi 5 dn c6y chuy,5n md

vin git

<lugc nhirng dac di€m hinh thSi cta mESCs.

- Nhfn bii5t du-o. c mESCs bing ^phuong^ph6p nhuQm

Alkaline

phosphatase

vd

marker ^dflc higu Oct3/4. mESCs sau 5 lAn cAy chuy6n vdn duong

tinh vdi

Alkaline phosphatase

vd

bi6u hiQn protein Oct3/4.

Ldi cim

on

Nghi6n ^ct?undy ^du-o.c thgc hi€n

v6i

^{sU h5}

trs kinh phi tir ^ac ^tai

Kc.04.01.04/06-10:

'Nghi6u

ctu

^phAn

lfp,

^nhdnnu6it6 bdo g5c nQi

md m4ch m6u ddy r5n tr6 so sinh vd tii bdo gtic

(10)

80

^{D.V. Dnc}^uh^nnk./ Tap chi Khoa hoc DHQGHN, Khoa hoc Tu nhi€n aii C6ng ngh€ 26 (2010) 71-82

ph6i chuQt. Theo

d6i

^c6ct6 bdo g5c trong co th6 nhAn trong didu

tri

bQnh suy triy .uu .hit5u x4 li€u cdn cht5t d dQng

vft

thi nghi€m,' du6i su chrl tri cria TS. Nguy6n Lai Thdnh - Trucrng Dai hoc Khoa hoc Tu nhi6n. Chring

tdi

bdy t6 ldng bitit on

tdi

BO Khoa hoc vd C6ng ngh6, Ban

Cht

nhi6m Chu'ong trinh KC-04 vd

chi

nhi6m dc tdiKC.04.0l/06-10.

Tni [Qu tham

khio

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M.

Katz, W.

Schmidt, R. Kemler, The in vitro development of blastocyst-derived embryonic stem cell hnes:

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t40l

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l42l

[43]

144l

Identification and culture of embryonic stem cells derived from inner cell mass of mouse blastocysts

Dang Van Ducl, Nguyen Lai Thanhl, Bui Viet Anhl, Do Doan Loi2

rFaculty

of Biologt, College of Science,I/NU, 334 Nguyen Trai, Hanoi, Yietnam 2Hanoi

Medical University,

I

^{Ton That}^Tung,^{Dong Da,}Hanoi

In Viebram, stem cell technologies, specially in embryonic stem cells (ESCs), are in progress

of

initiation. Current researches have focused mainly on adult stem cells and almost the results obtained have been just

initial

successes. kr this study, we established and improved the method for isolation and long term culture of embryonic stem cells derived from inner cell mass of mouse blastocysts (Mus musculus). The ESCs from inner cell ^massformed the colonies after isolation and passing. During the cultivation, we determined pluripotency of this t1pe of the cell with two makers alkaline phosphatase

and Oct3/4 franscriptional

factor

protein.

The

results

of

histochemical staining

with

alkaline phosphatase and immunofluorescence stain for Oct3/4 indicated that mouse embryonic stem cells after five passages with approximately 30 days of culture still remain the characters of stem cells.

Keywords: Embryonic stem cells, mouse embryonic stem cells, Alkaline phosphatase,Oct3l4.

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