Báo cáo " Tạo tấm tế bào sừng nhiều lớp trên màng collagen từ màng ối người " pot

Tgp chi Cong nghe Sinh hoc 7(2): 155-160, 2009
TAO JAM TE BAO
SUlVG
NHIEU
LOHP
TREN
MANG
COLLAGEN TlT
MANG
6I
NGlToa
Tran
Le Bao Ha', Tran Gong
Toai^
Hoang Nghia
Sffn^
'Tru&ng Dgi hoc Khoa hoc Tu nhien, thdnh pho Ho Chi Minh
^Truong
Dgi hoc
Ykhoa
Phgm Ngoc Thach, thdnh pho Ho Chi Minh
^'Vien Sinh
hoc nhiet
d&i
.
,;; ,i
,
^
TOMTAT
Bieu bi la lop ngoai cung cua da, cau thanh tit bieu mo vay phan ting biet hoa cuoi ciing. Bieu bi dong vai
tro bao ve dau tien cua ca the chong lai nhiing tac dong bat lgi cua moi tradng nhu cac tac nhan sinh hoc, hoa

hoc,
vat ly. Mo bieu bi gom nhieu lop te bao svmg co the dugc tao ra in vitro de umg
dimg
trong chiia tri cac ton
thuang da cap va man tinh. Trong nghien
ciiu
nay, tam te bao simg nhieu lop dugc tao ra tit te bao sirng nguai
truong thanh va mang collagen tir mang oi. Te bao simg dugc phan lap, nuoi, nhan trong moi truang khong
huyet thanh SFM (Serum Free Medium). Mang collagen dugc thu nhan ttr mang oi da dugc loai bo lop te bao
bieu mo va danh gia bang phuang phap nhupm HE. Sau khi dugc cay chuyen Ien mang collagen, te bao simg
dugc kich thich tang sinh va biet hoa
thaiih
nhieu lop bang
each
de tiep xuc khong khi. San pham tam te bao
simg nhieu lap dugc danh gia bang RT-PCR, hoa mo mien dich. Ket qua cho thay, mang oi da dugc loai bo
hoan toan lap bieu mo sau khi xit ly enzyme va ca
hpc.
Cac te bao simg da tang traang, tao 4 - 5 lap teen mang
collagen va bieu hien marker te bao simg da va chua biet hoa sau 7 ngay tiep
xiic
khong khi. Cac te bao simg
duang tinh vai marker tang sinh (p63) nam sat tren mang collagen, cac te bao ben fren la nhiing te bao sirng da
biet
hoa.
» « is
I ! • I
C
•
•

••
Tit khoa: Bieu
bi,
collagen,
mdng
oi,
moi truang khong huyet
thanh,
te bdo simg
I
MdDAU
Sy khep vet thuang can mgt vat lieu de hoi phuc
chiic nang hang rao cua bieu bi va sap nhap vao vet
thuang dang lanh. Hien nay, tu ghep vat da van la
"tieu chuan vang" de tai tao be mat cac vet thuang
lan. Tuy nhien, vimg da eho de ty ghep bi han ehe
frong cac vet thuang rgng. Dieu nay thuc day phat
trien cac lieu phap khac nhu la ky thuat ghep tam te
bao bieu bi nuoi cay (Cultured epithelial autograft -
CEA)(Raymundefa/.,
2005)
Cac tam te bao bieu bi nuoi cay thudng gom 3 -
5 ldp te bao va rat khd cam giii. Do do, can cd mgt
cong cu de thao tae tam te bao nay. De khac phuc,
mgt so vat lieu da dugc su dung de mang tam te bao
simg nhu mang collagen loai 1
ciia
bo, pHEMA
(poly(2-hydroxyethyl methacrylate)),
PIAAm

(N-
Isopropylacryamide),
PPS (plasma polymerised
fiinctional
surface) (Barbora et al,
2003;
Jones et al,
2003;
Masayuki et al, 2001; Zhu et al, 2005).
Mang li bao gom mgt lop dan te bao bilu md,
mgt mang ca ban, mgt ldp dac vo bao, lop nguyen
bao sgi va lap xdp ben duoi Mang ca ban
ciia
mang
6i gdm laminin 5, collagen loai IV, VII, XVII, tuang
ty vdi mang co ban eua da ve hinh thai va sieu cau
tnic (Nakamura et al, 2004) Mang di cd tinh khang
viem, khang xa hda, khang seo, kich thich bieu md
hda va tinh sinh mien dich thap (Hassan et al,
2008;
Oyama et al, 2003). Mang oi loai bieu mo da
dugc sit dung lam gia the frong cdng nghe md giac
mac,
sun, than kinh (Jin et al, 2007; Tseng, 2001;
Ueno
era/.,
2006).
Trong nghien
ciiu
nay, chiing tdi da sit dung

mang collagen tii mang di loai bieu md de lam gia
the nuoi te bao simg nham tao tam te bao simg nhieu
lap in vitro.
VAT LIEU VA PHUONG
PHAP
Doi
tu-ffng
nghien
cihi
Da lanh
ciia
benh nhan cd ehi dinh ghep da va
mang oi
ciia
san phu sinh mo.
Phuong phap thu nhan mau
Thu nhan mau da, mang di
ciia
san phu vita sinh
da dugc xet nghiem am tinh HIV, viem gan sieu vi B,
viem gan sieu vi C, giang mai, giii frong PBS
155
Trin Le Bao Ha et al
(Gibeo)
latih
cd khang sinh va chuyen vl phong thi
nghiem, thao tie frong vong 4 h.
Phirong
phap
xfr

ly mang oi (Hassan et al, 2008)
Mang oi duge rira frong dung dich PBS cho den
khi sach mau, sach eac md song khong can thiet. Sau
do,
lac mang oi frong dung dich
trj'psin
0,25%) -
EDTA 0,02% (Sigma) frong 30 phiit a
37°C
va dimg
mgt dung cu mem cao bo lap te bao bieu mo HAM,
franh lam va mang day. Hieu qua boc tach lap te bao
bieu mo mang oi dugc danh gia bang phuang phap
nhuom hematoxylin va eosin (nhuom HE).
Phuong phap phan lap
te
bao simg (Susan, 2003)
Mau da dugc rira
3
lan
frong
PBS va loai bo phan
thira (long, ma). Sau do, eat mau da
thatih
manh nho
va u vai Trypsin-EDTA (Gibeo) frong
18
h, 4°C. Lop
bieu bi va lap trung bi duge tach rai bang ea hgc, thu
nhan te bao simg rai tit lap bilu bi bing each dung

pipet de
huyIn
phu. Mat do tl bao,
t>;
le
%>
te bao
song dugc xac dinh bing each
dim
te bao sau khi
nhuom trypan blue.
Phuong phap nuoi, nhan tl bao sirng (Susan,
2003)
Te bao tach rai dugc nuoi frong moi truang sa
cap:
DMEM bo sung FBS (Fetal Bovine Seram),
EGF (Epidermal
Grov^fth
Factor), hydrocortisone,
cholera toxin (Sigma) vai mat do 3
x
10^
te
bao/cm^
be mat
dia
nuoi. Sau hai ngay, thay moi traang sa
cap bang moi traang khong huyet thanh SFM
(Seram Free Medium - Gibeo) danh cho te bao simg.
Tiep tuc nuoi a

37°C,
5%
CO2
eho den khi tl bao
tang
tradng
dat 70 - 80% be mat
dia
nuoi va ciy
chuyen.
Te bao sirng
Mang collagen
Loc cua
dia
long
Moi tru'd'ng nuoi
Hinh 1. Minh hoa phuang phap nuoi cay te bao trong dTa
I6ng.
Cac
tg
bao
sCrng
dugc phai ngoai khong
khi,
dinh du&ng
du'gc cung cap
tCr
moi tru'ang ben
duai
qua cac

16
lpc cua dTa
I5ng.
Phuofng
phap nuoi te bao simg tren mang
collagen
Mang collagen dugc frai fren day
ciia dia ling
(dia
insert) va cay chuyin tl bao simg len mang
collagen vai mat do 3
x
lO''
tl
bao/cm^bl
mat mang,
u a
37°C,
5%
CO2.
Moi traang dugc thay ben ngoai
va ben frong
dia
long
mli
2
ngay/lin.
Phuong phap tao tam nhilu
Idfp
tl bao simg

Khi te bao simg tang sinh kin bl mat collagen,
hiit
het moi
tradng
frong
dia ling
ra. Tilp tuc thay
moi
tracmg
ben ngoai dia
ling
mli ngay, keo dai 7
ngay.
Phuong phap xac dinh te bao simg bang RT-PCR
RNA
ting
dugc ly trich bing Trizol (Sigma). Sy
bilu hien gen duge danh gia bing One step RT-PCR
(Promega). Su bieu hien gen P-aetin nguai duge su
dung lam doi ehiing.
Trinh ty eua cac primer: p63 (5'- CAG ACT
CAA TTT AGT GAG -3' (sense), 5'- AGC TCA
TGG TTG GGG CAC -3' (antisense)), 440 bp;
involuerin
(5'-TCC TCC AGT CAA TAC CCA TC-
3'
(sense), 5'- CTT CAT TCC CAG TTG CTCA.
TC-3'
(antisense)), 373 bp; K14 (5'- ATG ATT
GGC AGC GTG GAG -3' (sense)

5'-
GTC CAG
156
Tgp chi
Cong
nghe
Sinh
hoc
7(2):
155-160,
2009
CTG TGA AGT GCT T -3' (antisense)), 390 bp; K5
(5'-
CCC AGT ATG AGG AGA TTG CCA ACC
-3'
(sense), 5'- TAT CCA GAG GAA ACA CTG CTT
GTG
-3'
(antisense)), 475 bp; P-actin (5'- CCA AGG
CCA ACC GCG AGA AGA TGA C -3' (sense), 5'-
AGG GTA CAT GGT GGT GCC GCC AGA C -3'
(antisense)), 587 bp (Adriana et al, 1998; Adriana et
al,
2001;
Mazlyzam et al, 2007; Virve et al, 2007).
Phan ling RT-PCR dugc thuc hien nhu sau: 45
phiit a
45°C,
3 phut d
94°C,

PCR 35 chu ki (45 giay
a
94°C,
30 giay d
55°C,
90 giay d
72°C),
ii
10 phut d
72°C
bang Thermocyeler cua Eppendorf. San pham
PCR dugc dien di fren gel agarose 1,5%.
Phuong phap nhan dien te bao simg con kha
nang tang sinh
Cac te bao simg cdn kha nang tang sinh frong
tam te bao dugc nhuom hda mo mien dich vdi
marker te bao goc bieu bi la
p63.
Sau khi tao ldp, tam te bao simg dugc cat lat va
nhudm vdi khang the p63 ddng 4A4. Ket qua dugc
quan sat va chup hinh dudi kinh hien vi quang hgc
Olympus.
KET QUA VA
BAN
LUAN
Ket qua nuoi, nhan te bao simg
Sau khi dugc phan lap va nuoi trong mdi tradng
khdng huyet thanh, cac te bao simg bat dau tao thanh
cum vao ngay 3-4. Sau dd, cac cum nay ldn dan va
hgp vao nhau tao thanh ldp dan te bao tren be mat

dia
nuoi sau 7 ngay.
Ket qua xir ly loai bieu mo mang oi
Sau khi xu ly vdi enzyme (trypsin-EDTA) 30
phiit va ca hgc (cao) toan bg ldp bieu md mang di da
dugc loai bd. Ket qua nay
phii
hgp vdi mgt sd edng
frinh da duge cong bd (Hassan et al, 2008; Ishino et
a/,
2004).
Ket qua nuoi te bao simg tren mang collagen
Sau khi xit ly, mang collagen dugc frai len
dia
long vdi mat bieu md dua len fren. Dieu nay giiip
eho tl bao simg dugc cay len
se
tiep xuc tryc tilp vdi
bl mat mang ca ban
eiia
mang li.
Kit
qua eho thay mang collagen kich thich tl
bao bam dinh nhanh ban. Mang ca ban cua mang li
gdm laminin 5, collagen loai IV, VII, XVII, tuang ta
vdi mang ca ban cua da ve hinh thai va sieu cau tnic
(Nakamura et al, 2004). Do dd, cac protein ngoai
bao nay se giup te bao bam dinh nhanh va di cu.
Sau 7 ngay nudi tren mang collagen, eac te bao
simg da tao thanh ldp dan.

Ket qua xac dinh te bao simg bang RT-PCR
Sau khi tao nhieu ldp fren mang collagen, cac
tam te bao dugc xac dinh bang phuang phap RT-
PCR vdi cac marker te bao simg.
Ket qua cho thay, cae te bao bieu hien ea 4
marker te bao simg, frong do cd 3 marker te bao
chua biet hda la p63, k5, kl4 va marker te bao da
biet hda la involuerin.
Cac te bao simg frong tam te bao in vitro bieu
hien duang tinh eac marker nhu cac te bao simg
trong md da ddi ehiing. Dieu nay cho thay cae te bao
dugc biet hda tao ldp van cdn giii tinh gde.
tao nhieu lop te bao
simg
tren mang Ket qua
collagen
Sau 7 ngay tiep xuc khdng khi, mau md duge
nhudm hda mien dich vdi khang the
p63.
Ddi ehiing
la mau da nguyen. Ca hai mau deu ed te bao duang
tinh vdi marker tang sinh, chua biet hda p63. Mau
tam te bao in vitro ed 4 - 5 ldp te bao simg vdi cac te
bao duong tinh p63 nam sat fren mang collagen, eae
te bao ben fren
la
nhiing te bao simg da biet hda. Ket
qua nay phu hgp vdi dac diem md hgc
eiia
da vdi cac

te bao gde bieu bi duong tinh p63 nam d ldp day,
ngay fren mang ea ban.
Sau khi tao ldp, tam te bao simg dugc tach ra de
dang khdi be mat
dia
nudi bang each keo lot mang
collagen. Nhu vay, mang collagen tit mang di la vat
mang ly tudng eho tam te bao simg.
KET LUAN
Mang di da duge loai bd hoan toan ldp bilu md
sau khi xir ly enzyme va ca hge, thu duge mang
collagen.
Cae te bao simg da tang trudng, tao 4 - 5 ldp tren
mang collagen va bieu hien marker tl bao simg da va
ehua biet hda sau 7 ngay tiep xue khdng khi. Cac tl
bao bieu hien marker te bao gdc bilu bi (p63) nim
ngay fren mang collagen.
Vdi ket qua thu dugc, hudng nghien cim ed thl
tien xa ban frong
iing
dung lam sang dl dilu tri cac
tdn thuang mit da fren benh nhan.
157
Trin Le Bao Ha et al
Hinh 2. A. Cac cum (40X) va lap dan te bao
sOng
trong dTa
nuoi;
B Cac cum
(100X)

va lap dan te bao
sOng
trong dTa
nuoi;
C. Mang ol nguyen ven (100X); D. Mang
61
da loai hoan toan lap bieu mo (100X). E. Te bao
sCrng
bam tren dTa nuoi sau 3 h
cSy
chuygn
(100X);
F.
T§
bao
sCrng
bam tren mang collagen sau 3 h cay chuyen (100X).
Hinh 3. Lap dan te bao
sOng
tren mang collagen (40X).
158
Tgp
chi
Cong nghe Sinh hoc 7(2):
155-160,
2009
M
1 2 3 4 5
Hinh
4. Ket qua

RT-PCR
cac
marker
cua
te
bao
sOng.
M.
Thang
marker;
1,
3,
5,
7,
9.
San
pham khuech
dai cac gen cua
t4
bao
sirng
nuoi
cay
tu'ong
iing
p63 (440 bp), k14 (390 bp),
k5
(475 bp),
involuerin
(373 bp),

p-actin
(587 bp);
2,
4,
6, 8, 10.
San
pham khuech
dai gen cua mau da
binh
thuang
doi
chung
tuang
u'ng p63, k14, k5,
involuerin,
p-actin.
Hinh
5.
Ket qua
nhupm
hoa
mo
mien
djch
vdi
khang
the
p63
mo
bleu

bi
tren
mang
collagen
(A) va
mau
da
doi
chCrng
(B)
(400X).
Cac te bao bat mau nau:
duang tinh
p63.
Ldi
cam on:
Chiing
toi
chdn thdnh
cdm cm
Benh
vien Chdn thuong chinh hinh, Binh vien
Phu sdn
Hiing Vuong
da
cung cdp
mdu da vd
mdng
oi
nguai

de tien hdnh nghiin
cuu
ndy.
TAI LIEU THAM
KHAO
Adriana
C,
Arzu
S,
Sandrine
W,
David FF, Stan
PCB
(2001) SPRR4,
a
novel comified envelope precursor; UV-
dependent epidermal expression
and
selective
incorporation into fragile envelopes.
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Cell
Sci
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3837-
3843.
Annie
Y,
Mourad
K,

Yunmei
W,
Emily
G,
Mark
DF,
Volker D, Nancy
CA,
Daniel
C,
Frank
M
(1998) p63,
a
p53 Homolog at 3q27-29, Encodes Multiple Products
viii'tx
Transactivating, Death-Inducing,
and
Dominant-Negative
Activities. Mot Cell 2 :
305-316.
Barbora D, Zuzana H, JiRi V, Radana, Zuzana K, JiRi M,
Martin
P
(2003) Reconstraction
of
epidermis
by
grafting
of keratinocytes cultured

on
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P,
Abolhassan
A
(2008) Properties
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Y,
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T,
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CJ,
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NJ,
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(2007) Human amniotic membrane as
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I,
James
SE,
Rubin
P,
Martin
R
(2003) Upward
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in
Integra
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preliminary report.

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MM, Fauziah O,
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MR, Saim L, Ruszymah
BHI
(2007)
Reconstraction of living bilayer human skin equivalent
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Inatomi T, Sotozono C, Shimizu Y, Kinoshita S (2004)
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MM (2003) Human placental amnion is a novel subsfrate
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319-330.
CREATION OF MULTILAYERED KERATINOCYTE SHEET ON COLLAGEN FROM
HUMAN AMNIOTIC MEMBRANE
Tran Le Bao
Ha''
*,
Tran Cong
Toai^
Hoang Nghia
Son^
'University of Natural Sciences, Hochiminh City
^Pham
Ngoc Thach University of Medicine, Hochiminh City
^Institute
of Tropical Biology
SUMIVIARY
Epidermis composed of terminally differentiated stratified squamous epithelium is the outermost layer of
the skin. The epidermis is the major barrier protect the body from inhospitable environment factors such as
biological, chemical, physical factors. Epidermis which consists of the multilayered keratinocytes could be
created in vitro for the freatment of acute burn injuries and chronic wounds. In this study, the multilayered
keratinocyte sheets were created from the adult human keratinocytes and the amniotic collagen membrane.
Human keratinocytes were isolated, cultured, amplified in seram free medium. Collagen membrane was
derived from human amniotic membrane of which the epithelium was removed and evaluated by HE staining.
After subcultured on collagen membrane, keratinocytes were stimulated for proliferation and differentiation to
create multilayer by airlifting. The multilayered keratinocyte sheet characteristics were evaluated by using RT-
PCR and immunohistochemistry. The results showed that epithelial cells were removed completely from the
human amniotic membrane by enzyme and mechanical treating. Keratinocytes proliferated and formed 4-5
layers on collagen. They also expressed some differentiation and proliferation markers at 7 days after airlifting.
The keratinocytes which were positive with proliferative marker (p63) lied closely on the collagen membrane
and upper

ofthe
cells differentiated.
Keywords:
Amniotic
membrane,
collagen,
epidermis,
keratinocyte,
serum
free
medium
' Author for correspondence: Tel: 0988575507; Fax: 84-8-38350 096; E-mail:
tlbha&.hcmuns.edu.vn
160