Hpl NGHI KHOA HQC CCiA Hpl H 6 A SINH Y Dt/qC H A N Q I V A C A C T I N H P H I A B A C tJ^N THLf XVlll
NuOl CJLY BIET H6A TE BAO GOC MANG 6\ THANH Ti BAO DA KERATIN
T 6 M TAT:
Td bdo g6c mdng Al Id cdc td bdo gdc da tidm ndng ChOng c6 thd du'p'c nudi cdy tdng sinh vd bi^t hda thdnh td bdo da bao gdm cdc td bdo keratin (keratinocyte), Mijc tidu cua dd tdi nghidn ci>u qud trinh bl$t hda td bdo gdc phdn ldp ti>
mdng di thdnh td bdo gldng td bdo da keratin Phi/o-ng phdp nghidn cCru: Nudi cdy td bdo trong mdi tm-dng DMEM bd sung vitamin C, BMP-4 vd cdc ydu td sinh tru'dng. Kdt qud nghidn citu:
Marker ciia td bdo da CK-14. CK-19 tdng Idn theo thdi gian Kdt ludn Td bdo gdc mdng di c6 thd bidt hda thdnh td bdo gidng td bdo da keratin
Ti> khda: T4 bio gdc ming 6l, t4 bio da keratin, biit hda. BMP-A, vitamin C, CK-14.
ABSTRACT:
DIFFERENTIATION OF AMNIOTIC MEMBRANE STEM CELLS INTO KERATINOCYTES Amniotic membrane stem cells are multipotent stem cells They can proliferate and differentiate into skin cells including keratinocytes The aim of research; study of differentiation of amniotic membrane stem cells into keratinocytes Research methods culture cell in DMEM medium supplemented with vitamin C, BMP-4 and the other growth factors Research results Markers of keratinocytes CK-14. CK-19 increased with time Conclusion Amniotic membrane stem cells can differentiate into keratinocytes.
Key words: Amniotic membrane stem ceffs, keratinocytes. diffemnb'atKin. BMP-4, vitamin C, CK-U.
I. O A T V A N OE:
Trong bai bao ddng tren tap chi trudc day chiing tdi da cdng bd ket qua nghien ciiu qua
* Bi mdn hda sinh - HQC viin quin y
" Trung tim nghiin ciru y sinh du^ hpc - HQC viin 72
PhQm Vdn Trdn*, DSMinh Trung**
trinh bi^t hda cua te bdo gSc mdng oi thdnh t^
bdo gan, l^ bdo t\iy [ I ] , Hi?n nay nhieu trung tdm nghiSn curu trong nudc vd Iren ih^ gidi dd thdnh cdng trong vi^c su d^ing t^ bdo goc trudng thdnh, tc bdo g6c phdi d^ bi?t hoa thdnh cdc I09I tc bdo khde nhau phyc v\i cho cay gh^p [2]. Tuy nhiSn ngu6n te bdo ndy cd hgn vd thudng gdp nhieu tranh cdi ve van dl dgo dire.
Mdng oi Id mpt sdn pham thudng bb di trong qud trinh sinh nd Id mgt nguon cung cap te bdo g6c Iy tudng [3]. Sir dyng t^ bao gdc mdng oi khdng gdp phdi nh&ng van dl ve dgo due, xd hgi. Cac te bao gdc phan lap tir mang oi cd tinh sinh mien djch thap, khong cd khd ndng ung thu hda vd cd khd ndng bi?t hda thdnh nhieu logi te bdo khac nhau [4].
Da ngucri vd dpng \at cd vii la mpt to chuc ddc bi?t bao gom nhOng quan \hk tk bao day ddc trong dd te bdo keratin con gpi la tl bao simg chiem iiVi 95%. Di^u d$c bi?t quan trpng la trong to chiic da cd cdc te bao goc bieu bi. Chung cd kha ndng tdng sinh manh truac khi bipt hda thdnh nhilu loai t l bao khac cua da ddng thdi ludn tdn tgi mpt nhdm tl bao cd kha ndng tdng sinh tren mang day. Tuy nhien trong cdc trudng hgp mat da, bdng nSng can cd bipn phap ghep da, ghep t l bao. Viec tim ra ngudn te bdo phyc vu cho phucmg ph£^
tri li?u te bdo gdc nga\ cang trd nen cdn thi^i Vi \d\. chiing tdi tien hdnh de tai vdi muc tieu nghien ciiu quy trinh biet hda t l bao gdc mang di thanh te bdo gidng te bdo da nhdm muc dich cd the sii dyng te bao nay san xudt da nhan tao hoac sir dyng cho cdy ghep.
quiny
Y HQC VI$T NAM T H A N G 8 • 2>2012
II, OOI TUONG VA PHUONG P H A P NGHIEN CCIU;
2.1. Phan ISp vi nuoi c^y bift h(5a te bao goc tir mang oi thinh te bio keratin
Quy trinh phan lap te bao goc tit ming oi da dir<jc chiing toi dSng tren t jp chi trudc day [1], Sau khi phan Ijp, t^ bao dugc nuoi c^y trong moi trucmg co bdn bao gom DMEM (Dulbecco's Modified Eagle's medium) c6 b6 sung them penicillin (50 U/ml).
streptomycin (50 ng/ml), L-glutamin (2 x 10- 3M), hu>C-l thanh bao thai be (10%) dong thai cay chuyen te bao 2 tu3n mpt !3n de duy tri trong ph6ng thi nghidm.
Bi?t hoa te bao goc thinh te bao da bang each nuoi cay ting sinh t^ bao 7 ngiy trong moi trucmg co ban. Quan sat thiy ti bao bao phu l(in dia nuoi cay. Sau do bo sung vao moi trudng ca ban cac ;cu to djnh huong biet hoa thanh te bao keratin bao gom 5 pg/ml insulin, 1 pM hydrocortison. 0,5 nM BMP-4 va 50
Bdng I: Cac moi dung de ch^y RT-PCR.
Hg/ml vitamin C. Thu hojch ti bao vio ngiy thli 1 (Nl) ngiy thii 7 (N7) vi ngiy thii 14 (nl4) sau khi bo sung yeu t6 bi$t h6a vio moi trubfng nuoi cay.
2.2. Ky thuft RT-PCR xie djnh bieu hivn ciia ARN thong tin (ARNtt):
Tich chiet ARN t6ng s6 tir cic te bao sfir dyng kit (Qiagen) sau d6 tign hinh t6ng h(;rp cDNA tit ARN (Kit - Fermentas). Phin ling PCR djnh luiyng dupc thyic hi?n tren miy LightCycler 1.5 (Roche Diagnostics) sir d\ing kit QuantiTect* SYBR* Green PCR (Qiagen). Sau khi lam bien tinh cDNA 15 phut t) 95°C. tir 40 dSn 50 chu ky PCR dugc thvc hi^n (15 giay: 95°C, 25 giay: 58°C vi 20 giay: 72°C). Nong dp ARNtt ciia timg dau an nghien ciiu dugc tinh toin dua tren ndng dp ARNtt ciia gen ribosom 18S. CJp chit moi dac hieu cho timg gen dugc mo ti trong bang sau.
Oct-4 (Octamer- bindinq protein 4}
Cytokeratine-19
Cytokeratine-14
Forward 5'-TGAGAAACGGCTACCACATC-3' Reverse 6'-TTACAGGGCCTCGAAAGAGT-3' Fo/warc/;5'-GAGGAGTCCCAGGACATGAA-3' Reverse, 5'-GTGGTCTGGCTGAACACCTT-3' Fonvard. 5'-AATATGAGGTCATGGCCGAGCAGA-3' Reveres 5'-TGTCTTCCAAGGCAGCTTTCATGC-3' Fofward 5-7"T"CT"GAACGAGArGCGrGAC-3' Reverse; 5'-GCAGC7-CAAT"CrCCAGGTTC-3'
2.3. Ky thuat Western blot
Ly giii te bio trong dung djch Tris-HCI 50 mmol/l, pH 7.5. Moi mau lay 10 pl chira 25 pg protein cho vio gieng. dien di tren gel acrylamide 10% (SDS-PAGE). Protein tren gel dugc chuyen len ming nitrocellulose, sau do dugc ii vai khang the chupt nhat (Sigma) khang OCT-4, CK-14, CK-19 cua ngudi. Protein phan irng vdi khang the thir nhat se dupc xac dinh bdi khing the thii hai gan vdi peroxydase (Sigma). Ket qua dupc bieu hien bang hinh anh tren phim sau khi tie dung vdi chat phat quang (ECL*. Amersham).
2.4. Ky thuat hoa mien dich te bao:
Hpl NtSHI KHOA HpC cClA Hpl H6A SINH Y DlJt?C HA NQI VA CAC TINH PHIA BAC l^N THtf XVlll Te bio dugc co djnh bang etanol 98% sau dd dupc ii vdi khing thl thir nhit khing vimentin, CK-14. CK-19 hojlc khing OCT-4. Khing thi thii hai dupc gin vdi chat huynh quang. Quan sit ti bio vi chgp hinh inh trgn kinh hien vi huynh quang.
III. KiT QuA NGHliN COU:
3.1. K^t qui nhu$m hda mign d|ch lc bio
Hinh I. Hinh anh nhugm hda mien djch te bio gdc ming di ngudi sau 7 ngiy nudi cay vdi cic diu in sinh hpc OCT-4, vimentin. CK-14, CK-19. DAPI: Nhupm nhan te bio.
Merge: Anh ldng ghep giiia hinh inh nhupm hda mien djch te bio ciia cic dau an nghien cuu vdi hinh inh nhupm nhan te bio.
3.2. Ket qua RT-PCR
Bleu hlpn ARNtt cua CK-14, CK-19 - Dau an te bdo goc blfl hoa thank le bdo giong te bdo keratin.
Sau 7 ngay nudi cay tren dTa plastic, nhdm cd bd sung trong mdi trudng nuoi cay BMP-4 vi vitamin C thay nong dp ARNtt ciia CK-14. CK-19 ting nh?. Sau 14 ngay nuoi cay. thay ting nong dp ARNtt cua cic dau an nay mpt cich rd r^'t (hinh 2). Ket qui phii hgp vdi bieu hien cua cic dau an niy d mirc dg protein xie djnh bang western blot (hinh 3).
II
01 2.5 .
^ t s i n
flrBn J J J I
' I Ngayl NgSy? Ng^yH
E ] a J]
mnh 2: Biiu hien ARNtI: (a) CK-14 vd (b) CK-19. Ndng dp ARNtt ting sau khi te bao dupc nuoi cay vdi vitamin C va BMP-4. Kit qui ARNtt dugc tinh toan so vdi nhdm chimg (arbitrary unit).
74
Y HQC VI$T NAM T H A N G 8 - 2/2012
3.3, Kit qua western blot: Bilu hi$n protein ciia OCT-4, CK-14 trong qui trinh bi^t hoa ti bio goc thinh ti bio keratin.
i ^^' y X
« M » «-•» - ' ^ 0CT4 (--45 kDa]
igpB HHgy • • • *
m
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t^tfu^ggt^t/M ActIn i
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Hinh 3: Bieu hi^n protein cda (a) 0CT~4 vd (b) CK-14. OCT-4 gidm dan Uong khi CK- 14 tdng dan theo thdi gian nudi cay. Actin du^c sir dyng de tham chieu l u ^ g protein dugc cho vao moi gieng di?n di.
IV. BAN LUAN:
Bi^t hoa te bdo goc mdng oi thdnh tc bao keratinocyte
Cac te bdo goc mdng di bieu hi?n nhieu marker te bao goc nhu octamer-binding transcription factor 4 (OCT-4), GATA-4.
hepatocyte nuclear factor-3p (HNF-3P)...
NhiJng yeu to nay cho thdy te bdo goc mang oi Cling la te bao goc da tiem nang. Trong khuon kho de tai nay, chiing toi dung ddu an OCT-4 de xdc dinh tinh goc ciia te bdo. De te bdo biet hoa thanh te bdo keratin, sau khi nuoi cay trong moi truong ca bdn duac 7 ngay, chung toi bo sung them vitamin C vd BMP-4.
Ndm 1996, Bagutti vd CS da thanh cong trong viec biet hoa te bdo goc phoi thanh te bdo CO bilu hien marker cua te bao bieu bi [5]. Cdp CK-8/CK-19 la nhiJng keratin ddu tien bieu hi?n trong qua trinh nuoi cay te bao phoi a ngay thii 15. Ti6p sau do Id biiu hi?n cua CK-14, CK-10 vd involucrin chi ra giai doan kk thuc biet hoa thdnh te bao keratin [5]. Thilu 131 integrin ldm anh huong den su biet hoa cua ti bao g6c thdnh te bao keratin, tuy nhien tac dung irc che nay co the lam dao ngugc nha them vao moi truong Keratinocyte Grow Factor (KGF) va
Epithelial Grow Factor-10 (FGF-IO), hai yeu to hda tan dugc tiet ra bdi cac nguyen bao sgi (fibroblast) cua da [5].
BMP-4 thugc nhom transforming growth factor-fi (TGF- B) dd dugc chung minh trong thvc nghi?m vd khd ndng djnh huong bi?t hoa te bao goc phoi thanh te bao keratin. Mpt so tdc gia da thanh cong trong biet hda te bao te bao keratin thanh to chiic giong bieu bi [7]. Cytokeratin K14 va CK-K19 Idn lugt bieu hien dong thai vai collagen IV, VII, laminin-1, -5, integrin a6p4, fibronectin vd nidogen [6]. Nicoden la dau an dugc san xuat boi nguyen bdo sgi cua da trong giai d o ^ sam cua su hinh thanh da khi phat trien phoi [7]. Su CO mat cua nicoden chiing to trong nhom te bdo biet hoa c6 nguyen bao sgi (fibroblast). Laminin-1 binh thuang chi co a fibroblast [7]. Ket qud chi ra rdng te bdo goc mang oi co the bi?t hda thanh te bdo da in vitro trong do BMP-4 va cac yeu to trung bi gift vai tro ddc biet quan trpng.
Su CO mdt ciia cdc te bdo goc da da tiem nang khu tru a trung bi, goc long va tuyen bd cho phep da tu lam mai lap bieu mo phii va cac phan phu ciia chiing [8]. Nhung te bao keratin co kha nang tu lam mai cao se co the la nguon te bao phuc vu trong y hpc tai tao 75
Hpl NGHI KHOA HQC CpA Hpl H 6 A SINH Y DU(?C
d$c bi$t trong ITnh vyc dilu trj bdng.
Qud trinh bi?t hda lii- bdo g6c thdnh ti bdo keratin c6 the chia thdnh hai giai do&n:
Giai dogn bi?t hda chiic ndng, tiic Id giai do9n md cac iC- bdo ghc bdt dau bieu hi^n cdc d^u an d$c hi?u ciia tc bdo keratin. Giai dogn thli hai Id giai dogn bi?t hoa ve hinh thdi, cdc te bdo keratin sdp xep tgo thdnh lo chiic trung bi vd bit'u bi. Nghidn ciru ciia chiing tdi thay.
6 ngdy thii 7 thi dd co sy bieu hi?n rd rang ciia CK-14. CK-19, vimentin d ca miic dp ARN vd protein. Sd dt chung toi chgn dong thdi nhieu dau an vi tren thyc te chua co dau an ndo thyc sy chi d$c hiai cho te bdo keratin. Khi te bdo goc bi?t hda thanh te bdo keratin thi bieu hi?n ciia cdc dau an ndy tdng len. Nhu vay ve chiic ndng, to bao goc dd trd thanh te bdo co chirc ndng ciia te bdo keratin.
Tuy nhien de te bdo goc phdt trien hodn todn thdnh te bdo keratin, tgo cac cau triic bieu bi, trung bi, can phdi co moi trudng ngogi bao dgc biet. Moi trudng ngogi bdo (extracellular matrix-ECM) cung cap cau triic khong gian CO vai tro quan trpng trong qua trinh tdng sinh, di chuyen vd biet hoa cua te bdo. Cam img biet hoa thdnh te bao keratin hieu qua nhat da dugc thu dugc khi te bao goc dugc nuoi cay tren gia dd co nguon goc tir cac nguyen bao sgi binh thudng ciia ngudi (HNF) va N1H-3T3 tl bao, cd hai deu co nguon goc trung mo [6]. Trong mo hinh nghien ciiu ciia chiing toi, te bao bieu hi?n K14 sau 7 ngay nuoi cay va tdng dan den thdi diem 14 ngay. Chiing loi mdi chi dirng lai d giai dogn cac te bao bieu hien cac ddu an ciia te bao keratin - giai dogn dau tien ciia qua trinh hinh thanh da.
Qua su bieu hi?n ciia dau an sinh hpc da chirng to cac te bao goc mang oi da co sir bi?t hoa thanh lc bao giong te bao keratin ciia da
HA NOI VA CAC TINH PH(A BAC LAN TH(S XVIII
ngudi. Nghifin ciru cua chiing toi cung phii hgp vdi cdc tdc gid khde. De tdi cdn dugc tilp tMC nghiSn cim.
T A I Llf U T H A M K H A O :
1. Phpm Vdn Iran vd c§ng sy (2011). Phan l§p vd nudi cdy bi?t hda te bdo gdc thanh te bdo gan vd tc bdo beta. Tgp chi y hpc Vietnam, s62/2011.38-43.
2. Korbling M, Estrov Z (2003). Adult stem cells for tissue repair a new therapeutic concept? N Engl J Med 349:
570-582.
3. Kubo M, Sonoda V, Muramatsu R, Usui M (2001). Immunogenicily of human amniotic membrane in experimental xenotransplantation. Invest Ophthalmol Vis Sci. 2001 Jun;42(7): 1539-46.
4. Ornclla Parolini, Francesco Alviano et all (2008). Concise review: Isolation and characterization of cells from human term placenta: outcome
5. Bagutti C. \^obus A. (1996).
DifTcixMitiiition of embr>'onal stem cells into keratinocytes: comparison of wild- type and beta 1 integrin-dcficient cells.
Dc\.Biol. 179: 184-196.
6. Corcaux C , Hilmi C. et all (2003).
Rcconstiluicd skin from murine embryonic stem ceils. Curr. Biol. 13: 849-53.
7. Albcrdam D (2004). Derivation of keratinoc\to progenitor cells and skin formation from embryonic stem cells. Int.
J. Dev, Biol, 48: 203-206.
8. Taylor G. et all (2000). Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell.
102:451-61.
