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
Trang 1T A O J A M T E B A O SUlVG N H I E U LOHP T R E N M A N G C O L L A G E N TlT M A N G 6 I
N G l T o a
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
I ! • I C • • ••
Tit khoa: Bieu bi, collagen, mdng oi, moi truang khong huyet thanh, te bdo simg I
M d D A U
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 l i 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 P H A P
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
Trang 2(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
Trang 3CTG 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 B A N 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
Trang 4Hinh 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
Trang 5M 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 K H A O
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 J Cell Sci 114:
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 polymer support - clinical
study Inter J Dermatol 42: 219-223
Hassan N, Habibollah P, Abolhassan A (2008) Properties
of HAM in tissue engineering Eur Cell Mater 15: 88-89
Ishino Y, Sano Y, Nakamura T, Connon CJ, Rigby H, Fullwood NJ, Kinoshita S (2004) Amniotic membrane as a carrier for cultivated human corneal endothelial cell
transplantation Invest Ophthalmol Vis Sci 45: 800-806
Jin CZ, Park SR, Choi BH, Lee KY, Kang CK, Min BH (2007) Human amniotic membrane as a delivery matrix for
articular cartilage repair Tiss Eng 13: 693-702
Jones I, James SE, Rubin P, Martin R (2003) Upward migration of cultured autologous keratinocytes in Integra
artificial skin: a preliminary report Wound Repair Regen
Trang 611:132-138
Masayuki Y, Mika U, Ai K, Chie K, Akihiko K, Terao O
(2001) Thermo-responsive culture dishes allow the intact
harvest of multilayered keratinocyte sheet without dispase
by reducing temperature Tissue Eng 7: 473-480
Mazlyzam AL, Aminuddin BS, Fuzina NH, Norhayati
MM, Fauziah O, Isa MR, Saim L, Ruszymah BHI (2007)
Reconstraction of living bilayer human skin equivalent
utilizing human fibrin as a scaffold Burn% 33: 355-363
Nakamura T, Yoshitani M, Rigby H, Fullwood NJ, Ito W,
Inatomi T, Sotozono C, Shimizu Y, Kinoshita S (2004)
Sterilized, freeze-dried amniotic membrane: a useful
subsfrate for ocular surface reconstraction Invest
Ophthalmol Vis Sci 45: 93-99
Oyama N, Bhogal BS, Carrington P, Gratian MJ, Black
MM (2003) Human placental amnion is a novel subsfrate
for detecting autoantibodies in autoimmune buUous
diseases by immuno-blotting Br J Dermatol 148: 939-944
Raymund EH, Markus D, Gilbert W, Bjoem S (2000)
Cultured human keratinocytes on type 1 collagen membranes
to reconstitute the epidermis Tiss Eng 6: 53-67
Raymund EH, Jurgen K, Ulrich K, Justus B, Alexander
DB (2005) Tissue engineering of cultiired skin substitutes
J Cell Mot Med 9: 592-608
Susan SY (2003) Current Protocols in Cell Biology John
Wiley Sons, Inc : 155-160
Tseng SC (2001) Amniotic membrane transplantation for
ocular surface reconstraction Biosci Rep 21: 481-489
Ueno M, Matsumura M, Watanabe K, Nakamura T, Osakada F, Takahashi M, Kawasaki H, Kinoshita S, Sasai
Y (2006) Neural conversion of ES cells by an inductive
activity on human amniotic membrane matrix Proc Natl Acad Sci USA 103: 9554-9559
Virve P, Jussi V, Tuula S, Leo T (2007) Effects of TGF-bl
on interleukin profile of human dental pulp and
odontoblasts Cytokine 40: 44-51
Zhu N, Warner RM, Simpson C, Glover M, Hemon CA, Kelly J, Eraser S, Brotherston TM, Ralston DR, MacNeil S (2005) Treatment of bums and chronic wounds using a new cell fransfer dressing for delivery of autologous
keratinocytes Eur J Plast Surg 28: 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