Nghiên cứu biệt hóa tế bào gốc trung mô máu cuống rốn người thành tê bào thần kinh, tế bào cơ tim và tế bào tiết insulin bằng dịch chiết mô

Tap chi Cong nghe Sinh hoc 6(4): 415-422, 2008

NGHIEN CITU BIT HOA TE BAO GOC TRUNG MO MAU CUONG RON NGU6I
THANH TE BAO THAN KINH, TE BAO CO TIM VA TE BAO TIET INSULIN BANG
BICH CHIET MO
Phum Van Pluic, Nguyen Bang Khoa, Triin Sao Kiem, Phan Kim Ngoc
Trufmg DO hoc Khoa hoc Ty' nhien, Ali hoc Quac gia thanh pha Ha Chi Minh
TOM TAT
Te bao gee co the biet hem dual tic Ong cita nhieu tic nhan khic nhau nhu hia chit, tic nhan vot
chat nen, chuyen gen, deng nuoi cay va dich chi& met. Trong do, Web chi& me dirge sir clang tucmg doi
phO bien; khong chi de chimg minh kha nang biet heta to bao goc do ma con nhrtm chimg minh kha nang
biet h6a dm no thanh kieu hinh cia mo khi ton tgi trong vi mot throng dm mo. Trong nghien ciru nay,
cac to bao gee trung me tir mau cutmg 'On nguth dime thin nghiem Wet h6a thanh to bao than kinh, ca tim
va to bao tier insulin bang (itch chiet tir nao thai chueit, tim heo va tuy chwjt con. Te bao goc trung mo fir
miu cueng ron dime thu nh'i'n va chirng minh theo cac quy trinh truerc day da cong bo. Kieu hinh to bao
sau khi biet hint duqc xac dinh dva vao hinh clang d.c trung, nhuOm 116a me va sv bleu hien gen chuyen
biet thong qua Ic'y thuit RT-PCR. Ket qui cho thay, cac to bao gOc trung me M man cueng rein co kha
nang biet hem thanh kieu hinh to bao cita me thu Bich chiet trong moi trtrong be sung Web chiet 6 ty le
thich hop. CS neng de 400 µg/ml protein dich chiet thu tir tuy chuOt, to bao gee biet h6a thanh cic cum
tuy dao vOi hieu qua cao nhat trong cac Wing do ldiao sat. Cic cvm tuy dao nay Neu hien cac gen chuyen
biet cho tuy nhu Glut-2, Ngn3, Pax4 va Insulin. C mot truing b8 sung 10% dich chiet nao, sv biet h6a
cixa to bao goc thanh cic neurosphere xay ra manh; clic to bao tir neurosphere bieu hien duang tinh v&i
nestin. Ci moi truiffng be sung 1% dich chiet tim, to bao gee trung mo thay del hinh clang gieng hinh dans
to bao ca tim va bieuhien gen cTnT dic hung cho to bao ca tim. NhAng ket qua nay cho thay, (Itch chiet
mO ci the lam thay doi kieu hinh to bao getc theo kieu With cia mo dich.
Tit khea: Biet hoa, cuang ran nguai, dich chiet ma, to biro ca tim, to bao goc, to biro than kinh
MO DAU
Te bao gee ten tai o hAu het cic me cita ca the
tnrong thanh. Chong c6 vai fro quan trong trong viec
duy tri can bang WM mo. Tat ca cac m8 trong co the
duqc cau thank tir cac to bao cochirc nang.pe trinh

bt tic dOng ter to bio c6 chirc nang, nhirng to bao gee
ton tai trong mei luon o cic vi tri chien lucre trong
niOt khoing khong gian rieng biet, gvi la 6 (niche). 0
to bio goc la vi moi truerng gilt!) cho to bao gee ton
tai tv lam moi - khong biet h6a. Khi khong gian ben
trong 6 thieu, to bio gee chui ra khoi 6; to day vOi
cic tic dOng to bao dA c6 chirc,nang se
chin
biet hint thanh kieu hinh nay. Dieu d6 gqi y rang cic
yeu to cia mo c6 tic dOng leo lii sv Wet h6a cua to
bao gOc (Collas et al., 2006; Hakelien et al., 2006).
Te bao goc tir mo ma ngueri c6 the biet hew
thanh to bao co kieu hinh giOng 4 bio tiet insulin
dual tic Ong cia dich chiet tuy (Lee et al., 2008),
thanh to bao ca tim dueri tic Ong cia dich chiet ca
tim (Gaustad et al., 2004; Schimrosczyk et al.,

2008). re bao gee trung mo tir thy xuang chuOt cimg
duqc chimg minh la oft kha nang biet h6a thanh to
bio tier insulin duai tic Ong cia dich chi& tuy
(Choi et al., 2005). Nguyen bio sqi tir thai chuOt
el-mg c6 the cam irng biet hew thanh 4 bao tiet
insulin khi nuoi trong moi triremg be sung dich chi&
to bao ung thu tiet insulin tuyen tuy (Hakelien et al.,
2006), hay nguyen bao sch 293T cimg ceo the bi Net
hee thanh to bao lympho T neu duqc cam img bii
dich chiet to bao T (Hakelien et al., 2002). Dich chi&
xuang bo duqc chimg minh c6 khi nang gay biet h6a
to bao goc trung mo nguai thanh to bao xuang (Woo
et al., 2006). Trong nghien cim dm Liu va (long We

gii (2005) cho thay, dich chi& tir nga rang c6 the
cam 1mg biet hew cac 4 bao hen than nhi rang thanh
cac 4 bao giOng nguyen bao nga rang (Liu et al.,
2005). Cac to bio goc phoi cling c6 the duqc biet hew
dyed tic Ong cha dich chiet thanh to bao cita me
dich nhu to bao khi quin (Qin et al., 2005).
Tren ca so cac ketxqua cia cac nghien ciru hen
day, nghien ciru nay tiep tic sir dwig dich chiet de
415


PhamVanPhilc et al.
kiem tra kha,nang biet hoa cUa bao gac,trung mo
mau cuong rein ngtrai thinh to bao gi8ng to bao than
kinh bang dich chiet nao thai chuet, thanh to bio ca
tim bang dich chiet tim heo con vi thanh to bao fiat
insulin bang dich chiet toy chuet chua truang thanh.
VAT LIEU VA PHLTdNG PHAP
Thu nhan to bito gOc trung ma mau men ran
ngtrai (hUCB MSC)
Mau cuong rein thu nhan tir cac sin pho da duqc
xet nghiem am tinh y6i HIV, HBV, HCV o Benh
vien Himg Vuang thanh pha He Chi Minh. Thu nhan
quan the 4 bao don nhan bang phucmg phap ly tam
ten gradient Ficoll-paque (Sigma) it toc a 2500
yang/phut, trong 5 phin. MSC duqc Lich khoi to bio
gec too mau trong van the re bao dam nhan bang
phuang phap nuoi cay va mai truang chon loc. Te
bio dam nhan sau khi thu nhan duqc huyen phu trong
moi truing nuoi cay IMDM (Sigma), 10% FBS

(Invitrogen, Gibco), trong birth nuoi cay (25 cm',
Nunc) sao cho dot mat a 3.105 4 bito/cm2 o dieu
kien 37°C, 5% CO2. Sau 48 h, cac MSC bit da'u barn
ten be mat birth nuoi, thay men twang de looi be cac
to bao khong barn (cac to bao chat va 4 bao giic to
mau), tiep toc nuoi den khi to bio dot mat a 70 80% dien tich be mat binh nuoi cay vii the de thay
moi truing la 4 ngay/lan. Cfic to bio thu /than ter Ian
cay chuyen thir 5 se duqc sir dong Wet h6a (Phan
Kim Ngoc et al., 2007; 2008).
Tyo (Itch chiet tyy
Toy chu'Ut sau khi thu nhan duqc cat nhuyen
trong dung dich PBS ba sung 10% FBS. Ly tam
3000 vong/phut trong 10 phut, thu nhan dich noi cho
eppendorf 1,5 ml sach. Tiep tic ly tam 12000
yong/phin trong 20 phut, thu ratan dich nen. Dich noi
duqc sir ding de b8 sung vao moi truing biet h6a
vii L-DMEM (Sigma), a cac nZing do protein tang
so cila dich chiet la 100, 200, 300, 400, 500 va 600
µg/ml. Vo tang dich chiet bang cich WC qua ming
loc 0,2 gm. Moi truing co ba sung dich chi&
t duqc
tit lonh trong toi da 7 ngay sau khi pha.
Tao dich chiet nao
Nao thai chuet 18 dpc duqc tach, u lonh va nghien
bang cod, ding nhat nao trong dung dich PBS va
ethanol de dot the tich la 1,5 ml. Dich nao dong nhat
duqc dong lonh qua dem trong nitrogen long, sau do,
giai dong chain bang cach u it 4°C. Sau khi tan di,
dich nao duqc ly tam a 3000 yang/phut trong 5 phin
416

va thu dich noi. Dich noi age ba sung yeti huyet
thanh theo tk le 1:1; sau do, loc bang ming loc 0,2 gm
va bao quan o —20°C cho sir dong trong 1 wan.
Tyo dich chiet ca tim heo
Tim heo tir 2 - 3 thing tu6i duqc can de xac dinh
klufn lucmg. N6 duqc chuyen vao dia chira sin dung
dich PBS/Gentamycin va cat nho. Sau do, ly tam him
hqp ma' ca tim vita cat de loot be PBS, AM them 1 ml
trypsin 0,25%/100 mg m8, khuay mau Ito bang 'ca
khuay tiro toc a 200 yang/phut, trong 30 phtit,
36,5°C. Sau do, thu dich noi, ly tam 3000 yang/phut
trong 10 phut de thu can 4 bio, tai huyen phi" can
bang 1 ml moi truong, at it 4°C. Te bao ca tim duqc
phi ye bang song sieu am & buem song trung birth
theo chu 16, phat xung trong 30 giay, ngimg 30 giay;
lien toc trong 30 phut. Sau khi ly giai, dich d8ng nhat
duqc ly tam 12.000 vong/phut trong 15 phut, it 4°C
va thu dich noi. Dich noi duqc dong lath trong
nitrogen long qua am; sau do giai dong nhanh va
loc qua ming 0,2 gm. Sir dong trong 1 tuan.
Cam ung bit hoa Wing dich chiet
hUCB MSC dang phat trien phi' kin chitng 70%
be mat dong co nuoi duqc thay men tramg biet hem
tuang img cho vies cam img biet h6a thanh to bao
tiet insulin, to bao co tim va bao than kinh la moi
truing nuoi be sung dich chiet tuyen toy, mei ca tim
va mei nao. Men twang biet hem duqc thay vii the
da 4 ngay/lan.
MCA tnr&ng biet h6a hUCB MSC thanh to bio tiet

insulin: L-DMEM (Sigma), be sung 100, 200, 300,
400, 500 va 600 µg/ml protein dich chiet tong so.
M6i tnrang biet hem hUCB MSC thanh to bao
giang to bao than, kinh: IMDM (Sigma) 2% FBS
(Gibco), bit sung lin lugt 1, 5, 10 va 15% dich chiet
nao thai chuet theo quy trinh ten.
Med twang biet has hUCB MSC thanh 4 bao ca
tim: IMDM (Sigma) 2% FBS (Gibco) ba sung 0,5, I,
5 va 10% dich chiet.
Te bao sau khi biet h6a tir to bito MSC duqc xac
dinh thong qua so thay den hinh dong, nhuem h6a
mei va RT-PCR vii cac marker ac trong.
Nhuam hoa ma
Nhuam dithizone (Sigma): binh nuoi to bao sau
khi cam img biet hem duqc do be moi truing, rira hai
Ian bang
co dinh bang ethanol tuyet ai trong
15 phut. Sau do, ink to bao duqc it trong thuac
nhuOm dithizone trong 30 phut it 37°C; do bo thuac


Tgp chi eonk frighe Sinh hpc 6(4): 415-422, 2008
binePBS 'ita quay sat so hinh
nhuOm; rira lai 2
thanh phirc hqp maikki trong'cac to bao tiet insulin
va cum to bio giang tuY dad.
Phan img RT-PCR
RNA tang se. &roc tach bang Trizol (SigmaAldrich) theo huong clan cua nha san xuat
dinh
Wong bang may quang phe UV/vis. De chay PCR, 2

lig RNA tong so duqc chuyen thanh cDNA sir dung
SuperScript II reverse transcriptase (Promega) v6i

oligo (dT) (Promega,' USA) va random hexamer
primers (Promega). PCR duqc tien hanh sir dung Taq
DNA polymerase (Promega). Tat ca phin img PCR
duqc tien hanh sir dung may lua'n nhiet PCR
(EppendorO. San pham duqc phan tich dien di ten
gel agarose 3%. Ten va trinh to cac primer, kich
thy& cua san phim PCR, chu Icy nen hanh va nhiet
de lai cho mai cap mai duqc het ke trong bang 1.
Gen 13-actin sir dung nhu met doi chimg Mai (internal
control).

Bang 1. Cac primer sir dung trong nghien ciru.
Gene

Size (bp)

Insulin

263

Ngn3

313

Pax4

496

Glut-2

298

Beta actin

587

Nestin

495

cTnT

250

Sequence

GCAGCCTTTGTGAACCAACA
GTTGCAGTAGTTCTCCAGGTG
GGTAGAAAGGATGACGCCTC
CCGAGTTGAGGTCGTGCAT
AGGAGGACCAGGGACTACCGT
TTTAGGTGGGGTGTCACTCAG
GTACAATGACAGAAGATAAG
TGCTACTAACATGGCTTTGA
CCAAGGCCAACCGCGAGAAGATGAC
AGGGTACATGGTGGTGCCGCCAGAC
AGAGGGGAATTCCTGGAG

CTGAGGACCAGGACTCTCTA
OGCAGCGGAAGAGGATGCTGAA
GAGGCACCAAGTTGGGCATGAACGA

KET QUA VA THAO LUAN
Bitt h6a to bio hUCB MSC thanh to ban tit
insulin

6 hail htt cite to thi nghie'm v6i cac ming dO djch
chiet lchac nhau, cac to bio hUCB MSC den thay d8i
hinh clang sau khi thay mai truang Wet h6a 24 h.
M8i trirang biet hea v6i 100, 200, 500 va 600 ug/m1
djch chi& lam to bao bong len be mat dung cu nuoi
va chet nhieu sau 24 va 48 h nuoi. Trong khi d6,
tnrong biet h6a veri nOng d6 protein djch chi&
tong so la 300, 400 µg/ml, se to bao chet it Mn di, so
thay doi hinh dang xay ra chair, nhieu to bao co lai

moi

va tap hop thanh nhieu cum giong nhu toy dao vao
ngay 10 (Hinh 2A, B).
So phan img cua 4 btlo khi be sung dich chi& co5

Annealing

Cycles

temperature
45

30

45

30

45

30

43

30

60

30

53

35

60

35

the do nhieu ca the Ichac nhau. Met se tac gia cho
rang co the so phan img cua to bio .1i bat lqi
djch

chiet lam thay dei ip suat
thauSdo thanh phan
protein b8 sung vao); mat so cho rang de la phan
img do cac thank phan trong djch chiet (chit
là
protein) gay tac Ong. Met se gia thiet cho rang
rang c6
the thanh phan DNA trong djch chiet tac Ong len so
bieu hien gen to bio. Nhung ca the cu the cua van de
nay chua duqc chimg minh r8 rang; c6 le phu thuec
vao timg twang hqp.
Trong nghien ciru nay, so phan ung cua to bao
khi bo sung dich chiet vai protein thap (100 vi 200
µg/ml) va cao (500, 600 µg/ml) lam cho 4 bac) bong
len va chet nhieu Wang to nhu trong 18 dei chung
khong be sung djch chiet (mai tnrang L-DMEM)
Wei tnrong L-DMEM , moi twang ngheo dinh
clueing duqc sir dung de gay stress cho so biet h6a.
417


Phan Van Ph& et al.
Cac to bit) hUCB MSC hau nhu khong the song va
phat trien,ducm trong m8i truang nay khi khong be,
sung huyet thanh. 0, m8i truing bo sung 100, 200
ug/m1 protein ton so djch chiet co the chua dap Ong
dti Minh phan chat can duet de duy tri su phat then
cua cac hUCB MSC nen to bao chet. Trong khi d6,
mai truing be sung 500, 600 µg/ml thi thanh phan
protein qua cao lam thay dOi ap suit them thau va to

bao ding chet. Trong do, 6 hai ming di) 300 va 400
ug/m1 protein djch chiet, thi s8 luting to bao chet rat
it, hau het cac to bio tiep t lc phat teen va thay doi
hinh clang theo th6i gian. Trong nghien dm nay, day
la hai ming d8 thick hop that.

cac neng d8 tren deu co su xuat hien cac clan
VC*, bao giong tny dao va dieu nay khans xay ra 6 18
doi chimg. IChi nhuem voi dithizone, tat ca cac clue
to bio giong tny dao deu bat man thuoc nhueim va
nhieu to bao don ding bat mau do. Khi dem s8 lucmg
clue to bao va to bio ducrng tinh thuoc nhuOm
dithizone cho they 6 m8i truang biet h6a voi 400
.tg/m1 protein tong so tit dich chiet thi hieu qua biet
hoa cao that voi 13,3 gun to bao ducmg tinh thuoc
nhuOm/thi truing (Hinh 1A).
Khi kiem tra RT-PCR 618 biet h6a be sung 400
ug/m1 protein djch chiet, cho they to bao bieu hien
Glut-2, Ngn3, Pax4 vi Insulin (Hinh 1B).

14

■

12
10
8
6
4
2

0
100

200

300

A

400

500

600

Ladder Beta actin Control Glut-2 Ngn3 Control Pax4 Control Insulin

B

Hinh 1. (A) Riau qua biet Ma 0 cac ntong dO dich chiet khac nhau. (5' ding de 400 pg/ml, hiOu qui biet hoa
xay ra cao nhat Va 6 nong dO 100 hay 600 pg/ml thi than nh6t. (B) Ket qua chay RT-PCR xac djnh Si,, bieu
hion mot se gen chuyen biOt cho to bac) tint insulin. Kat qua cho they, to bao sau khi biet hoa 'Au hien cac gen
quan trong cho to bao tiet insulin nhu, Glut-2, Ngn3, Pax4 va Insulin.
Bitt hos hUCB MSC thinh to bar) giOng to ban
than kinh
KM cam Ong biC't hem vai mei tnrang be sung
djch chiet, so phin Ong cue to bio vii moi tnrong
nay co khic nhau 6 cac neng de djch chiet ,khfic
nhau. Nhan xet chung la so phat trien cna to bao
them lei so v6i i6 dei chimg (nuoi trong mai truing

418

IMDM 10% FBS). Trong do, a to be sung djch chiet
1 vi 5% so phat trier chant.' va gin nhu it thy cac
tic deng pita djch chiet. Vio ngay 15 new, hau nhu
town be to bio trong 218 nay bj bong va chef town b6
sau 15 ngay; vio ngay 7 c6 xuat hien cac to bio co
hinh dang giOng 4 bac) than kinh; tuy nhien se luting
rat it, ten met binh nu& cay 25 cm2 chi c6 5 - 6 to
bio. 6 18 15% dich chiet, to bao ding chet nhieu,


Tap chi Cong nghe Sinh hQc 6(4): 415-422, 2008
vao ngay 7 Ming xuet hien nhieu 4 bio co hlnh clang
giong, to bao than kinh veri so lucmg it va den ngay
15, hau het to bao chat. (5 lo 10% dich chiet, to bao
chet ttrcmg doi theo th6i gian, den ngay, 3 da co su
hien nhieu to bao veri hinh dang rat giong van
cac to bao than kinh nhu neuron (Hinh 3A,B,C); so
hrqng cac to bao nay tiep tuc tang len cho den ngay
15. Tiep,tuc nu6i cay cac to bio tren den ngay 21,
hau het to bao chet.
Cac to bao hUCB , MSC thuang duqc phat then
trong mei tnremg „c6 bo sung 10 - 20% FBS, mot so
nghien cim con be sung EGF va bFGF. Khi biet h6a,
nhArn tao stress de cam img bier h6a, mei trtrang nuoi
cay sir dung ming deo huyet thanh thap (2%); va khi
do, trong mei truang nay to bao chet den do thieu
nhieu thanh phan kich thich tang twang quan trong
trong FBS. Nen khi be sung dich chiet a 1, 5, 10 va

15% deu they co su giant tang twang cua hUCB MSC
so ,v6i
chimg. 0 ruing dO 5%, dich chiet, bao
chet it nhet va cho hieu qua thay doi hinh dang re, ref,
a', le day là Ong di, phi, hop nhat von Itrong chat
trong dich chiet be sung vao lucmg chat mat di khi
giam Ming do huyet thanh. Phan tram didh chiet qua
cao (15%) se lam thay doi ap swat them that, dm mei
tin:mg, nen lam to bao chet nhieu. Trong mot so lo thi
nghiem 6 non,g TO 10% dich chiet c6 xuat hien cac
cim to bao giong,nhu,cac neurosphere hinh thanh kin
nu6i cay to bao gee than kinh.
Khi chay RT-PCR danh gia su bieu hien gen
nestin (dac trong cho to bao than kinh) cho thay cac
to bio thu nhan tir mAu biet hoa tied dich chi& 10%
duang tinh von marker nay sau 10 ngay cam irng
biet hoa (Hinh 3D).
Bit hoa hUCB MSC thinh to bio co tim
Khi be sung mei truerng cam ling bier hem veri
cac Ming dO dich chi& khac nhau deu gay cac phan
img cita to bao din yeti mei tnrang mai. Cac phan
Ung duqc ghi nhan la su bong Uric to bao, su chet, su
co cum va hlnh thanh cac cim to bao; ngoiti ra ming
c6 str hinh thanh cac to bao co hinh dang giong to
bio ca.
Su bong tree bao tang den tir 0,5, 1, 2,5, 5 va
10% dich chiet bbee sung va a nOng 40 2,5, 5 va 10%
dich chiet, heu nhu toan be to bao chet,vao ngay 7 sau
khi cam img. Ming do 0,5 va 1 % to bito,phat trier,
cham so van den chUng, so lucmg to bio chet it va bit

deu hinh thanh cac cam to bao vao ngay 14 - 15 ngay
(Hinh 4A), va hen het to bao chet vao ngay 40.
Tir ngay 21, a lo biet hoa bang 1% dich chiet, co su

xuAt hien met so to bao co hinh dang giting 4 bio co
(Hinh 4B).
Trong suet 40 ngay theo doi, tit ca cac nieu
cac 18 deu khong xuat hien cac to bao co dap. Su
xuet hien cac to bao co bap IA mot bang chirng manh
ma nhieu tac gia a tien hinh thanh ming de bier hoa
to bao MSC thanh to bio Ca tim bang 5-azacystidin.
(XiaoHong Li et al., 2007; Xu et al., 2008).
Mau 4 bac, biet h6a sau 21 ngay a to be sung 0,5
va 1% dich chiet duqc ti'en hanh RT-PCR veri gen
marker cTnT
wing cho to IA° ca tim) cho they
chi a 10 veri 1% dich chiet, to bao co bieu hien gen
nay (Hinh 4C).
Vi thinh phan dich chi& mei chua kiem sok het,
ben canh thanh phan chinh la protein thi dich chiet
can nhieu thanh phan khic ma quan trong la DNA,
RNA. Cling c6 the Fie thuyet rang su cam img bier
hoa la do thanh phan, DNA, RNA trong, dich chiet
bien nap vao to bao g8c; va khi do, su bieu hien gen
hay hinh dang dat duqc la do su bieu hien cim nhung
DNA va RNA nay. ,Va neu nhu hien tuqng nay xity
ra thi su thay doi kieu hinh tren co the la tam than va
DNA va RNA ngoai lai chi ten tai trong to bao mot
than gian ngin. Nghien dm nay chua giai quyet duqc
van de nay.

KET LU IAN
Cac to bao gee la nhimg to bio c6 kha nang
chuyen doi, biet hoa thanh cac to bao co chirc nang
theo dich chiet cim ,m8 dich. Trong dich chiet cue
mei, nhieu thinh phan protein ngoai bac, va lion bio
ton tai. Nhung thanh phan do cling co the lit you to
gay cam img biet Mia to bio gtic theo lcieu hinh cim
m0 ay.

CS ruing deo 400 ttg/m1 protein dich chi& thu tir
fay chuOt chua twang thanh, to bac, g8c trung m8 thu
tir man cueing fon ngtrai co the, biet hoa thanh cac
cam My deo veri hieu qua cao nhat trong cac ming dO
khao sat sau 10 ngay cam img. Cac cum qty. deo nay
bieu hien cac gen chuyen bier cho to bao tiet insulin
nhu Glut-2, Ngn3, Pax4 va Insulin. 6 min truong bo
sung 10% dich chiet nao thai chuOt, su biet hoa cim
to bao goc trung m8 thanh cac neurosphere xay ra
manh sau 10 ngay cam ling. Cac to bio tir
neurosphere nay bieu hien ducmg tinh vai nestin.
min trtrerng bo sung 1% dich chiet tim heo con, to
bao gOc trung mei thay del hinh dang giong hinh
Bang to bio co tim sau 21, ngay cam img va bieu hien
gen cTnT d4c trong cho to bao ca tim.
419


Pham Van Phac et al.

Hinh 2. (A) Te bao g6c trung m6 sau khi thu nhan va khong cam 0,ng biM hea (X20); (B) Te bao g6c trung me

sau khi biM h6a 10 ngay trong mei trifeng biet h6a chira 400 pg/ml protein dich chiM &vac nhuem yeti
dithizone (X10). Cac to bao hUCB MSC sau khi thu nhan co hinh dung giOng vai nguyen bao sqi. Sau khi biet
hoa, cac to bao co va hinh thanh cum to bao gieng yen toy deo. Cac cum nay bat mau de khi nhuem vbi thuec
nhuem dithizone.

O
Actin DOi chieng Nestin
Hinh 3. hUCB MSC duvc biet h6a thanh to bao giOng to bao than kinh
bang dich chiet. (A) Cum to bao grang neurosphere vao ngay 5; (B) Cac
to bao CO scyi dal Ong neuron xuat hien vao ngay 7; (C) Cac to bao co
nhieu nhanh vai 1 nhanh dai giOng axon xuet hien vao ngay 10. (Ket
qua trong mau be sung dich chiM 10%) (Hinh chup 6' vat kinh X20). (D)
KM qua chay RT-PCR vai gen marker Nestin cho to bao than kinh. Ket
qua cho they to bao bieu hien gen nestin, trong khi do to bao kh6ng
cam irng biM h6a (dei chirrig) kh6ng bleu hien.

N6ng d6 1%

by

M Actin

MT cTnT DC

N6ng d6 0,5% Kh6ng cam Ong
MT Actin cTnT DC Actin cTnT DC

600
300

Hinh 4. Ket qua biM haa hUCB MSC thanh to bao ca tim bang dich chiet. (A) Com to bao hinh thanh trong to
thi nghiem be sung 1% dich chiet (de phong dal X10); (B) Te bao co hinh clang giOng to bao co, tim &roc ghi
nhan vao ngay 21 sau khi bit h6a 0, to thi nghiem be sung 1% dich chiet (de phOng clai 40X); (C) KM qua RTPCR cho maTh to bao 0, hai le be sung 0,5 va 1% dich chiet cho they chi re 1% CO sty bieu hien dm cTnT.
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Tap chi Cong nghe Sinh hpc 6(4): 415-422, 2008
Ltd cam
Chung toi xin chin thanh cam an Benh
vien Phu san Hung Vuvng thanh pho HO,Chi Minh
dd cung cap matt mau cu Ong
'
ran nguai de tien hanh
nghien cieu nay.
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RESEARCH ON DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD
DERIVED MESENCHYMAL STEM CELL INTO NEURAL-LIKE CELL, CARDIAC
MYOCYTE, INSULIN PRODUCING CELL BY TISSUE EXTRACT
Pham Van Phuc, Nguyen Dang Khoa, Tran Bao Kiem, Phan Kim Ngoc.
University of Science, Vietnam National University, HCM City
SUMMARY
Stem cells can differentiate into functional cells by a lot of agents, such as chemical, physical
stimulating, matrix contact, gene transferring, co-culture with functional cell and tissue extract. Among
them, tissue extract can be commonly used to differentiate stem cells. Differentiating stem cells by tissue

Author for correspondence: Tel: 84-8-38397719; Fax: 84-8-38967365; E-mail:
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Pham Van Phtic et aL
extract not only proves differentiating capacity of that extract but also demonstrate the fate of these stem
cells which live in that tissue microenvironment. In this research, human umbilical cord blood derived
mesenchymal stem cells were differentiated into neural like cells, cardiac myocytes and insulin
producing cells using extract from murine fetal brain, porcine fetal heart tissue and immature mouse
pancreas. Mesenchymal stem cells were derived from human cord blood using previous published
procedure. Differentiated cell's phenotype was determined by specific morphology, histochemistry
staining and the expression of specific genes by RT-PCR. The results showed that tissue extract can be
used to alter the fate of stem cells to functional cells by using medium with suitable ratio of tissue
extract. At 400 tg/ml total protein of pancreas extract, mesenchymal stem cells could differentiate into
islet with the most efficiency. Islet cell expressed some specific genes such as Glut-2, Ngn3, Pax4 and
Insulin. In medium with 10% brain extract, stem cells differentiated into neurospheres that expressed
nestin gene. In medium with 1% myocardial extract, stem cells changed the morphology to myocardial
cell's morphology and expressed cTnT gene.
Keywords: Cardiac myocytes, differentiation, human umbilical cord blood, neural like cells, stem cells,
tissue extract

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